extfieldsofstudy: Chemistry Medicine

text:
Gαi Signaling Promotes Marginal Zone B Cell Development by Enabling Transitional B Cell ADAM10 Expression

The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how Gi signaling affects this fate decision is unknown. Here, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gαi proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gαi-deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gαi-deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gαi signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gαi nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gαi nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gαi signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development.

The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how Gi signaling affects this fate decision is unknown. Here, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gαi proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gαi-deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gαi-deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gαi signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gαi nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gαi nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gαi signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development.
Keywords: heterotrimeric g-protein, notch2, aDaM10, B lymphocyte, marginal zone inTrODUcTiOn The fetal liver and the bone marrow support B1 and B2 lymphocyte development (1,2). Fetal liver hematopoietic progenitors give rise to long-lived B1 B lymphocytes, which predominately reside in serosal lined cavities and in the spleen. As hematopoiesis shifts from the fetal liver to the bone marrow, B1 B cell production largely ceases, and bone marrow progenitors produce B2 cells, which have two developmental fates that are not realized until the newly formed B cells traffic to the spleen. These recent splenic arrivals are termed transitional B cells and they express B220, CD19, IgM, and CD93 (3). They must undergo a second tolerance checkpoint and will eventually mature to a follicular (FO) or a marginal zone (MZ) B cell (4). FO B cells reside in lymphoid follicles and continuously recirculate between lymphoid organs. MZ B cells do not recirculate but can shuttle between the splenic MZ and follicles (5). They have a limited antigen receptor repertoire, largely specialized to recognize encapsulated bacteria and polysaccharide antigens in a T cell-independent fashion (6).
Precisely when the B2 cell developmental fate is assigned in the spleen has been controversial (7)(8)(9). Bone marrow-derived B cell progenitors enter the marginal sinus and red pulp of the spleen and are termed transitional B cell type 0 (T0) cells (10). T0 cells migrate from the MZ sinus into the splenic white pulp, where they acquire surface IgD becoming T1 cells. T1 cells that gain CD23 are termed transitional B cell type 2 (T2) cells and their survival is highly BAFF dependent (11). Based on high or low expression of CD21, T2 cells are divided into T2-MZ and T2-FO, respectively (3). As designated, these cells are biased to become FO or MZ B cells; however, the commitment to a MZ or FO B cell phenotype likely occurs before the T2 stage. A recent study exploring the loss of MZ B cells in thousand-and-one amino acid kinase (TAOK) 3-deficient mice found that wild-type T1 cells bearing surface ADAM10 exclusively differentiated into MZ B cells, while ADAM10 surface-negative T1 cells gave rise to both FO and MZ B cells (12).
A variety of genetic mutations bias B2 B cell differentiation toward either a MZ or FO B cell fate (13)(14)(15)(16)(17)(18). These mutations largely impact Notch2, BCR, or BAFF signaling. A MZ fate requires the engagement of Notch2 on transitional B cells by Delta-like 1 (DL1). Splenic fibroreticular cells located in the white pulp express DL1 and their genetic ablation blocks MZ B cell development (19). Ligand binding to Notch2 induces an ADAM10-dependent cleavage that allows a subsequent intramembranous cleavage by γ-secretase. ADAM10 −/− mice lack MZ B cells (14,20). The ADAM10 and γ-secretase-mediated cleavages releases the intracellular domain of Notch2 (NICD2), which enters the nucleus to interact with DNA-binding proteins to activate Notch2 target genes. Notch2 along with nuclear factor-κB (NF-κB) activation downstream of the BAFF receptors instructs a MZ fate (21). BCR signaling during positive selection may also impact the B2 cell fate decision as weak signals promote MZ B cell development while stronger signals bias the cells toward a FO B cell, perhaps by tuning the sensitivity of the cells to Notch2 signaling (15,22). The loss of Gαi proteins in developing B cells severely reduces MZ B cell numbers; however, whether or how this loss affects the MZ versus FO B cell fate decision is unknown (23,24).
