extfieldsofstudy: Medicine Biology

text:
Involvement of Cell Cycle Elements, Cyclin-dependent Kinases, pRb, and E2F·DP, in B-amyloid-induced Neuronal Death*

Previous evidence by others has indicated that a variety of cell cycle-related molecules are up-regulated in brains of Alzheimer’s disease patients. The significance of this increase, however, is unclear. Accordingly, we examined the obligate nature of cyclin-dependent kinases and select downstream targets of these kinases in death of neurons evoked by B-amyloid (AB) protein. We present pharmacological and molecular biological evidence that cyclin-dependent kinases, in particular Cdk4/6, are required for such neuronal death. In addition, we demonstrate that the substrate of Cdk4/6, pRb/p107, is phosphorylated during AB treatment and that one target of pRb/p107, the E2F·DP complex, is required for AB-evoked neuronal death. These results provide evidence that cell cycle elements play a required role in death of neurons evoked by AB and suggest that these elements play an integral role in Alzheimer’s disease-related neuronal death.

Alzheimer's disease (AD) 1 is a neurodegenerative disorder marked by progressive loss of memory and impairment of cognitive ability. Patients with AD display neuropathological lesions including amyloid plaques, neurofibrillary tangles, and eventual neuronal loss in brain regions associated with cognitive function (1). The amyloid plaque, an invariant pathological hallmark of AD, is composed of B-amyloid (AB), a 39 -43-amino acid residue hydrophobic peptide that assembles into insoluble aggregates (2,3). It is derived by alternative proteolysis from an integral membrane localized B-amyloid precursor protein (BAPP) (4). The importance of AB in progression of AD is underscored by the identification and linkage of mutations of BAPP and the presenilins in familiar cases of AD (5)(6)(7). Presenilins are transmembrane proteins thought to be involved in BAPP processing and trafficking (4). Additional support for the importance of AB in AD comes from reports demonstrating the neurotoxic properties of aggregated AB (7) The mechanism by which AB causes neuronal death is not well understood.
Recently, several groups have reported abnormal up-regulation of a variety of cell cycle proteins in brains from AD patients (8 -10). However, it is unclear whether these deregulated cell cycle events contribute to neurodegeneration in AD or whether they are a byproduct of a stressed brain. The cell cycle is a tightly regulated process controlled by sequential activation of cyclin-dependent kinases by up-regulation of its obligate activating cyclin partner (11). Generally, it is thought that Cdk4/6 and cyclin D1 complex regulate the G 0 to G 1 transition, whereas Cdk2/cyclin E and Cdk3 control G 1 to S transitions. Finally, Cdk2/cyclin A and Cdc2/cyclin A are believed to control G 2 and M progression. One important target substrate for the CDKs is the tumor suppresser, retinoblastoma protein (pRb), which is phosphorylated by activated Cdk4/6/cyclin D (13). Once hyperphosphorylated, Rb is released from the transcription factor complex E2F⅐DP, which then activates genes required for S phase transition (14). Interestingly, it is up-regulation of several of the above discussed cell cycle control elements that have been reported in brains of AD patients. They include cyclin B, D, and E, Cdc2, and Cdk4 (8 -10). In addition, increased Cdc2 activity has also been shown in AD brains (9).
In this report, we test the hypothesis that cell cycle elements play an obligate role in neuronal death evoked by AB. We provide support that certain cell cycle-related elements, Cdk4/6 and E2F⅐DP, are required for AB-induced neuronal death and that this signaling pathway may be a required component of neurodegeneration in AD.

EXPERIMENTAL PROCEDURES
Generation of Recombinant Sindbis-Recombinant sindbis virus expressing FLAG epitope-tagged DN Cdk2, 3, 4, and 6 as well as FLAGtagged mutants of DP1 containing either the E2F-binding domain or DNA-binding domain deletions were constructed as described previously (15,16). Control nonexpressing vectors were generated by eliminating the initiating codon of each inhibitor and in the case of Cdk3, Cdk4, and Cdk6, introducing a premature stop codon. All mutations, deletions, and FLAG tags were introduced by polymerase chain reaction as described previously and confirmed by sequencing (15,16). Viral particles were generated by in vitro transcription and transfection into baby hamster kidney cells and titered by plaque assay as described previously (15,16).
Culture and Survival of Neuronal PC12 Cells-PC12 cells were differentiated by treatment with nerve growth factor as described previously (17). The cells were plated onto 96-well plates (coated with collagen I) at a density of 5,000 or 20,000 cells/well. Prior to addition of peptides, cultures were rinsed once with RMPI 1640, and the medium was changed to RMPI 1640 plus penicillin/streptomycin AB (1-40, Bachem CA, Torrence, CA) was added to the neuronal PC12 cell cultures at a concentration of 100 g/ml. TUNEL and LDH assays were performed according to the manufacturers instructions (TUNEL, in situ cell death detection kit, Roche Molecular Biochemicals; LDH, Promegas CytoTox 96 nonradioactive cytotoxicity assay kit).
Culture and Survival of Cortical Neurons-Rat cortical neurons were cultured from embryonic day 18 rats as described previously (16). The neurons were plated into 24-well dishes (approximately 200,000 cells/ well) coated with polylysine in serum-free medium (N2:Dulbecco's modified Eagle's medium (1:1) supplemented with 6 mg/ml D-glucose, 100 g/ml transferrin, 25 g/ml insulin, 20 nM progesterone, 60 M putres-cine, and 30 nM selenium). For studies involving virally mediated expression, two days after initial plating, the neurons were infected at a multiplicity of infection of approximately 2 and incubated overnight. Three days after initial plating, the medium was exchanged with serum-free medium supplemented with pre-aggregated AB and flavopiridol (obtained from Hoechst Marion Roussel or the National Cancer Institute) where appropriate. AB was preaggregated by incubation incubation in serum-free medium at a concentration of 0.2 mg/ml at 37°C overnight. At appropriate times of culture under the conditions described in the text, cells were lysed, and the numbers of viable cells were evaluated as described previously (16). All experimental points are expressed as a percentage of cells plated on day 0 and are reported as the means Ϯ S.E. (n ϭ 3).
Western Blot Analyses-Cortical neurons were dissociated and cultured as described above. At appropriate times during AB treatment or 24 h after infection (expression analyses), the neurons were harvested in sample buffer, and 50 g of protein were loaded onto SDS-polyacrylamide gels and transferred onto nitrocellulose membrane as described previously. Blots were probed with anti-phospho-Rb antibody (New England Biolabs, 1:1000 dilution), anti-cyclin D1 (Santa Cruz, 1:300 dilution), or anti-FLAG antibody (10 g/ml; VWR).

Inhibition of AB-evoked Death by the Pharmacological CDK
Inhibitor Flavopiridol-The study of neuronal death in AD in vivo is made difficult by the lack of significant neuronal loss in mice models of AD expressing mutant BAPP or presenilins and the difficulty in genetic manipulations of neurons in vivo. Given this, we utilized an in vitro model of AB-induced neuronal death of cultured embryonic day 18 cortical neurons and neuronally differentiated PC12 cells. Although these systems may not completely reflect the in vivo processes involved in AD, we feel that such studies provides valuable insight into ABinduced death signaling events. Reports from many groups as well as our own experience has indicated that aggregated AB of various lengths, including 1-42, 1-40, and 25-35, cause the apoptotic death of CNS neurons (18,19). As shown in Fig. 1, AB , treatment results in the death of cortical neurons (70% death at 36 h). Equivalent treatment of neuronal PC12 cells also results in an increase in TUNEL positive neuronal PC12 cells and LDH release (Fig. 2).
To test for the obligate role of certain cell cycle elements in neurodegeneration, we reasoned that inhibition of such regulatory signaling would lead to neuronal survival. Because CDKs are both up-regulated in AD brains and play a central role in cell cycle progression, we first assessed the ability of the pharmacological CDK inhibitor flavopiridol to protect neuronal PC12 cells and cortical neurons from AB-evoked death. Fla- has been shown to inhibit CDK activity with high specificity and is less effective against epidermal growth factor receptor kinase and protein kinases C and A (20). Flavopiridol reduced TUNEL staining ( Fig. 2A) and LDH release (Fig. 2B) in neuronal PC12 cells treated with AB. Similarly, flavopiridol protected primary cortical neurons from AB-evoked death (75% survival with flavopiridol cotreatment versus 25% in the controls; Fig. 1). Most importantly, the concentration of flavopiridol required for neuroprotection (Fig. 1B) is the same dose reported to be required for inhibition of cell cycle progression (21). As shown in Fig. 3, cortical neurons treated with AB alone showed profound blebbing and shrunken phase bright morphology and degenerated processes, whereas neurons cotreated with flavopiridol demonstrated a flatter, more phase dark morphology typical of live cortical neurons.
Requirement for Cdk4 and 6 in Death of Cortical Neurons Evoked by AB-Although the above studies suggest the importance of CDKs in AB-evoked neuronal death, we also targeted CDKs using a more molecular biological approach to identify individual CDKs critical for regulating neuronal death. To do this, we expressed kinase dead dominant negative (DN) forms of CDKs via the recombinant sindbis virus. We have previously utilized this neuronotropic viral vector to target genes to both sympathetic and cortical neurons and to evaluate neuronal apoptotic signaling mechanisms (15,16). The DN CDK constructs were FLAG epitope-tagged to permit detection of the expressed protein. Expression of the CDKs were confirmed by Western blot analyses (Fig. 4A).
Expression of both DN Cdk4 (Fig. 5B) and DN Cdk 6 ( Fig.  5C) protected cortical neurons from death evoked by AB, whereas DN Cdk2 (Fig. 5A) and DN Cdk3 (data not shown) did not. To control against the effects of the virus, we infected cultures with recombinant virus containing each respective DN CDK construct with the initiation codon deleted and a premature stop codon inserted. No effect on neuronal survival was observed with the control viruses. These results suggest that Cdk4/6 play an important role in neuronal death evoked by AB.
Phosphorylation of pRb/p107, a Substrate of Cdk4/6 -The only reported substrates of Cdk4/6 activity is pRb and its re- lated family member p107 (12,13,22). If, as our pharmacological and DN CDK expression studies suggest, Cdk4/6 activity is required for death of neurons, we would predict that phosphorylation of pRb/p107 would increase during death of cortical neurons evoked by AB. To test this, we utilized an antibody directed against the phospho-epitope of pRb at serine 795 and its equivalent site in p107, sites known to be phosphorylated by Cdk4/6. As would be expected if Cdk4/6 was active, there was a transient increase in pRb/p107 phosphorylation 4 h after AB treatment of cortical neurons (Fig. 6).
Because an up-regulation of cyclin D1 may increase Cdk4/6 activity, we next determined whether an elevation in cyclin D1 protein could be detected during AB treatment of cortical neurons. Cyclin D1 protein was readily detectable in cultures of untreated control cortical neurons by Western blot analyses. However, cyclin D1 levels remained relatively constant during the time course of AB treatment (Fig. 6).
Requirement for E2F⅐DP Complex in Death of Cortical Neurons Evoked by AB-Phosphorylation and subsequent release of pRb/p107 would lead to E2F activation (12)(13)(14). Accordingly, we examined whether inhibition of E2F⅐DP results in increased neuronal survival. To test this, we expressed a mutant form of DP1, an obligate binding partner to the E2F family members, which contains a deletion in the DNA-binding domain (DP1 103-126) (23). This construct has been previously shown to act in a dominant negative fashion to inhibit cell cycle progression and E2F activity (23). As shown in Fig. 7, expression of DN DP1 protected cortical neurons from AB-induced death (75% survival with DN DP1 versus 35% without). No protective effect was observed with the control "stop" viruses or a mutant form of DP1, which has the E2F-binding domain deleted (DP1 233-272). The latter construct does not inhibit E2F transactivation (23) and was used as a control. Both DP1 constructs (DP1 103-126 and DP1 233-272) were FLAG epitope-tagged, and expression was confirmed by Western blot analyses (Fig. 4B). This findings taken together with the Cdk4/6 data suggest that Cdk4/6 activity may act to induce AB-evoked neuronal death via the actions of E2F⅐DP1 members. gesting that elements that normally control cell cycle progression in proliferating cells may also modulate neuronal death. Accordingly, we examined 1) whether CDKs play a required role in death of cortical neurons evoked by AB and 2) whether certain signaling elements downstream of CDKs (pRb and E2F⅐DP) are modified and/or required for death.
Involvement of CDK in Death of Neurons Evoked by AB-We show presently that the CDK inhibitor flavopiridol and expression of DN Cdk4/6 but not DN Cdk2 or 3 block death of PC12 cells and/or cortical neurons evoked by AB. These results imply that Cdk4/6 activity is an obligate signaling component of AB-induced neuronal death. In support of this observation, several groups have suggested that CDKs may play an important role in neuronal death. For example, NGF deprivation leads to increased Cdc2 activity and cyclin B expression in neuronal PC12 cells (24) as well as elevated cyclin D1 transcript levels in sympathetic neurons (25). Cyclin D1 protein is also increased in cis-platinum-treated sensory neurons (26). Furthermore, expression of the CDK inhibitor p21 is required for survival of differentiated neuroblastoma cells (27), and the CDK inhibitor p16 protects these cells from death caused by trophic factor deprivation (28). Pharmacological cell cycle blockers suppress death of sympathetic and cortical neurons evoked by trophic factor deprivation (21,29) and/or DNA damaging conditions (17,30). Finally, virally mediated expression of CDK inhibitors such as p16 and p27 and of dominant negative forms of Cdk4 and 6 inhibit the death of neurons because of the above mentioned apoptotic stimuli (15,16).
In contrast to the up-regulation of cyclin D1 in AD brains and in several other neuronal death paradigms (see above), we do not observe increased expression of this protein with AB treatment. Cyclin D1 is readily detectable in our untreated control cultured cortical neurons. The failure to see an increase in cyclin D1 levels may reflect on the embryonic nature of our culture system and suggests that activation of Cdk4 may occur via post-translational mechanisms. In support of this, we have observed an increase in cyclin D1-associated kinase activity in dying cortical neurons evoked by DNA damage (15). This increase is observed without change in levels of cyclins, CDKs or CDK inhibitors. 2 CDK activation may proceed through a different mechanism in the adult brain, perhaps requiring cyclin and/or CDK synthesis. role in apoptosis as well as cell cycle control. For example, overexpression of pRb in several cell contexts results in increased survival (31)(32)(33), whereas loss of Rb is associated with increased death (34,35). In addition, mice lacking in pRb display massive neuronal loss during development (36). Finally, phosphorylation of Rb has been shown to occur in dying neurons treated with cis-platinum (26) and camptothecin. 3 One consequence of the phosphorylation of pRb/p107 is release from and activation of E2F⅐DP complexes (12,13). Accordingly, we hypothesized that E2F⅐DP may play a required role in death of cortical neurons evoked by AB. Interestingly, several groups have suggested that E2F, like pRb, may play a role in cell death. For example, overexpression of E2F in a variety of non-neuronal cells triggers apoptotic death (37,38). In addition, we have observed that overexpression of E2F1 induces death of neurons. 4 To test our hypothesis, we expressed a DN binding form of DP1, an obligate binding partner to E2F, wild type, and deletion mutants of DP1, which do not display DN characteristics (23). In support of the involvement of E2F⅐DP complexes in neuronal death evoked by AB, we demonstrate that expression of DN DP1 inhibits death, whereas the non-DN deletion constructs had no effect.

Rb and E2F⅐DP1 as Mediators of Neuronal Death Evoked by AB-We
At present the means by which E2F⅐DP may mediate neuronal death are unclear. The fact that the DN DP1 construct contains a deletion of the DNA-binding domain suggests a critical role for such activity in mediating neuronal death (23). The apparent requirement for this DNA-binding domain in death signaling suggests the importance of transcriptionally mediated events. Recent reports, however, suggest that E2F may mediate death in certain proliferating systems through a transcriptionally independent mechanism that requires the E2F DNA-binding domain (39).
Although we favor a mechanism by which pRb/p107 mediates death through release from E2F⅐DP complexes and DN DP1 blocks death through inhibition of E2F⅐DP1 activity, other possibilities must be considered. For example, pRb is known to interact with multiple proteins including c-Abl (40). In addition, E2F and DP are also reported to physically interact with p53 both in vitro and in proliferating cells (41,42). The existence of such interactions in neurons and/or their potential function/importance are unknown and cannot be ruled out.
The findings presented here represent a significant insight into the potential mechanisms underlying the loss of neuronal function and viability in the brains of those suffering from Alzheimer's disease. The action of amyloid to induce a state of neuronal de-differentiation, indicated by an attempted return to cell cycle, may be an early event in the progressive loss of neuronal and synaptic function. In this context, intervention and reversal of this process may lead not only to an alteration in the progression of cognitive decline in the Alzheimer's patient, but the potential to restore neuronal function and improve cognition.


extfieldsofstudy: Biology Medicine

text:
Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors*

Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such transfected cells are bona fide phagosomes, capable of fusion with endomembranes, of luminal acidification, and of controlling the growth of microorganisms. The aim of the current study was to determine whether COS-1 and Chinese hamster ovary cells, rendered phagocytic by expression of human FcγRIIA receptors, express the cellular machinery required to support phagosomal maturation. Immunolocalization studies demonstrated that early endosomes, as well as late endosomes and/or lysosomes, fuse sequentially with phagosomes in the transfectants. Microfluorescence ratio imaging of particles labeled with pH-sensitive dyes revealed that maturation of the phagosome was accompanied by luminal acidification. The drop in pH, which attained levels comparable to those reported in professional phagocytes, was prevented by inhibitors of vacuolar-type H+-ATPases. Optimal phagosomal acidification required elevation of cytosolic [Ca2+], suggesting that it results from fusion of endomembranes bearing proton pumps. Moreover, the transfected cells effectively internalized live bacteria. Opsonization was essential for bacterial internalization, implying that it occurred by FcγRIIA-mediated phagocytosis, as opposed to invasion. Uptake into phagolysosomes was associated with inhibition of bacterial growth, due at least in part to the low intraphagosomal pH. These studies indicate that the biochemical events that follow receptor-mediated particle internalization in cells transfected with FcγRIIA receptors closely resemble the process of phagosomal maturation in neutrophils and macrophages. FcγRIIA-transfected cells can, therefore, be used as a model for the study of additional aspects of phagocyte biology.