In B cells, the absence of Gαi2 markedly reduces the available pool of Gi proteins, and the additional loss of Gαi3 depletes it, disabling chemoattractant receptor signaling (24). Surprisingly, the absence of the three highly expressed B cell chemoattractant receptors CXCR5, CCR7, and EBI2, whose combined loss completely disrupts the splenic lymphoid architecture, did not cause a loss of phenotypic MZ B cells (25). Two G protein-coupled receptors (GPCRs) have been linked to MZ B cell positioning and retention, the cannabinoid receptor CB2 and the sphingosine 1-phosphate receptor 1 (S1PR1). Genetic targeting CB2 reduced MZ B cell numbers, while the loss of S1PR1 had no impact (5,(26)(27)(28). Despite the reduction in MZ B cells, the loss of CB2 did not affect BCR or Notch2 signaling (26). Targeting another GPCR, GPR97, caused an expansion of MZ B cells at the expense FO B cells, perhaps by affecting NF-κB activity (29). Together, the GPCR gene targeting data suggest that the loss of Gαi in B cells causes the loss of MZ B cells due to their failure to be retained in the MZ, rather than impacting MZ B cell development.
In this study, we tested whether the loss of Gαi proteins directly affected MZ B cell development. We show that despite the direct exposure to Notch2 ligand expressing cells, neither Gαi-deficient nor pertussis toxin (PTX)-treated progenitor B cells properly differentiate to MZ B cells. Favoring an intrinsic defect in Notch2 signaling, the Gαi-deficient transitional B cells have low levels of surface ADAM10 and of Notch2 target genes. Furthermore, treating B cell progenitors with PTX causes a partial T1-T2 transition block and inhibited the normal increase in ADAM10 that occurs on transitional B cells. The significance of these results is discussed.

animals
C57BL/6J and C57BL/6J vav1-cre mice were obtained from Jackson Laboratory. Mice with targeted deletion in Gnai3 −/− and Gnai2 −/− , and Gnai2 fl/fl mice were kindly provided by Dr. Lutz Birnbaumer (NIEHS, NIH) and backcrossed more than 12 times to C57BL/6J mice. Gnai2 fl/fl mb1-cre and Gnai2 fl/fl vav1-cre mice were obtained by crossing the appropriate cre expressing strain with the Gnai2 fl/fl mice and backcrossing to obtain the desired genotype. ric8 fl/fl mice on a mixed background have been described previously and were backcrossed 10 times on to C57BL/6J. The C57BL/6 mb1-cre mice were kindly provided by Dr. Michael Reth (University of Freiburg, Germany). For those experiments that directly compared WT and gene targeted mice, littermate controls were used when possible. Otherwise age and sex matched mb1-cre, vav1-cre, ric8 fl/fl , or Gnai2 fl/fl mice served as controls. All mice were used in this study were 6-14 weeks of age. Mice were housed under specific-pathogen-free conditions. All the animal experiments and protocols used in the study were approved by the NIAID Animal Care and Use Committee at the National Institutes of Health.

rna isolation and real-Time qPcr
Splenic B-cell subsets were sorted, and RNA was isolated using the TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions. Complementary DNA was synthesized using oligo(dT) and Omniscript RT (Qiagen) from 500 ng or 1 µg of total RNA. The real-time PCR primers used to amplify genes are listed below while those for Dtx1, Id2, Id3, Bcl3, Btk, Hes1, and Rpl27 were from Qiagen (QuantiTect primer QT00097139, QT01038870, QT00248185, QT00247583, QT00102179, QT00313537, and QT02328683, respectively). Real-time PCR was performed using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) following the Rotor-Gene SYBR Green PCR kit (Qiagen) protocol. All results are expressed as 2 −ΔΔCt , where ΔΔCt = (Cttarget − CtRpl27) for treated samples-(Cttarget − CtRpl27) for control cells. Ct stands for cycle threshold. Data are shown as the mean ± SEM.