Phagocytosis of invading microorganisms by leukocytes represents a vital component of host defense against infection
. Microbes are taken up into a phagocytic vacuole, which matures upon fusion with endomembrane compartments including endosomes and lysosomes (4 -6). Transfer of soluble and membrane-associated components from these compartments confers microbicidal activity to the phagosome (7).
The initial steps of the phagocytic sequence involve recognition and binding of microbial pathogens, a process enhanced by soluble components of serum, including complement fragments and immunoglobulins. These serum factors, known as opsonins, are recognized by receptors on the phagocyte cell membrane, such as the complement receptor 3 and immunoglobulin Fc receptors. Recent studies have begun to elucidate the signaling pathways involved in this complex series of events (for reviews, see Refs. 3 and 8 -10). Binding of antibody-opsonized particles to the phagocyte surface initiates activation of nonreceptor tyrosine kinases of the Src family and of p72 syk that are important for the phosphorylation of tyrosine residues within the Fc receptor complex (11,12). Spatial grouping ("clustering") of p72 syk appears to be a key step in the early phase of phagocytosis (13) and is accompanied by an accumulation of additional phosphorylated proteins in the vicinity of the phagocytic particle, resulting in activation of Ser/Thr (14,15) and phosphoinositide kinases (16,17), and a rise in free cytoplasmic Ca 2ϩ (18 -20). These events are associated with the formation of an actin-rich cup encircling the nascent phagosome, leading to internalization of the particle (3,(21)(22)(23).
Definition of the detailed molecular mechanisms triggered by individual receptors is complicated by the co-expression of multiple receptor types in phagocytes. Not only are both complement and Fc receptors present in neutrophils and macrophages, but several different members of the Fc receptor family co-exist in these cells. To overcome this difficulty, several laboratories have opted to express individual, well defined receptors in nonphagocytic cells, which are devoid of endogenous opsonin receptors (10,13,17). Remarkably, heterologous expression of single subtypes of Fc receptors, such as the Fc␥RIIA receptor, suffices to induce effective internalization of IgGopsonized particles (10).
Such heterologous models are attractive not only because they afford analysis of individual receptors but also because the cells used for expression are more amenable to transfection than natural phagocytes. This facilitates the introduction of cDNAs encoding other molecules of interest, and this model can be used to study multiple aspects of phagosomal formation, maturation, and bactericidal activity. To date, however, the use of heterologous expression systems has been limited to the analysis of the early steps in the signaling cascade (10,20,24). This is due, in part, to the fact that the fate of phagosomes in cells transfected with opsonin receptors is not known. It is not clear whether such phagosomes undergo a maturation process that resembles that reported for professional phagocytes.
The aim of the current study was to determine whether nonmyeloid cells, rendered phagocytic by expression of Fc␥ receptors, possess the cellular machinery required for phagosomal maturation. Immunofluorescence was used to monitor the interaction between endomembrane compartments and the nascent phagosome in COS-1 and Chinese hamster ovary (CHO) 1 cells transfected with human Fc␥RIIA. In addition, microfluorescence ratio imaging of zymosan particles labeled with pH-sensitive dyes was used to study the development of phagosomal acidification.
Bicarbonate-free RPMI 1640 medium was buffered to pH 7.3 with 25 mM Na-Hepes. The sodium-rich medium used for incubation of intact cells contained 140 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgCl 2 , 1 mM CaCl 2 , and 10 mM Hepes (pH 7.4). PBS consisted of 140 mM NaCl, 10 mM KCl, 8 mM sodium phosphate, 2 mM potassium phosphate, pH 7.4. The Na ϩ -rich medium used in the Na ϩ loading experiments contained 140 mM NaCl, 5 mM glucose, 15 mM Hepes, pH 7.4. The Na ϩ -free medium was identical except that Na ϩ was substituted by N-methyl-Dglucammonium. The K ϩ -rich medium had the same composition as Na ϩ medium, but NaCl was replaced by KCl. In all cases, the osmolarity was set to 290 Ϯ 5 mosM with the major salt. Dulbecco's modified Eagle's medium was obtained from Life Technologies, Inc. Fetal bovine serum was obtained from Life Technologies, Inc. and heat-inactivated by incubation at 56°C for 30 min prior to use in cell culture.
Monoclonal antibodies to lysosome-associated membrane protein 1 (LAMP-1) and LAMP-2 were obtained from the Developmental Studies Hybridoma Bank, maintained by the University of Iowa and Johns Hopkins University School of Medicine (Baltimore, MD). Mouse monoclonal antibodies against CD63 were generously provided by Dr. A. J. Verhoeven (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service). FITC-conjugated donkey anti-mouse IgG, antirabbit IgG, and anti-rat IgG were obtained from Bio-Rad. Cy3-conjugated donkey anti-rabbit IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Sheep RBC and rabbit antibody to these cells were from Cappel.
Labeling and Opsonization of Zymosan Particles-Zymosan particles (Sigma) were preswollen in PBS for 30 min prior to labeling. The swollen particles were sedimented by centrifugation, washed twice, incubated in PBS (pH 8.5) containing either 2 mg/ml of FITC or 1 mg/ml each of the succinimidyl ester of Oregon Green and FITC for 30 min at 37°C, and then washed several times with PBS (pH 7.2). The labeled particles were next opsonized by incubation with human IgG (Baxter) for 1 h at 37°C followed by several washes to remove unbound IgG.
Phagocytosis Assay-COS-1 and CHO cells were stably transfected with Fc␥RIIA cDNA as described (25). These cells (called hereafter COS-2A and CHO-2A, respectively) were plated onto 25-mm coverslips and grown to 70 -80% confluence in Dulbecco's modified Eagle's medium containing 10% serum. The phagocytic ability of these cells was assayed by incubating opsonized zymosan with cells in the presence of the impermeant, fluid-phase marker Lucifer Yellow. To synchronize phagocytosis, cells were cooled to 4°C in the presence of opsonized zymosan and the particles were allowed to adhere for 30 min. Lucifer Yellow (2 mg/ml) was then added, and the cells were warmed to 37°C for 1 h. After this period, the cells were again cooled and washed, and phagocytosis was determined by counting those cells that had internalized yeast along with the fluorescent fluid phase marker Lucifer Yellow. To ensure that the dye did not leave the phagosome, the cells were kept cold throughout the visualization period by using a cooled microscope stage and precooled slides.
Transfection and Labeling of Tfn Receptors-The cDNA encoding the human Tfn receptor cloned in the pCDM8 vector was a kind gift of Dr. J. Bonifacino (National Institutes of Health, Bethesda, MD). Transfection of CHO cells with the human Tfn receptor was accomplished using Fugene 6 (Roche Molecular Biochemicals) as instructed by the manufacturer. To study endosome-phagosome fusion, cells transfected with the human Tfn receptor were serum-starved for 1 h in Dulbecco's modified Eagle's medium and then allowed to internalize opsonized RBC for 15 min. Extracellular RBC were then lysed by hypotonic shock with water, and the cells were subsequently incubated with 50 g/ml tetramethylrhodamine isothiocyanate-labeled Tfn for 10 min. The cells were then cooled to preclude further membrane traffic and observed with epifluorescence and Nomarski optics.
To monitor the kinetics of endosome-phagosome fusion, the cells were first labeled with tetramethylrhodamine isothiocyanate-Tfn as above, chased for 7 min to clear surface-bound Tfn, and then allowed to internalize opsonized RBC for 15 min. The cells were then incubated further for periods of up to 30 min and analyzed.
Measurement of Cytosolic Calcium ([Ca 2ϩ ] c )-To measure cytosolic free calcium, the cells were loaded with fura-2 by incubation with 2 M of the precursor ester for 20 min at 37°C. Fura-2 ratio fluorescence measurements were performed on a Nikon Diaphot TMD microscope (Nikon Canada, Toronto, Ontario, Canada) equipped with a 100-W xenon lamp, a shutter/rotating mirror/fiber optic assembly (RatioMaster, Photon Technologies Inc., South Brunswick, NJ), a Fluor ϫ 40/1.3 oil-immersion objective, and a high sensitivity photometer (D-104, PTI) interfaced to a Dell Pentium computer via a 12-bit A/D board (Labmaster, National Instruments). The cells were alternately excited at 340 and 380 nm while the emission at 510 nm was recorded. Photometric data were acquired at 10 Hz using the Felix software (PTI). The microscope was equipped with a separate light source for long wavelength transillumination (Ͼ620 nm), a 580-nm emission dichroic mirror, and a separate video camera (MTI 72, Dage-MTI, Michigan City, IN), to allow continuous Hoffmann-enhanced visualization of the cells during the fluorescence measurements. Calibration was performed as described previously (26). Where indicated, cells were also loaded with 10 M of the ester form of BAPTA by incubation for 20 min at 37°C.
Measurement of Cytosolic pH (pH c )-To measure pH c , COS-2A cells were grown to 60 -70% confluence on 25-mm coverslips and loaded with 2 M of the ester precursor of BCECF for 20 min at 37°C. Fluorescence of BCECF was measured essentially as described previously using the PTI system detailed above, with the filter combination described earlier (27). Acute acid loading was accomplished by the ammonium prepulse technique (28). Calibration of the fluorescence ratio versus pH was performed for each experiment by equilibrating the cells in isotonic K ϩ -rich medium buffered to varying pH values (between 7.45 and 6.0) in the presence of the K ϩ /H ϩ ionophore nigericin (5 M). Calibration curves were constructed by plotting the extracellular pH, assumed to be identical to the cytosolic pH under these conditions, against the corresponding fluorescence ratio (29).
Measurement of Phagosomal pH (pH p )-For these studies, zymosan particles were labeled with FITC alone or in combination with Oregon Green, as indicated. COS-2A or CHO-2A cells plated onto 25-mm coverslips and grown to 70 -80% confluence were overlaid with opsonized, fluorescently labeled zymosan particles (Ӎ1 ϫ 10 6 /coverslip) and allowed to interact for 1 h. The coverslip was placed in a thermostatted Leiden holder on the stage of a Zeiss IM-35 microscope with a ϫ 63, 1.4 numerical aperture oil-immersion objective. Measurements of pH p were obtained through the combined application of video microscopy and fluorescence ratio imaging, as described previously (29). Calibration of fluorescence ratio versus pH p was obtained by fixation of cells with 1.6% paraformaldehyde for 20 s followed by permeabilization with 0.2% Triton for 1 min. Preliminary experiments indicated that treatment with paraformaldehyde did not affect the fluorescence properties or pH sensitivity of the probe. After washing, the pH of the bathing medium was varied, and the corresponding fluorescence ratio was recorded.
Immunofluorescence Microscopy-Immunofluorescence studies were performed essentially as described (26). For localization of the lysosomal markers CD63, LAMP-1, and LAMP-2, cells (with or without opsonized zymosan treatment) were fixed with cold methanol (Ϫ20°C) for 15 min. The cells were then washed with ice-cold PBS and labeled with primary antibody for 2 h at room temperature or overnight at 4°C. The cells were then washed with PBS and labeled with Cy3-labeled secondary for 1 h at room temperature, washed, and mounted using Slow Fade (Molecular Probes, Eugene, OR).

RESULTS
Assessment of pH p -To examine the molecular events involved in the intracellular processing of microbes in nonprofessional phagocytic cells, we utilized COS-1 and CHO cells stably expressing the human Fc␥RIIA receptor (COS-2A and CHO-2A, respectively) as model systems. These cells have been shown to be capable of internalizing IgG-opsonized particles, such as sheep RBC (25,30).
The plasma membrane of most mammalian cells, including leukocytes, is virtually devoid of vacuolar (V-type) proton pumps or ATPases. In phagocytes, acidification of the phagosomal lumen requires the insertion of V-ATPases through fusion with endosomes and/or lysosomes. Therefore, as an initial approach to establish whether phagosomal maturation occurs in COS-2A and CHO-2A cells, we measured pH p . We utilized microfluorescence ratio imaging of yeast particles (zymosan) labeled with pH-sensitive fluorescent dyes. Most previous studies of pH p have utilized particles covalently labeled with fluorescein. Although this probe has a large quantum yield and exquisite pH sensitivity, its pK a (Ӎ6.5) limits the measurements to the moderately acidic range. As illustrated in Fig. 1 (triangles), the responsiveness of this dye below pH 5.5 is marginal. Preliminary observations using FITC-labeled yeast revealed that the acidification of the phagosome might exceed the limit of sensitivity of this probe. To extend the range of sensitivity of our determinations, we used a second probe, namely Oregon Green 488 (pK a Ϸ 4.7). Following covalent attachment to zymosan particles, the emission of this dye is linearly sensitive to pH in the 4.0 -5.5 interval (Fig. 1, crosses), complementing the range of sensitivity of fluorescein. Therefore, for all subsequent experiments, we used particles that were dual-labeled with FITC and with the succinimidyl ester of Oregon Green. The resulting particles display sizable fluorescence changes throughout the pH 4.0 -7.5 range (Fig. 1, circles). Fig. 2 shows representative measurements of pH p in COS-2A cells that were allowed to internalize dual-labeled zymosan particles. The pH of the particles associated with the cells was heterogeneous: some were acidic, whereas others had a pH that approximated that of the external medium (Fig. 2B). As not all the particles that associate with cells become internalized, it was essential to identify those particles that were within phagosomes. Differential interference contrast microscopy could not reliably discriminate between extra-and intracellular particles ( Fig. 2A). Accordingly, two physiological criteria were developed to distinguish internal from external particles. First, abrupt changes in extracellular pH should affect the fluorescence ratio of extracellular particles but should have little acute effect on intraphagosomal particles (Fig. 2D, squares). Second, the pH of particles within phagosomes should be elevated upon addition of NH 4 Cl, which traverses the plasma and phagosomal membranes as NH 3 and becomes protonated in the phagosomal lumen, whereas extracellular particles should remain unaltered. As shown in Fig. 2, particles that were found to be in a near neutral environment responded rapidly to changes in external pH (Fig. 2D, circles) but showed no responsiveness to NH 4 ϩ (Fig. 2, C and E, circles). Conversely, acidic particles responded abruptly to addition of NH 4 ϩ (Fig. 2, C and E, triangles) but only slowly and moderately to extracellular pH change (Fig. 2D, squares), thereby fulfilling the criteria established for intraphagosomal particles. These observations imply that under the conditions of our studies, virtually all intraphagosomal particles are acidic. When measured 1 h after addition of the particles, the intraphagosomal pH averaged 4.84 Ϯ 0.11 (n ϭ 20). Fig. 2E illustrates an experiment in which particles were added to the cells during the course of the observation, in order to capture the development of the acidification process. As illustrated by the triangles, acidification was complete within 5 min, maintained for at least 20 min, and recovered readily from the addition of NH 4 ϩ . Similar results were obtained using CHO-2A cells (not illustrated).
Role of the V-ATPase in Acidification-Phagosomal acidification in professional phagocytes is mediated largely by V-ATPases (31,32). To ascertain whether a similar mechanism is involved in the heterologous transfectants, COS-2A cells were allowed to internalize labeled zymosan and then treated with concanamycin, a potent and selective inhibitor of V-ATPases (33). As before, the phagosomal location of the particle was first verified (Fig. 3A), and the inhibitor was then added. As shown in Fig. 3A, concanamycin triggered the gradual dissipation of the phagosomal acidification. The slow course of alkalinization, which was not accelerated by using higher doses of the inhibitor, implies that the phagosomal membrane is comparatively tight to H ϩ equivalents. This contrasts with the faster dissipation rates observed in endosomes (34) and is compatible with the markedly more acidic pH of phagosomes, which approached 4.5 in some experiments (e.g. Fig. 3B). A similar rate and extent of dissipation was recorded using bafilomycin (Fig. 3B), a different macrolide antibiotic also reported to inhibit V-ATPases selectively (35). When added prior to phagocytosis, the inhibitors precluded phagosomal acidification (not shown). Neither bafilomycin nor concanamycin affected the fluorescence of extracellular particles. Jointly, these experiments confirm that the marked acidification of the phagosomal compartment of COS-2A (and also CHO-2A cells; not illustrated) is mediated by V-ATPases, as is the case in professional phagocytes.
Whereas most mammalian cells do not express V-ATPases in their plasmalemma, active proton pumping has been reported in the surface membrane of osteoclasts and in certain epithelia, including the renal proximal tubule. Because COS-2A cells are derived from the kidney, and because the acidification of the phagosome developed rapidly, it was important to consider whether V-ATPases are constitutively present and active in the plasma membrane of these cells. To this end, the cytosolic pH of COS-2A cells was measured with the indicator dye BCECF. Neither bafilomycin (Fig. 4)  Preswollen zymosan particles were covalently labeled with 2 mg/ml either Oregon Green (crosses) or fluorescein (triangles) or with 1 mg/ml each (circles). The labeled particles were exposed to different pH values over a range of 3.5-8.0, and the ratio of the fluorescence with excitation at 490/440 nm was determined as described under "Experimental Procedures." loaded by an NH 4 ϩ prepulse. Na ϩ and HCO 3 Ϫ were initially omitted to preclude recovery by electroneutral ion exchange. Under these conditions, the cells recovered slowly from the acidification, but the rate of recovery was unaffected by the addition of bafilomycin. By contrast, a rapid alkalosis was triggered by reintroduction of Na ϩ , due to initiation of Na ϩ /H ϩ exchange. These observations imply that V-ATPase activity is not detectable on the surface membrane of COS-2A cells and suggest that the vigorous bafilomycin-sensitive proton pumping observed in the phagosome required recruitment of V-ATPases from other compartments.
Fusion of Phagosomes with Endosomes and Lysosomes-Because the preceding evidence is consistent with import of V-ATPases by the phagosome, we sought to identify the putative endomembrane compartment(s) that may be the source of the proton pumps. In professional phagocytes, phagosomes fuse initially with early endosomes, identified by the presence of Tfn receptors and Rab5, and subsequently with late endosomes and lysosomes (5,6). We therefore initially analyzed the distribution of Tfn receptors in CHO-2A cells before and after phagocytosis of zymosan. To increase the number and affinity of the receptors for human Tfn, CHO-2A cells were transiently transfected with human Tfn receptor cDNA. These cells were incubated with fluorescent human Tfn for 1 h at 37°C, washed, and incubated for an additional 7 min in culture medium to allow internalization of surface-bound Tfn and delivery to early endosomes (36). As reported for other cells, Tfn receptors are present in endosomal vesicles throughout the cytosol, with accumulation in the juxtanuclear region (Fig. 5A), where recycling endosomes are thought to concentrate (36).
To assess the interaction between phagosomes and early endosomes, CHO-2A cells were allowed to take up opsonized RBC and, when phagocytosis was completed, Tfn was added. After 10 min, some of the phagosomal membranes were lined by Tfn receptors (Fig. 5C), indicating fusion of early endosomes with the phagosome. Not all the RBC were surrounded by labeled membranes, which may reflect asynchrony in the internalization and fusion process. Note that in these experiments phagocytosis preceded binding of Tfn, ruling out internalization of surface Tfn along with the nascent phagosome. These data indicate that at least a fraction of the phagosomes formed are capable of fusing with early endosomes. Similar results were obtained in COS-2A cells using zymosan particles or when pulsing with Tfn and a 7-min chase preceded phagocytosis (not shown).
The next stage in the maturation of the phagosome in professional phagocytes is fusion with late endosomes and ultimately with lysosomes. This event was analyzed next. Because some of the marker proteins such as the LAMPs are expressed in both late endosomes and lysosomes, no effort was made in our studies to distinguish between these compartments. The distribution of three markers was compared in cells before and 1 h after exposure to zymosan. As shown in Fig. 6, CD63, LAMP-1, and LAMP-2 all display a random punctate distribution in untreated COS-2A cells (Fig. 6, A, D, and G, respectively). Following internalization of zymosan particles, which were labeled with a different fluorophore to facilitate co-localization (Fig. 6, C, F, and I), some of the endo/lysosomal markers were mobilized and fused with the phagosomal membrane (Fig. 6, B,  E, and H). Note that only a fraction of the adherent zymosan particles became internalized (solid arrowheads). These findings of spatial co-localization indicate that late endosomes and/or lysosomes also fuse with phagosomes in cells heterologously transfected with the Fc␥RIIA receptor.  4 Cl was added to the medium at pH 7.4. E, acidification of a zymosan particle upon internalization (triangles). For comparison, the pH of a particle that remains external throughout is illustrated. The occurrence of internalization was tested by addition of 40 mM NH 4 Cl.
The sequential nature of the fusion of endosomes and lysosomes was analyzed in detail. Early endosomes were loaded with Tfn and, after chasing the surface-bound Tfn, phagocytosis was initiated by exposing the CHO cells to opsonized RBC for 15 min. After varying periods of time, the reaction was stopped, and the presence of Tfn and LAMP-1 in phagosomes was assessed as above. The results of three similar experiments are summarized in Fig. 7. Tfn is present in a large fraction of the phagosomes at the earliest time tested (15 min after initiation of phagocytosis) and rapidly decreases thereafter, becoming virtually undetectable after a further 30 min. By contrast, the fraction of phagosomes stained for LAMP-1 is modest after 15 min but increases progressively. Most of the phagosomes were LAMP-1-positive 30 min after phagocytosis was completed. These results bear remarkable resemblance to the course of phagosomal maturation in monocytes/macrophages (see, for example, Ref. 5).
Role of Calcium in Phagosomal Acidification-There is disagreement concerning the requirement for calcium in the process of phagosomal maturation. In neutrophils, fusion of phagosomes with lysosomal like granules that express CD63 is thought to be strictly dependent on elevated [Ca 2ϩ ] c (19). By contrast, phagosome to lysosome fusion was unaffected when [Ca 2ϩ ] c transients were prevented in macrophages (37). This discrepancy may reflect differences between neutrophils and macrophages in the regulation of phagosome to lysosome fusion and from the fact that multiple and different receptors are activated during phagocytosis. We therefore used the heterologous transfection system to reanalyze the [Ca 2ϩ ] c dependence of phagosomal maturation.
As reported earlier (20), engagement of Fc␥RIIA induced a rapid and transient elevation of [Ca 2ϩ ] c in the Fc␥RIIA transfectants (Fig. 8A). Suspension of the cells in Ca 2ϩ -free medium, followed by depletion of the intracellular stores using thapsigargin, abrogated the [Ca 2ϩ ] c response to a subsequent addition of IgG-opsonized particles (Fig. 8B). The response to receptor cross-linking was similarly eliminated by preloading the cells with the chelating agent BAPTA (Fig. 8C). Under these conditions, particle internalization was unaffected, as reported for professional phagocytes (38). Calcium independence of phagocytosis is also in accord with the observation by Odin et al. (20) that different regions of the Fc␥RIIA mediate calcium flux and particle internalization.
BAPTA was applied in parallel experiments to assess phagosomal acidification, used as an index of maturation. As shown in Fig. 8D, chelation of intracellular Ca 2ϩ diminished but did not prevent the development of phagosomal acidification. Phagosomal pH averaged 4.7 Ϯ 0.1 in control (n ϭ 17) and 5.2 Ϯ 0.18 in BAPTA-treated (n ϭ 12) cells. Because the products of hydrolysis of the acetoxymethyl ester of BAPTA can have nonspecific deleterious effects, we also evaluated the need for Ca 2ϩ using thapsigargin. Prevention of the [Ca 2ϩ ] c transient by depletion of the stores resulted in an even more pronounced inhibition of the acidification, which reached only 5.7 Ϯ 0.2 (n ϭ 12) (Fig. 8D). Jointly, these experiments indicate that Fc␥RIIA receptor-mediated phagosomal maturation is partly dependent on increased [Ca 2ϩ ] c .
Functional Significance of Phagosomal Acidification-As illustrated in Fig. 2E, phagosomal acidification develops shortly after particle internalization. It has been speculated that the initial phase of acidification, likely induced by insertion of V-ATPases from early endosomes, is required for the successful fusion of phagosomes with lysosomes. This notion is based on findings using weak bases or ionophores to dissipate endomembrane pH, with resulting inhibition of phagosome to lysosome fusion (39). However, the agents used to alter pH concomitantly affect the volume and ionic composition of acidic compartments. Such secondary effects complicate the interpretation of the role of pH gradients in fusion. To analyze the effect of dissipation of pH on the fusion of phagosomes with lysosomes, without simultaneously inducing swelling and/or ionic replacement, we inhibited the V-ATPase with concanamycin or bafilomycin prior to the induction of phagocytosis. As shown in Fig.  9A, the V-ATPase inhibitors had no significant effect on the ability of the transfected cells to internalize IgG-opsonized particles. More importantly, neither concanamycin nor bafilomycin altered the fusion of phagosomes with LAMP-1 or LAMP-2-containing vesicles (Fig. 9B). Parallel determinations ascertained that, when added prior to phagocytosis, the V-ATPase blockers completely prevented the development of phagosomal acidification (not shown). We conclude that acidification of the phagosomal vacuole is not required for effective phagosome to lysosome fusion.