chemotaxis assays
Chemotaxis assays were performed using a transwell chamber (Costar). Briefly, splenic B cells were immunostained for B cell subsets with fluorochrome-conjugated antibodies against B220, CD21, and CD23; washed twice, resuspended in complete RPMI 1640 medium, and added in a volume of 100 µl to the upper wells of a 24-well transwell plate with a 5-µm insert. Lower wells contained various concentrations of CXCL12 or CXCL13 (R&D Systems) in 600 µl of complete RPMI 1640 medium. The numbers of cells that migrated to the lower well after 2 h incubation were counted using a MACSQuant flow cytometer (Miltenyi Biotec). The percent migration was calculated as the numbers of cells that migrated into the lower part of the chamber divided by the total number of cells in the starting cell suspension, and multiplying the results by 100.

imagestream (is): labeling, acquisition, and analysis
The splenic B cells were stained with B220 PE conjugated, CD21 e450 conjugated and CD23 PE-cy7 conjugated for 10 min then fixed using 2% paraformaldehyde. The p65 subunit of NF-κB was visualized by indirect labeling. The primary Rabbit polyclonal NF-κB/p65 antibody (SantaCruz Biotechnology) was diluted 1:50 in 3% BSA, and 0.1% Triton X-100 in PBS and added to 1 × 10 6 fixed cells. Samples were incubated for 2 h at room temperature. Primary antibody was washed and 1:200 dilution of secondary Alexa488-conjugated donkey anti rabbit IgG antibody (Jackson ImmunoResearch Laboratories Inc.) was added and incubated at room temperature in the dark for 1 h. Secondary antibody was removed, and cells resuspended in 100 µl PBS. Just prior to running on the IS, all samples had Draq5 (Cell Signaling) added (20 nM final concentration), to visualize the nucleus. At least, 20,000 events were collected for all samples on an IS MarkII using 488, 405, and 642 nm laser excitations. Cell populations were hierarchically gated for single cells that were in focus and were positive for both Draq5 and p65. Then, based on Draq5 intensity histogram, cells were gated on those that were in G0 and G1/S phase to avoid cells that have a disrupted nuclear membrane. After those gates were applied at least 1,000 cells per group were acquired (follicular: B220 + CD23 + CD21 − , transitional: B220 + CD23 − CD21 − , and MZ: B220 + CD23 − CD21 + ). Following data acquisition, the spatial relationship between the NF-κB and nuclear images was measured using a feature (ratio translocation) calculated in the IDEAS software package. The "Ratio translocation" feature, is the ratio of NF-κB intensity in the nuclear mask over NF-κB intensity in the whole cells (calculated using the brightfield mask) expressed as the percentage. Nuclear mask was defined based on Draq5 intensity where a signal was considered specific when it was a least 20 times higher than the background. The shift in the distribution between two populations (e.g., control versus treated cells) was tested using the Fisher's Discriminant ratio calculated using Graphpad Prism software. A cell was considered with a nuclear p65 NF-κB when the "Ratio translocation" was greater than 60%.

statistics
In vivo results represent samples from three to nine mice per experimental group. Results represent mean values of at least triplicate samples. SEMs and p values were calculated with t-test or two-way ANOVA using GraphPad Prism (GraphPad software).