Bacteriostatic Effect of Fc␥RIIA Receptor-transfected Cells-
The results above indicate that Fc␥RIIA receptor-transfected cells can effectively recapitulate the processes of phagocytosis and phagosomal maturation. We therefore considered whether, like professional phagocytes, the transfected cells could limit the growth of bacteria. For these studies, CHO-2A cells were exposed to viable Escherichia coli for the indicated periods; the cells were then washed extensively, exposed to gentamicin for 1 h at 37°C (to kill any remaining extracellular bacteria), and lysed with Triton X-100 (to release intracellular bacteria), and the lysates were plated on LB agar plates for bacterial quantitation. As shown in Fig. 10A, the transfectants were able to effectively internalize bacteria, but only after opsonization with an E. coli-specific antibody.
To assess the fate of the bacteria following internalization by CHO-2A cells, we compared their growth within the cells to that of bacteria that were released from phagosomes by treatment with detergent 1 h after ingestion, and suspended freely in culture medium. As illustrated in Fig. 10B, intracellular bacteria failed to replicate, and in fact their number decreased, although the extent of the reduction did not attain statistical significance. Over the same period of time, bacteria released from the cells shortly after ingestion proliferated actively, their number increasing by over 7 orders of magnitude. This indicates that the Fc␥RIIA receptor-transfected cells exerted an Any adherent extracellular RBC that were not internalized were then lysed hypotonically. Next, the cells were incubated with labeled Tfn for 10 min, and the reaction was terminated by washing in ice-cold medium. The cells were visualized using fluorescence (B) and Nomarski (C) optics. Arrows point to RBC that colocalize with Tfn.

FIG. 6. Co-localization of lysosomal markers with internalized particles in COS-2A cells.
A and B, localization of CD63 revealed by indirect immunofluorescence using Cy3-labeled antibodies. In A, the cells were otherwise untreated, whereas in B, the cells were allowed to internalize FITC-labeled zymosan particles for 1 h prior to fixation. The position of a particle that co-localizes with CD63 is indicated by the solid arrowhead, whereas unstained particles that were not internal- The precise mechanism underlying bacteriostasis remains undefined, but phagosomal acidification is most likely to contribute to this effect, because the rate of replication of E. coli grown in culture broth decreases sharply below pH 5 (Fig. 10B, inset). As reported above, the phagosomal pH in Fc␥RIIA receptor-transfected cells can reach 4.84. We attempted to establish more directly whether phagosomal acidification was essential for the bacteriostatic action of the CHO-2A cells. However, the concentrations of bafilomycin and concanamycin required to inhibit phagosomal acidification directly and effectively killed E. coli. Moreover, prolonged exposure to these and other agents that dissipate endomembrane acidification proved toxic for CHO-2A cells. DISCUSSION The goal of this study was to determine whether nonmyeloid cells, rendered phagocytic by expression of human Fc␥ receptors, possessed the cellular machinery that would allow functional maturation of the phagosome. Our data demonstrate that not only can such cells internalize opsonized particles rapidly and efficiently in a ligand-dependent manner but that the phagosome becomes progressively acidified by recruitment of V-ATPases from endomembrane compartments. Moreover, we observed that when actively dividing E. coli are internalized by this receptor-mediated pathway, they promptly cease to proliferate. Failure of the bacteria to grow within the transfected cells was not due to opsonization or to the phagocytic process itself, because E. coli released from phagosomes shortly after internalization proliferated actively in suspension. Instead, it is likely that the conditions generated within the phagosome upon fusion with lysosomes limit the growth of the bacteria. Lysosomal hydrolases and the low luminal pH probably contribute to this effect.
Fc receptors were shown earlier to modify the fate of intracellular vacuoles that encapsulate invading Toxoplasma. This microorganism generates a parasitophorous vacuole that neither fuses with lysosomes nor becomes acidic (24). When the vacuole contains active Fc␥RIIB1 or B2 receptors, fusion with lysosomes and acidification develop (24). Importantly, unlike Fc␥RIIA, the B1 and B2 isoforms are unable to trigger phagocytosis of IgG-coated inert particles, such as RBC (10,40,41). This implies that signals generated by interaction of Toxoplasma with other cellular components are required for Fc␥RIIB1 or B2-induced phagosomal formation and maturation. In contrast, cross-linking of Fc␥RIIA suffices to induce phagosome formation (25) and to trigger its fusion with endosomes and lysosomes. This differential behavior has been attributed to the presence of an immunoreceptor tyrosine-based activation motif (ITAM) or ITAM-like sequence in the cytoplasmic tail of Fc␥RIIA receptors. Such an ITAM-like sequence is not present in human Fc␥RIIB1 or B2 (42)(43)(44)(45). Concomitant phosphorylation of both ITAM tyrosine residues of Fc␥RIIA is thought to promote the attachment and subsequent activation of p72 syk via its dual SH2 domains (12,46,47). Clustering of p72 syk is in turn sufficient to promote phagocytosis (13), and failure to activate this kinase may account for the inability of Fc␥RIIB1 and B2 to initiate phagocytosis. It is likely that signals other than the activation of p72 syk must contribute to phagosomal maturation, inasmuch as Fc␥RIIB1 and B2 facilitate lysosomal fusion and acidification, at least in the case of Toxoplasma-induced vacuoles (24).
As in the case of macrophages, phagosomes formed by IgGopsonized particles in Fc␥RIIA-transfected cells fused sequentially with early endosomes and subsequently with late endosomes and lysosomes (Fig. 7). The resulting phagolysosomes acidified to levels comparable to those reported in macrophages (48,49). The acidification was reduced but not eliminated by chelation of intracellular calcium or by depletion of intracellular calcium stores. In this regard, the transfected cells resemble neutrophils, in which prevention of calcium transients inhibits phago-lysosomal fusion (19), but they differ from macrophages, in which no effect was noted (37). It remains unclear whether these two closely related cell types utilize fundamentally different fusion mechanisms, or whether methodological differences account for the apparent discrepancies. It is noteworthy, in this regard, that fusion systems heretofore thought to be calcium-independent are being found to require the divalent arrowhead, 500 nM thapsigargin was added. Next, zymosan was introduced (solid arrowhead). C, cells preloaded with the Ca 2ϩ chelator BAPTA were suspended in Ca 2ϩ -free medium, and zymosan was added where indicated by the solid arrowhead. D, calcium dependence of phagosomal acidification, measured by ratio imaging as described in Fig. 2. Otherwise untreated cells (control, n ϭ 17) were compared with cells pretreated with BAPTA (n ϭ 12) or thapsigargin as in B and C (n ϭ 12). Data are means Ϯ S.E. then allowed to bind zymosan particles for 15 min on ice, followed by a 30-min internalization period at 37°C. Where appropriate, the inhibitors were present throughout. The cells were then fixed and permeabilized, and the distribution of LAMP-1 and LAMP-2 was studied by indirect immunofluorescence. Co-localization with the zymosan particles was assessed as in Fig. 6. Data are means Ϯ S.E. of three separate experiments.
cation (50,51). The calcium change occurs rapidly in a localized juxtamembrane region and can only be abrogated using high concentrations of rapid chelating agents (50,51). It is therefore conceivable that a requirement for calcium transients may have been overlooked in some macrophage studies.
In macrophages, fusion of phagosomes with lysosomes has also been reported to be disrupted by agents and conditions that dissipate intraorganellar acidification (39). Unexpectedly, no such inhibition was detected in the transfected cells. This may point to a fundamental difference in the underlying fusion mechanism, but it may alternatively reflect secondary effects of the procedures used to dissipate the acidification. In our experiments, the formation of a pH gradient was prevented by inhibition of the V-ATPase, whereas in previous studies, weak bases or ionophores were used. In the presence of an active ATPase, the earlier methods lead to progressive swelling of phagosomes and other acidic organelles and can even produce ATP depletion. These effects are not expected to occur in bafilomycin or concanamycin-treated cells. The method used herein is more conservative, and therefore, the role of pH in phagosome to lysosome fusion in leukocytes warrants reconsideration.
In summary, transfection of Fc␥RIIA into nonmyeloid cells was observed to reconstitute not only particle internalization but also phagosomal maturation and acidification. Such reconstituted phagolysosomes are capable of limiting the growth of internalized microorganisms. Additional microbicidal mechanisms of professional phagocytes, such as the respiratory burst oxidase and pore-forming peptides like the defensins are lacking in the Fc␥RIIA receptor-expressing cells, and they could potentially be co-transfected to evaluate their individual contribution to bacterial killing. The present findings, therefore, further extend the use of Fc␥ receptor transfection systems for the study of the biology of phagocytosis.


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Regulatory Sequences of the Mouse Villin Gene That Efficiently Drive Transgenic Expression in Immature and Differentiated Epithelial Cells of Small and Large Intestines*

Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent β-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted β-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa.

Transgenic mice are routinely used to study the molecular and cellular basis of normal and pathological states in intestinal mucosa (1)(2)(3)(4)(5). The major limitation regarding the targeting of exogenous transgenes in this tissue is that the epithelium of the mouse intestinal mucosa is renewed every 2-5 days (6 -8). The epithelial cells arise from multipotent stem cells functionally anchored at the base (more precisely in the lower third) of the proliferative compartment of the epithelium, the crypts of Lieberkü hn. These crypts display a monoclonal organization because they are each derived from a single progenitor cell (9). Descendants of stem cells multiply in the middle portion of each crypt (10) and gradually differentiate into four principal cell types. In the small intestine, absorptive enterocytes (constituting Ͼ80% of the epithelial cells), mucus-producing goblet cells, and enteroendocrine cells migrate upward from the crypts to the apex of surrounding villi (whose colonic counterparts are hexagonal-shaped cuffs) (11), where they become apoptotic and are exfoliated into the gut lumen (12). In contrast, antimicrobial peptides secreting Paneth cells migrate to the bottom of the crypts, where they reside for about 20 days (13).
Given the remarkable protective effect of this epithelium, it is not surprising that most previous studies aiming to induce neoplastic transformation in intestinal mucosa of transgenic mice have failed (14,15). In these reports, the use of promoter sequences that direct oncogenes in nonproliferating enterocytes located in the upper third of crypts produce only minor phenotypic abnormalities without tumorigenic consequences in the gut epithelium, suggesting that the residence time of these villus-associated cells may not be sufficient for the oncogenes to exert their effects. Furthermore this suggests that transgenic mouse models of neoplasia may require an efficient targeting of oncogenes in crypt stem cells or their immediate descendants.
Villin is a cytoskeletal protein that is mainly produced in epithelial cells that develop a brush border responsible for absorption as in the digestive apparatus (epithelial cells of the large and small intestines) and in the urogenital tract (epithelial cells of the kidney proximal tubules). Because it is expressed in the proliferative stem cells of the intestinal crypts (16,17), it is believed to be an early marker for committed intestinal cells. The multiple levels of regulation control villin gene activity during mouse embryogenesis (18 -20) and account for the strict pattern of tissue-specific expression observed in adults. Moreover, the expression of the villin gene in intestinal epithelial cells is conspicuously maintained in their corresponding carcinomas (21)(22)(23)(24).
The specific expression pattern of villin suggests that it is an appropriate candidate for the characterization of regulatory sequences that could allow targeting of heterologous genes into a selected population of cells in the mouse digestive tract. With this goal in mind, the human villin gene has been isolated and characterized (25). A 2-kb 1 5Ј-flanking region has been found to contain sufficient regulatory elements to promote tissue-specific expression of a reporter gene in intestinal and renal cell lines (26). In transgenic mice, this regulatory region is able to drive the expression of the human Ha-ras oncogene in the tissues in which the endogenous gene is actively transcribed. However, low levels of expression were observed that did not trigger malignant tissue appearance into the gut of these animals. 2 These observations led us to further analyze an extended genomic region of the mouse villin gene with the goal of map-ping additional elements localized 5Ј and/or 3Ј and involved in promoting high levels of transgenic expression in the intestinal mucosa. Here we report the analysis of tissue-specific expression of the mouse villin gene using: (i) DNase I-hypersensitive sites assays, (ii) transient transfection assays, and (iii) transgenic mice.

EXPERIMENTAL PROCEDURES
Cell Culture and ex Vivo Transient Transfection-Human colon carcinoma enterocyte-like CaCo2 cells, pig kidney proximal tubules-derived LLCPK1 cells, and distal tubules-derived Madin-Darby canine kidney cells were cultured as described (26). Cells cultures were cotransfected using 15 l of Lipofectin reagent (Life Technologies, Inc.) with 5 g each of ␤-galactosidase reporter plasmid construct and the control plasmid pRSVLuc, which contains the luciferase gene under the control of the Rous sarcoma virus promoter. Cells were harvested 48 h later, and cell extracts were assayed by chemiluminescent detection of both ␤-galactosidase (Galacto-Light, Tropix, Inc.) and luciferase (Luciferase Assay Kit, Tropix, Inc.) activities using a luminometer (Berthold). ␤-Galactosidase activity (light units) was corrected for variations in transfection efficiencies as determined by luciferase activity. All transfections were repeated at least three times. Results are expressed as fold induction over that of the vector without promoter, pBasic.
Primer Extension Analysis-Total RNA was isolated from mouse intestine with RNA NOW reagent (Biogentex). For primer extension assay, 2 ng of 32 P-labeled oligonucleotide probe (5Ј-GAGTGGTGATGT-TGAGAGAGCCT-3Ј) complementary to nucleotides ϩ81 to ϩ103 of the murine villin cDNA (GenBank TM accession number M98454) was hybridized with 30 g of total RNA at 60°C (0.25 M KCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) for 90 min. Transcription with 5 units/l of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) was carried out at 37°C for 90 min in 300 l of a solution containing 75 mM KCl, 3 mM MgCl 2 , 50 mM Tris-HCl (pH 8.3), 10 mM dithiothreitol, 0.75 mM deoxynucleoside triphosphates, 75 g/ml actinomycin D, and 0.3 units/l RNasin. The primer extension products were separated by electrophoresis in denaturing 8% polyacrylamide gels. The full-length extension product (105 nucleotides) was obtained by comparison with the length of the comigrating sequencing reaction products. A primer extension control experiment was performed on the same total RNA preparation using a 32 P-labeled oligonucleotide probe (5Ј-CATAGTTCTCGTTCCGGT-3Ј) complementary to nucleotides ϩ63 to ϩ80 of the mouse intestinal fatty acid-binding protein cDNA and generating an 81-nucleotide extension product (27).
DNase I-hypersensitive Site Analysis-Tissues from 30 mice were used per assay of intestine, kidney, liver, and spleen. Nuclei preparation and DNase I digestion were performed as described (28) with minor modifications. Nuclei were digested without or with 20 to 160 units of DNase I (DPRF Worthington) for 10 min at 0°C. 10 g of purified DNA was digested overnight with restriction enzyme (BamHI or BglII). The DNase I-hypersensitive sites were analyzed by Southern blotting using the BglII-PstI probe (0.5 kb) (as indicated in Fig. 2).
Plasmid Construction-All constructs described were subcloned into the pBluescript II KS vector (Stratagene) with fragments isolated from a DASHII phage containing a 16.3-kb region (9 kb upstream and 7.3 kb downstream from the translation initiation codon) of the mouse villin gene (kindly furnished by G. Tremp, Rhône-Poulenc Rorer) (29). The pD1 construct (as described in the legend to Fig. 3B) was prepared by ligating a BamHI fragment of 5.1 kb (1.8 kb upstream from the ATG translation initiation codon of the mouse villin gene, subcloned 5Ј to the nuclear localization signal-␤-galactosidase gene-SV40 polyadenylation site, using a polymerase chain reaction (PCR) strategy) at the BamHI site in a plasmid containing the 3.7-kb region of the mouse villin gene (immediately 5Ј to the 1.8-kb region described above). The pA1 and pA2 (containing an internal 1-kb deletion) constructs have resulted from several steps based on the BstEII sites present in the 3.7-kb region described above and in a plasmid containing the 3.5-kb region of the mouse villin gene (immediately 5Ј to the 3.7-kb region). The pC1 and pC2 constructs were derived from the pA1 and pA2 plasmids cut with ApaI and re-ligated, respectively. To generate the pB1 construct, a BglII fragment (480 bp) from the 3.5-kb region described above was excised and cloned into the KpnI site of the pC1 plasmid. The pA3, pB3, and pC3 constructs correspond to the pA1, pB1, and pC1 deleted from the intron 1 (see Fig. 3B). The sequence between the transcription initiation start site and the translation initiation codon, excluding the intron 1, was deduced from that of the murine villin cDNA and was introduced into the BglII-NcoI sites of the pC1 construct by using a dimerized oligodimer made of a coding-strand oligonucleotide (5Ј-GAT-CTCCCAGGTGGTGGCTGCCTCTTCCAGACAGGCTCGTCCAC-3Ј) and a noncoding-strand oligonucleotide (5Ј-CATGGTGGACGAGCCTG-TCTGGAAGAGGCAGCCACCACCTGGGA-3Ј), resulting in the pB3 construct. The pA3 and the pC3 constructs were derived from the pB3 plasmid by ligating an ApaI fragment (3.1 kb) and a BglII fragment (480 bp), both from the 3.5-kb region described above, at the ApaI site in the pB3 plasmid, respectively. Subcloning steps were confirmed by DNA sequencing.
Transgenic Mice Generation-The transgenes digested with XhoI-NotI were injected into the pronuclei of the fertilized eggs of the B6/D2 mice in collaboration with the Service d'Experimentation Animale et de Transgénèse, CNRS (Villejuif, France). Mice carrying transgenes were first identified by PCR of genomic DNA to confirm the presence of the ␤-galactosidase gene and then analyzed by Southern blotting to determine the copy number of the integrated transgene. Each founder animal harbored one copy of the transgene per genome. Small intestine, colon, kidney, stomach, liver, heart, lung, thymus, brain, spleen, and muscle were dissected from transgenic mice and either prepared for total RNA extraction or embedded to perform cryosections.
Reverse Transcription-PCR Analysis-Total RNA was isolated with SV Total RNA Isolation System (Promega). 20 ng of pd(N) 6 random primer (Pharmacia) were hybridized with 2 g of total RNA at 70°C for 10 min in distilled water. Reverse transcription with 200 units of Moloney murine leukemia virus reverse transcriptase (SuperScript II, Life Technologies, Inc.) was carried out at 37°C for 90 min in a 20-l solution of 1ϫ First Strand Buffer (Life Technologies, Inc), 10 mM dithiothreitol, 0.5 mM deoxynucleoside triphosphate, and 0.4 units/l RNasin. 2 l of the resulting cDNAs were amplified by PCR reaction in 50 l for 40 cycles. Each cycle consisted of 60 s at 94°C, 60 s at 51°C (for transgene and villin) and 57°C (for TFIID), and 30 s at 72°C. For the transgene primers, 5Ј-CAACTTCCTAAGATCTCC-3Ј coding strand and 5Ј-ATTCAGGCTGCGCAACTGTT-3Ј noncoding strand were used, generating a 250-bp product. For villin amplification 5Ј-CAACTTC-CTAAGATCTCC-3Ј coding strand primer and 5Ј-GCAACAGTCGCTG-GACATCACAGG-3Ј noncoding strand primer were used, generating a 473-bp product; for TFIID amplification 5Ј-CCACGGACAACTGCGTT-GAT-3Ј coding strand primer and 5Ј-GGCTCATAGCTACTGAACTG-3Ј noncoding strand primer were used, generating a 220-bp product. Onefifth of the PCR product was run on an ethidium bromide-containing agarose gel.

Determination of the Transcription Start
Site-To determine the transcriptional start site of the mouse villin gene, total RNA was isolated from intestine and analyzed by primer extension assay using an oligonucleotide complementary to the mouse villin cDNA downstream of the ATG initiation codon. The efficiency of the reaction was confirmed by primer extension of the mouse intestinal fatty acid-binding protein gene (fabpi) from the same RNA preparation (27). Analysis of the fabpi extension product on a sequencing gel by comparison with a sequence ladder (Fig. 1A) revealed a strong signal band of a size of 81 bp as expected. The extension product of villin was 105 bp, indicating that the transcriptional start site (an adenosine residue subsequently designed as nucleotide ϩ1) was 57 nucleotides upstream of the translation initiation codon of the murine villin cDNA (Fig. 1B). Comparison of the genomic sequence encompassing 9 kb upstream from the ATG initiation codon with the cDNA sequence, position of splice site consensus sequences in the 9-kb genomic sequence (Fig. 1B), and determination of the transcription start site reveal that the mouse villin gene has one transcription start site that is separated from the ATG initiation codon by a 5.5-kb intronic region (Fig. 1C).
DNase I-hypersensitive Sites in the Mouse Villin Gene-To characterize the key regulatory regions involved in the specific control of villin expression, we have mapped the DNase I-hypersensitive sites (31) in the mouse villin gene (along a region extending 9 kb upstream and 4.4 kb downstream from the translation initiation codon, as represented in Fig. 2A). The chromatin form of the mouse villin gene in different tissues (intestine, kidney, liver, and spleen) was submitted to limited DNase I digestion and subsequently digested with the appropriate restriction enzymes. Using BglII digestion and a 0.5-kb probe homologous 5Ј of the 7.5-kb BglII fragment (Fig. 2B), two sets of DNase I incubation-related fragments were detected, migrating at 5.5 and 2.7 kb, and corresponding to hypersensitive sites designated as HS I (located at approximately ϩ5.5 kb downstream from the transcription start (ϩ1) site, just upstream from the ATG initiation codon) and HS II (located at approximately ϩ3 kb downstream from the (ϩ1) site), respectively. HS I was observed in nuclei isolated from intestine, kidney, and liver, whereas HS II was only present in intestinal tissue. No specific hypersensitive sites were detected in nuclei isolated from spleen. The presence and location of these hypersensitive bands were confirmed by hybridization with the 0.8-kb probe ( Fig. 2A) homologous to the 3Ј end of the 7.5-kb BglII fragment (data not shown). Using BamHI digestion and the 0.5-kb probe (Fig. 2C), five sets of DNase I-treated nucleirelated fragments were detected, migrating at 3.4, 4.3, and 4.7 kb and approximately 10 and 15 kb, corresponding to the hypersensitive sites HS II, HS III (located at Ϫ0.5 kb upstream from the (ϩ1) site), HS IV (located at Ϫ1 kb upstream from the (ϩ1) site), HS V (located at Ϫ10 kb upstream from the (ϩ1) site), and HS VI (located at Ϫ15 kb upstream from the (ϩ1) site), respectively. HS III was observed in nuclei isolated from both intestine and kidney, whereas HS IV was only present in intestinal tissue as HS II. The hypersensitive sites HS V and HS VI were only present in liver tissue (in which villin is weakly expressed) and were located far upstream from the transcription start site in regions (i) that have not been subcloned and (ii) that could belong to an adjacent gene; for these reasons, these hepatic-specific hypersensitive sites were not analyzed further. As for BglII digestion, no specific hypersensitive sites were detected in nuclei isolated from spleen. Using other independant restriction digestions (EcoRI and HindIII) and the 0.5, 0.8, and 1.25 kb probes (as represented in Fig. 2A), similar results were obtained (data not shown).
In conclusion, four major distinct DNase I-hypersensitive sites (HS I to HS IV) were shown to be present in the region extending from Ϫ1 kb to ϩ5.5 kb in respect to the transcription start site (Fig. 3A) of the mouse villin gene. These sites were detected in intestine (HS I to HS IV), kidney (HS I and HS III), and liver (HS I), tissues in which villin is expressed, but they were not found in spleen, a tissue that does not produce villin. These findings correlate with the tissue-specific control of villin gene expression and suggest that the putative critical regulatory elements lie within these regions. HS II and HS IV were only detected in intestine and are probably associated with tissue-specific transcription factor-binding sites involved in the positive control of villin gene intestinal expression.
Analysis of Promoter Activity by Transient Expression-To test the effects of the segments containing the DNase I-hypersensitive sites (Fig. 3A) on transcriptional activity and to define more precisely the element(s) controlling villin gene expression in the intestine, segments were subcloned upstream of a promoterless LacZ plasmid (Fig. 3B). The construct pA1 contained all the subcloned regions downstream from the ATG initiation codon, encompassing the four DNase I-hypersensitive sites (HS I to HS IV) described above and the 5.5-kb intronic sequence, intron 1. Plasmids pA2 and pA3 were identical to pA1 except for the presence of intestine-specific hypersensitive site HS II and intron 1, respectively. Plasmid pB1 and plasmid pC1 were similar to plasmid pA1 but lacked the regions extending from Ϫ480 bp to Ϫ3.5 kb and Ϫ100 bp to Ϫ3.5 kb according to the transcription start site, respectively. Plasmid pC2 was identical to pA2 but lacked the region extending from Ϫ100 bp to Ϫ3.5 kb. Plasmids pB3 and pC3 were identical to pB1 and pC1 except for the presence of intron 1, respectively. The plasmid pD1 was identical to pA1 except for the presence of the transcription start site and the region extending upstream from this site. The plasmid pBasic, which does not contain a promoter or enhancer, and a pControl plasmid, which possesses the SV40 promoter, were also tested in each experiment.
Transient transfections with these recombinant plasmids were performed in CaCo2 and LLCPK1 cell lines, which express villin, and in Madin-Darby canine kidney epithelial cells, in which no villin expression is detected. Transcription from the villin promoter was measured by assaying ␤-galactosidase activity in extracts made from the transfected cells, and the results were expressed as fold induction over that of the promoterless vector, pBasic (Fig. 3C). High levels of ␤-galactosidase activity in the pControl transfected cell lines (CaCo2 cells, 50-fold over that of pBasic; LLCPK1 cells, 98-fold) demonstrated the presence of efficient general transcription/translation mechanisms in these cells (data not shown). Very low levels of ␤-galactosidase activity in pD1 both transfected cells compared with pBasic transfected cells showed that the transcription start site was necessary for an efficient specific transcription of the reporter gene and that nonspecific transcription was not initiated elsewhere in the villin regulatory sequences. The construct pA1 expressed the ␤-galactosidase gene at the highest level in CaCo2 cells (8- decreased ␤-galactosidase expression in CaCo2 cells (2-fold over pBasic) to about 25% of that of pA1, demonstrating that a major element that confers intestinal activity was confined within this fragment. Similar results were obtained when the region upstream from the transcription start site (encompassing HS III and HS IV) was almost wholly deleted with or without HS II (pC1 and pC2, respectively). The deletion of the intronic region alone (pA3) or in combination with deleted sequences upstream from the transcription start site (pB3 and pC3 extend only from Ϫ480 and Ϫ100 bp, respectively) affected to a lesser extent ␤-galactosidase expression in the same intestinal cells (5.5-fold over pBasic), with a decrease to only about 65% of that of pA1, demonstrating that the regulatory elements that lay within 100 bp were sufficient to promote transcription in cultured cells. However, the level of ␤-galactosidase activity increased strongly when the plasmids pA3, pB3, and pC3 were transfected in LLCPK1 cells (10-, 44-, and 45-fold over pBasic, respectively), showing that the absence of the first intron, in combination with the lack of intestine-specific HS IV, was able to promote transcription in a kidney cell line. This would suggest that negative elements that confer repression in kidney transcription are confined in these elements.
To test specificity, the villin promoter-related constructs were transfected in Madin-Darby canine kidney cells, which do not express villin. After transfection, these cells showed only base-line levels of ␤-galactosidase activity when compared with pBasic-related activity (data not shown), demonstrating that the villin regulatory sequences were unable to promote efficient transcription in nonexpressing villin cells and that consequently the expression of the reporter gene in CaCo2 and LLCPK1 cells is specifically dependent upon these regulatory sequences. Taken together, these results from transient transfection of cultured cells demonstrate that (i) the mouse villin genomic sequence, extending from Ϫ3.5 to ϩ5.5 kb, specifically directs an efficient level expression of the ␤-galactosidase reporter gene in intestine-derived cells, (ii) this level is dramatically reduced when the intronic intestine-specific hypersensitive site HS II or the region upstream from the (ϩ1) site is deleted, (iii) lack of the entire first intron seems to partially restore the intestine-related ability to promote transcription, and (iv) lack of the entire first intron in combination with intestine-specific hypersensitive site HS IV is correlated with a strong increase of ability in promoting transcription in kidneyderived cells.
Analysis of Transgene Expression in Mice-Because the Ϫ3.5 to ϩ5.5-kb region of the mouse villin contained the enterocytelike-specific promoter/enhancer activity in transient transfection assays, we examined the ability of this region to drive intestine-specific expression of the ␤-galactosidase reporter gene in transgenic mice. Five founder animals that contained the pA1 construct as a transgene were obtained. The founder mice were analyzed for mRNA reporter gene expression in several adult tissues by reverse transcription-PCR analysis. From the same cDNA samples, products encoding ␤-galactosidase, villin, and TFIID were analyzed. The PCR assays enabled only the detection of spliced transcribed mRNA, excluding that from genomic DNA itself, by means of an exon connection strategy by combination of a 5Ј PCR primer from within the mouse villin promoter sequence just upstream of the splice donor site and the 3Ј primers from within the ␤-galactosidase gene or the villin gene. For each founder, no reporter gene expression was detected in the tissues in which villin mRNAs were not detected using the PCR assay (Fig. 4). For all founder mice, the reporter gene transcription was detected along the cephalocaudal axis of the gut (duodenum, jejunum, ileum, proximal, and distal colon) following the intestine-specific expression of the villin gene (Fig. 4). In the kidney, the transgene was only transcribed in one founder of five animals obtained (Fig.  4). TFIID mRNA was present in all samples from tissues in which the reporter gene expression could not be detected (Fig.  4), confirming the quality of RNA from these tissues.