Models of gα i Deficiency and loss of MZ B cells
To better understand the role of Gαi proteins in MZ B cell development in mice, we used several different mouse lines. Mice with a B cell-specific loss of Gαi2 were generated by crossing Gnai2 fl/fl mice to mb1-cre mice. Double knockout (DKO) mice, which lack both Gαi2 and Gαi3 in their B cells were developed by crossing Gnai3 −/− mice to the Gnai2 fl/fl mb1-cre mice (24). We also crossed the mb1-cre mice to the ric8 fl/fl mice. Ric-8A functions as a chaperone and guanine nucleotide exchange factor for a subset of G protein α subunits including Gαi. The B cell specific loss of Ric-8A causes a marked reduction in B cell expression of Gαi2 and Gαi3 (30). Finally, we deleted Gnai2 in hematopoietic cells using vav1-cre, which results in the loss of Gnai2 at all stages of B cell development (31). To compare MZ development in these various mouse strains, we prepared spleen cells from them and analyzed cells in the B220 gate for their expression of CD21 and CD23. MZ B cells are CD21 high and CD23 low , while FO B cells are CD23 high and CD21 int ( Figure 1A). As we have previously noted MZ B cell development is largely intact in the Gnai3 −/− mice, markedly reduced in the mice with a B cell specific deletion of Gnai2 and in Gnai2 fl/fl vav1-cre mice, and severely reduced in the DKO and ric8 fl/ fl mb1-cre mice ( Figure 1A). We confirmed the loss of MZ B cells in the DKO mice by alternatively immunostaining with CD23 and CD1d. As expected we observed a sharp reduction in the B220 + CD23 low and CD1d high cells in the DKO mice ( Figure 1B). Besides the reduced % of MZ B cells, there is also an overall loss of B cells in the spleen as has been previously documented (Gnai2 fl/ fl mb1-cre, Gnai3 −/− , Gnai2 fl/fl vav1-cre, ric8 fl/fl mb-1cre, and DKO mice have approximately 92, 94, 88, 72, and 50%, respectively, of the number of splenic B cells in control mice) (24,30,31). A detailed picture of the % of cells among the different splenic B cell subsets in the various Gαi-deficient mice is shown in Table 1.

gα i -Deficient Transitional B cells have similar levels of nuclear p65 and Phosphorylated Bruton's Tyrosine Kinase (pBTK)
Since the strength of BCR signaling in transitional B cells is linked to the MZ versus FO B cell fate decision (8,32), we checked whether we could find evidence of enhanced BCR signaling in Gαi-deficient transitional B cells, which would help explain their lack of MZ B cells. First, we used IS cytometry to examine the nuclear localization of p65 NF-κB in transitional and FO B cells from WT mice and from mice whose B cells largely lacked Gαi2/3 (ric8 fl/fl mb1-cre). We found that approximately 38, 47, and 45% of the WT transitional, FO, and MZ B cells, respectively, had nuclear p65. However, these results did not differ from those obtained with the B cells fractions deficient in Gαi proteins (Figures 1C-E). Next, we assessed the status of BTK by examining the levels of BTK phosphorylated on Y223 in transitional and FO B cells. Located in the BTK SH3 domain, this is an autophosphorylation site, whose phosphorylation occurs following Src kinase Y511 transphosphorylation and that can be induced by IgM crosslinking (33). Normalized to the WT T0 cells, the WT T1 and T2, and FO cells had a 19, 53, and 48% increase in pBTK, respectively ( Figure 1F). Compared to their respective wild-type fractions, the DKO T0 and T1 fractions had an approximately 12% decrease in pBTK, the DKO T2 cells had a similar level, while the DKO FO B cells had a 35% increase. Overall, these results do not support a role for enhanced BCR signaling in the Gαi-deficient B cell transitional B cells as a contributing factor to the lack of MZ B cells in these mice.

Decreased notch signaling in B cells lacking gα i expression
Upon entering the splenic white pulp, T0 B cells acquire surface IgD and develop to T1 B cells (10). However, Gαi-deficient B cells have difficulty entering the white pulp (24). Consistent with this defect, the Gαi-deficient B cells exhibit a partial T0 to T1 transition block. This can be visualized by flow cytometry by gating on the B220, IgM, and CD93-positive cells and plotting IgD versus CD23. Such profiles show an increased ratio between the T0      and T1 cells from the Gnai2 fl/fl mb1-cre, ric8 fl/fl mb1-cre, and DKO mice as compared to the same ratio using cells from the WT and Gnai3 −/− mice (Figure 2A). Furthermore, the Gαi-deficient T1 and T2 transitional B cells expressed a lower level of surface IgD than did the WT transitional B cells (Figure 2B). A failure to properly enter the white pulp could limit Gαi-deficient B cells from contacting the Notch ligands present on CCL19-expressing follicular fibroblasts (19). To assess whether the Gαi-deficient B cells had received inadequate Notch signals, we first checked CD21 expression on transitional and FO B cells from WT and DKO mice. A known Notch2 target gene, the CD21 gene has binding sites for Rbp-J, a DNA-binding protein to which the NICD binds (34). Consistent with an early impairment in Notch signaling, the DKO transitional B cells had reduced levels of CD21 expression compared to control cells ( Figure 2C). Next, we used RT-PCR to check the expression of several other Notch target genes including hairy and enhancer of split-1 (Hes1), Deltex1 (Dtx1), and inhibitor of DNA binding 2 (Id2). Hes1 is a basic helix loop helix transcription factor activated by Notch signaling, while Dtx1 is a ubiquitin ligase that regulates Notch signaling (35). Id2 and Id3 inhibit the DNA-binding activity of E proteins, transcription factors important for B cell development and differentiation (36). The control transitional B cells (T0-T2) expressed higher mRNA levels for Dtx1, Id2, Id3, and Hes1 than did the DKO cells, while Btk and Bcl3 mRNA levels were similar ( Figure 2D) To determine whether the Gαi-deficient B cells express normal levels of Notch2, we first examined B cell lysates by Notch2 immunoblotting, which revealed no differences in the level of unprocessed Notch2 or the N2-Ext domain (Figure 2E). To assess Notch2 surface expression, we used flow cytometry. We found similar levels of Notch2 expression on WT and DKO bone marrow progenitors (Figures 2A-E) and recirculating B cells ( Figure 2F) (data not shown). Next, we checked various splenic B cell populations (Figure 2F). WT and DKO T0, T1, and FO splenic B cells also expressed similar levels. We did note a slight enhancement in Notch2 expression on the DKO T2 cells, and a reduced level on the DKO MZ precursor (MZP) cells. Thus, the reduction in Notch2 target genes noted in the Gαi-deficient B cells cannot be explained by inadequate levels of Notch2 expression at the plasma membrane.
To test whether we could restore MZ B cell development by ensuring access to Notch ligands, we used a previously describe in vitro assay of splenic B cell development (37). Non-adherent bone marrow cells that had been cultured for 4 days with Il-7 (>95% B220 + CD19 + CD93 + ) were plated on a stromal cell line that expresses a Notch ligand (OP9-DL1) or not (OP9), in the presence of BAFF. Phenotypic T0, T1, and T2 cells were evident 2 days later, and phenotypic FO and MZ shortly thereafter (data not shown, see below). By 7 days, approximately 15-25% of the recoverable cells had a MZ B cell phenotype. As expected, few if any MZ B cells appeared in the OP9 cultures. By contrast, directly plating the Gαi-deficient B cell progenitors on the OP9-DL1 cells did not rescue normal MZ B cell development (Figures 3A,B). These data argue that the Gαi-deficient transitional B cells are less sensitive to Notch ligands. gα i nucleotide exchange, but not gβγ signaling, is needed for MZ B cell Development in OP9-Dl1 cultures To provide further evidence that Gαi signaling is needed for MZ B cell development we made use of PTX, which ADP ribosylates Gαi proteins preventing receptor triggered Gαi nucleotide exchange. This inhibits Gβγ subunit release from the heterotrimeric G-protein, thereby blocking the activation of downstream effectors of Gαi-GTP and Gβγ (38). When we treated bone marrow B cell progenitors with PTX, prior to plating them on OP9-DL1 cells, we found a sharp reduction in the appearance of phenotypic MZ B cells. Initially, we added PTX during the final 24 h of the IL-7 culture, but subsequently, showed that a 2-h exposure had a similar inhibitory effect (Figure 3C). To assess the importance of Gβγ subunit release, we employed Gallein, a small molecule inhibitor of many Gβγ triggered signaling pathways including chemotaxis (39). We first established that Gallein inhibited B cell chemotactic responses (Figure 3D). It did, as a 20-µm concentration reduced B cell chemotaxis to either CXCL12 or CXCL13 by approximately two-thirds. However, repeatedly adding a similar concentration to the OP9-DL1 B cell cultures did not adversely affect MZ B cell development (Figure 3E). This result argues that a Gβγ effector function unaffected by Gallein treatment, or that a Gαi-GTP effector helps mediates MZ B cell development. Since the loss of Gαi can affect intracellular cyclic adenosine monophosphate (cAMP) levels by allowing the unopposed action of Gαs, we assessed the impact of raising intracellular cAMP levels in the OP9-DL1 cultures. IBMX increases cAMP by inactivating cAMP phosphodiesterases while the β-adrenergic receptor agonist terbutaline raises cAMP via Gαs-mediated activation of adenylyl cyclases. While both compounds reduced the appearance of MZ B cells, neither approached the efficacy of PTX (Figure 3F). We also tested the impact of limiting CXCR4 or CB2 signaling by adding specific antagonists, AMD3100 and AM630, respectively (40,41). However, both compounds had a negligible impact on MZ B cell development in the OP9-DL1 system (Figure 3F). We did note that adding CXCL12 slightly raised the % of MZ B cells at the termination of the culture although the difference did not reach significance. One caveat in interpreting these experiments, not only the progenitor B cells, but also the OP9-DL1 cells were exposed to the different compounds. However, this is unlikely an issue in the PTX experiments, as we exposed the progenitor B cells to PTX prior to adding them to the OP9-DL1 cells. Finally, to assess whether the loss of Gαi proteins altered cAMP signaling in vivo, we checked phosphorylated protein kinase A and phosphorylated CREB levels in the WT and DKO transitional B cells by flow cytometry (Figure 3G). We found no significant differences between WT and DKO transitional B cells in this regard. Thus, while elevated cAMP levels may adversely affect MZ B cell development they are unlikely to account for the lack of MZ B cells when B cells lack Gαi proteins.

reduced aDaM10 expression in gα i -Deficient Transitional B cells
Diminished Notch2 target gene expression following direct exposure to Notch2 ligands suggests defective Notch2 processing in the Gαi-deficient cells. We focused on ADAM10, whose activity releases the extracellular domain of Notch2 allowing γ-secretase to release the NICD2. The ADAM10 metallopeptidase functions not only to cleave the extracellular domain of Notch2 but also other surface membrane proteins including CD23 and ICOS ligand (42,43). Consistent with a defect in ADAM10 sheddase activity, we found elevated levels of both membrane proteins on the Gαi-deficient T2 cells compared to WT cells ( Figure 4A). Immunoblotting ADAM10 in cell lysates from WT and DKO B cells revealed that the DKO B cells had reduced levels of ADAM10, and impaired ADAM10 processing ( Figure 4B). Maturation of ADAM10 requires removal of the prodomain by either Furin or PC7. The prodomain functions as a molecular chaperone important for the localization of the catalytic domain to the cell membrane (44). The DKO B cell lysates had 50% less processed ADAM10 compared to the WT B cell lysates. To assess ADAM10 surface expression, we used flow cytometry comparing both bone marrow and splenic B cell subsets. This analysis showed a reduction in ADAM10 expression on the DKO bone marrow Fr. B/C, Fr. D., and Fr. E cells and on DKO splenic T0 and T1 cells, and MZPs ( Figure 4C). Of note, the Gαi-deficient T2 cells had elevated CD23 and ICOS ligand levels despite having ADAM10 levels equivalent to the WT T2 cells. This may reflect the reduced ADAM10 levels on the Gαi-deficient T1 cells or reduced ADAM10 activity despite a similar expression levels.