To examine the precise cellular distribution of transgene expression within the tissues, cryostat sections of small intestine, colon, and kidney were prepared and subsequently stained for ␤-galactosidase enzyme activity. Similar results were obtained by immunofluorescence analysis of ␤-galactosidase expression (data not shown). For four of five transgenic mice, a heterogeneous pattern of expression in small intestine and colon was observed. This heterogeneity might be due to mosaicism because we examined founder animals. The expression was confined to the nucleus of the epithelial cells, as expected because the ␤-galactosidase gene contains a nuclear localization sequence signal (Fig. 5). The staining was detected by a stronger signal in the villi migrating cells when compared with the crypt cells, of both small intestine (Fig. 5A) and colon (Fig. 5C) epithelium, thus confirming that the Ϫ3.5 to ϩ5.5-kb region of the mouse villin gene is able to recapitulate precisely the cellular pattern of expression, along the crypt-villus differentiation axis, of the endogeneous villin gene (17). A continuous labeling of all cells of the crypt (Fig. 5, B and D) was observed, suggesting the expression of the transgene in the stem cells (10). It is noteworthy that the intensity of the ␤-galactosidase staining was similar to that of intestinal sections from chimeric animals which possess a ␤-galactosidase gene integrated at the villin locus by homologous recombination procedure (32), indicating that the Ϫ3.5 to ϩ5.5-kb region of the mouse villin gene was able to promote intestinal transcription as efficiently as the mouse villin gene itself. In the kidney of the founder mouse in which the transgene was detected by reverse transcription-PCR, the staining was only observed in the epithelial cells of the proximal tubules where the villin gene is expressed (data not shown). The founder animals were able to transmit the transgene to their offspring with a similar pattern of ␤-galactosidase expression (data not shown). In our attempt to direct an efficient expression of the reporter gene in the intestinal epithelium with shorter regulatory sequences, plasmids pA3, pB3, and pC3 were used to generate transgenic mice, because these constructs display efficient levels of ␤-galactosidase activity in intestine-derived CaCo2 cells. The presence of the transgene assessed by ␤-galactosidase staining procedure was observed in three of the four independent lines of pA3 transgenic mice generated. These three lines expressed the reporter gene only in the small intestine (in both the immature and differentiated epithelial cells along the crypt-villus axis), and all three lines failed to express the transgene in the other tissues tested, particularly noteworthy is the lack of expression in the colon and the kidney (data not shown).
These results demonstrate that (i) the 3.5-kb regulatory region upstream from the transcription start site of the mouse villin gene is necessary and sufficient to sustain expression strictly in small intestine of transgenic mice, (ii) the first intron of the mouse villin gene is required for colon and kidney expression in transgenic mice. Concerning the pB3 and pC3 transgenic mice, no transgene expression was observed in all tissues examined, including small intestine, colon, and kidney. Thus, the key cis-acting elements of the villin gene required for intestinal and/or kidney-related expression of transgene(s) in transgenic mice are not located only within the region encompassing Ϫ480 bp upstream from the transcription start site, as observed in the cultured epithelial cells. DISCUSSION In this report, we demonstrate that cis-acting sequences located within a 9-kb region (-3.5 to ϩ5.5 kb from the start site of transcription) of the mouse villin gene are sufficient to direct both correct tissue-specific and high expression level of the ␤-galactosidase reporter gene in transgenic mice, when compared with the endogenous gene (19). Reporter gene expression is detected in the whole intestinal tube and appropriately restricted to epithelial cells along the crypt-villus axis of both small intestine and colon. In addition, these regulatory elements can maintain a gradient of ␤-galactosidase gene expression from the crypts of Lieberkü hn to the tips of villi that precisely reproduce the gradient exhibited by the murine villin gene (17). Similarities between transgene and endogenous gene expression were also noticed as judged by a comparison with the staining intensity of ␤-galactosidase activity in intestinal sections from our transgenic mice and mice in which the reporter gene has been inserted at the natural villin locus by homologous recombination (32).
In the kidney, for only one animal of five analyzed, mouse reporter gene expression was restricted to epithelial cells of the proximal tubules recapitulating the villin expression pattern in this tissue. This suggests that transcriptional mechanisms specifying gene expression to intestine and kidney tissues are in the Ϫ3.5 to ϩ5.5-kb region of the mouse villin gene and that those related to kidney may be sensitive to positional effects. Indeed it is known that the transgene expression is dependent on the site of chromosomal integration and can be influenced by regulatory regions in the vicinity, presumably acting on chromatin conformation (33). The construct that entirely lacks the first intron of 5.5 kb but that harbors 3.5 kb 5Ј to the start site of transcription of the mouse villin gene, placed in front of the ␤-galactosidase gene, restricts the in vivo expression of the reporter gene only into the epithelial cells along the crypt-villus axis of the small intestine. The extinction of the reporter gene expression in the kidney might be due to strong positional effects, as reported above, whereas the extinction related to the colon might be due to the absence of regulatory elements of the intron 1, such as the intestine-specific DNase I-hypersensitive site HS II.
Constructs harboring only the first 480 and 100 bp 5Ј to the start site of transcription, in combination with the lack of the first intron, placed in front of the ␤-galactosidase gene, both failed to drive intestine-specific and kidney-specific expression of ␤-galactosidase, suggesting that the intestine-specific DNase I-hypersensitive site HS IV localized just upstream from the 480 bp might play an important role in promoting reporter gene expression into the epithelial cells of the small intestine. Thus, distinct and separable regulatory elements in the mouse villin gene may direct transgene expression along the cephalocaudal axis of the gut: the regulatory elements required for transgene expression in the small intestine might be localized in the 3.5-kb region (i.e. the HS IV site) upstream from the transcription start site, whereas those necessary for the colonic expression might be localized in the first intron (i.e. the HS II site).
The inability of shorter regulatory sequences of the mouse villin gene to direct correct expression of the reporter gene in the whole intestine of transgenic mice might also be explained by spatial rearrangement of chromatine structure due to the lack of the entire first intron. In fact, the results described here are reminiscent of those of the adenosine deaminase gene (34) and the aldolase B gene (35), in which elements located in the first intron are required for transgene expression in vivo, because they may contain cis-acting tissue-specific enhancer elements and/or elements involved in promoting decondensation of the chromatin structure, allowing the accessibility for transcription factors and RNA polymerase. Further investigations will be required to elucidate the precise role of the first intronic region of the mouse villin gene.
To explain the discrepancy seen in the ability of the mouse villin gene regulatory elements to promote transcription of the reporter gene in cell cultures versus transgenic animals, we may argue that the regulation of gene expression in the intestinal epithelium occurs as cells differentiate and migrate along the crypt-villus axis. This process depends on the contacts that these cells maintain with other neighboring cells on one hand and with the extracellular matrix on the other hand (36). Thus, an ex vivo system as the intestine-derived CaCo2 cell line used in our study, is limited by its weak ability to recapitulate the temporal and spatial complexities of this epithelium and emphasizes the importance of using in vivo models to define a function for specific regulatory sequences (37,38).
Previous studies carried out in transgenic mice to map transcriptional regulatory elements responsible for intestinal expression have been performed using cis-acting sequences of genes expressed in villus associated-enterocytes of small intes- tine (4, 5, 38 -40). In some of these cases, precocious activation in the crypts in combination with extended expression in the colon occurs in an inappropriate manner. Thus, to our knowledge, the 9-kb regulatory region of the mouse villin gene represents the only characterized cis-acting sequences reported today that allow the expression of a heterologous gene in small intestine and colon epithelial cells of transgenic mice reproducing with great fidelity the tissue-specific and cell-specific pattern of expression when compared with that of the endogenous gene itself. In addition, the mice lines that drive a transgenic expression exclusively restricted to the intestinal mucosa could already be studied after selection of those that will not display expression into the kidney because of the positional effects.
The ability to target genes of interest in transgenic mice following the villin-restricted pattern of expression and particularly in the crypt stem cells should lead to the development of targeted genes in animal models. Experimental mouse models reproducing several steps of human colorectal carcinogenesis (a possible genetic pathway has been proposed by Fearon and Vogelstein (41)) could for instance be obtained by efficiently targeting the associated oncogenes or mutated tumor suppressor genes to colonocytes using the villin regulatory region. Another use could lie in the establishment of new cell lines derived from the digestive tract by targeting a thermosensitive SV40 T antigen to the crypt-resident progenitors of intestinal cells, as used in other systems (42)(43)(44).