In the study of Taok3 −/− mice (12), the reduction of ADAM10 expression on Taok3 −/− T0 and T1 cells was shown by gating on CD19 + CD93 + IgM + spleen cells, plotting ADAM10 expression versus CD23, and focusing on the ADAM10 + CD23 − cells. A similar strategy shows the reduction in ADAM10 expression on the DKO cells as compared to the WT cells ( Figure 4D). Because certain tetraspanins (TSPAN) can interact with ADAM10 and modify ADAM10 activity (45), we gated on CD19 + CD93 + IgM + cells and analyzed ADAM10 versus CD81 (TSPAN28), CD63 (TSPAN30), CD53 (TSPAN25), or CD9 (TSPAN29). Of the different TSPANs, only CD81 proved useful as we found fewer ADAM10 + cells in the CD81 + subset of Gαi-deficient transitional B cells when compared to wild-type cells (Figure 4D). Finally, we checked the status of pTAOK1-3 by flow cytometry using a monoclonal antibody that recognizes phosphorylated S181 in TAOK1 and 2, and S177 in TAOK3 (Figure 4E). We found a small reduction in the amount of phosphorylated TAOK1-3 in the DKO T0 and T1 cells. Thus, the poor ADAM10 expression on T0 and T1 cells in the spleen helps to explain the defective MZ B cell development in mice whose B cells lack Gαi2 or Gαi2 and Gαi3.
Both cXcr4 engagement and anti-igM crosslinking increase TaOK, BTK, and creB Protein Phosphorylation in Transitional T0 and T1 cells Next, we compared the effect of CXCL12 and anti-IgM on the phosphorylation status of TAOK proteins in various B cell subsets with a focus on transitional B cells. To do so, we exposed wild-type B cells to either anti-IgM or to CXCL12 and used flow cytometry to monitor pTAOK proteins in the different splenic B cell subsets (Figure 5A). We found that a 15-min exposure to either stimulus increased pTAOK1-3 in T0 and T1 cells, although anti-IgM proved more potent. Anti-IgM stimulation also raised the pTAOK1-3 levels in T2, MZP, and MZ B cells while CXCL12 did not. For comparison, we checked the same stimuli on the levels of pBTK and pCREB. Mice lacking BTK have a defect in the T2 to FO B cell transition, but do not have defective MZB cell development (46). We found that anti-IgM treatment triggered increases in pBTK levels in all B cell populations apart from FO B cells, while CXCL12 increased pBTK levels in all the B cell populations except for T0 and FO B cells. In each instance, CXCL12 triggered a lower, and less sustained response than did BCR crosslinking (Figure 5B). In non-hematopoietic cells, CREB phosphorylation has been linked to ADAM10 processing (47). We found that anti-IgM and CXCL12 increased pCREB levels in all the splenic B cell subsets although again BCR crosslinking led to higher, more sustained responses ( Figure 5C). These results indicate that chemoattractant receptor and BCR signaling activate a partially overlapping set of signaling intermediates in transitional B cells.
cXcl12 or Bcr crosslinking raises aDaM10 levels on WT T0/T1 B cells, but not on gα i -Deficient T0/T1 B cells In the previous study of Taok3 −/− mice, the analysis of wild-type, transitional B cells following BCR crosslinking ADAM10 translocated from the cytosol to the plasma membrane as assessed by immunocytochemistry (12). We performed a similar analysis although we followed ADAM10 membrane expression on transitional B cells by flow cytometry after exposure to anti-IgM or CXCL12. We found that both stimuli triggered increased ADAM10 expression on a subset of T0 and T1 B cells, while ADAM10 levels rose variably on T2 B cells, and not at all on FO B cells in response to the same stimuli. Shown are flow cytometry patterns from an experiment where anti-IgM induced a particularly strong increase in ADAM10 expression ( Figure 6A, and data not shown). Next, we compared the response of WT and Gnai2 fl/fl vav1-cre B cells to CXCL12 or anti-IgM. As expected, the Gαi-deficient B cells failed to mobilize ADAM10 to the cell surface following stimulation with CXCL12; however, surprisingly anti-IgM treatment also failed to raise ADAM10 expression on the Gαi-deficient T0/T1 B cells. By contrast, anti-IgM did increase ADAM10 expression on T2 B cells from the same Gαi-deficient mice (Figure 6B). To confirm these results, we assessed whether blocking Gαi signaling in WT transitional B cells would also inhibit anti-IgM-induced upregulation of ADAM10, which it did (Figure 6C). In contrast to the Gnai2 fl/fl vav1-cre T2 cells, the PTX-treated WT T2 did not respond to antigen receptor crosslinking. As expected, PTX treatment reduced the CXCL12-induced increases in pBTK levels in the T0, T1, and T2 cells (Figure 6D). In contrast to the sharp reduction in ADAM10 expression following exposure of the transitional B cells to anti-IgM, pBTK levels were minimally affected by the PTX treatment ( Figure 6D). Thus, the loss of Gαi proteins or Gαi signaling not only impairs the upregulation of ADAM10 on T0/T1 B cells in response to CXCL12 but also following BCR crosslinking.

The increase of aDaM10 levels in OP9-Dl1 cultures Depends Upon gα i signaling
To further test the impact of BCR and Gαi signaling on developing wild-type transitional B cells, we used bone marrowderived B cell progenitors and the OP9-DL1 system. Following culture with IL-7 for 4 days, the bone marrow cells were 95% CD19 + B220 + CD93 + . These cells lacked CD21 and CD23 expression, one-third expressed surface IgM, and of those one-half expressed IgD. Approximately 3% of the CD19 + CD93 + IgM + cells expressed ADAM10 (Figure 7A). These cells were plated on OP9-DL1 cells in the presence of BAFF. Prior to plating we treated some cells with PTX for 2 h or not. To others cultures, we added CXCL12, anti-IgM, anti-IgM plus CpGs, or Ibrutinib, which inhibits Bruton's tyrosine kinase (48). We analyzed the cells 2 days later. First, we assessed the relative percentage of T0, T1, and T2 cells finding that PTX, anti-IgM, anti-IgM plus CpGs, and Ibrutinib to varying degrees inhibited both the T0-T1 and T1-T2 transition. By contrast, the addition of CXCL12 had no effect ( Figure 7B). Next, we analyzed the impact of the same stimuli on ADAM10 expression on developing T0 and T1 cells. We found that PTX, anti-IgM, and anti-IgM plus CpGs caused a reduction in the % of CD81 + ADAM10 + cells, while CXCL12 and Ibrutinib had no effect (Figures 7C,D). These results indicate that Gαi signaling supports ADAM10 expression on T0 and T1 B cells, providing a mechanism by which either constitutive Gαi signaling or the engagement with GPCR ligands can promote MZ B cell development. By contrast, the persistent exposure of progenitor and transitional B cells to surface Ig crosslinking interferes with T0-T1 and T1-T2 transitions and blocks ADAM10 membrane expression.
Finally, we confirmed the importance of Gαi proteins in ADAM10 expression by checking the acquisition of ADAM10 by developing WT and Gαi-deficient B cells in the OP9-DL1 system. Following culture with IL-7 for 4 days, bone marrow cells from either control (Gnai2 fl/fl or mb1-cre) mice or from the DKO mice were plated on OP9-DL1 cells in the presence of BAFF. We analyzed the cells prior to plating on the OP9-DL1 cells, also one (data not shown), and 2 days after plating. Representative flow cytometry patterns demonstrating CD93 versus CD19 expression, IgM versus CD23, IgD versus CD23 on IgM + gated cells, and ADAM10 versus CD81 on T0, T1, and T2 cells are shown (Figures 8A,B). Following culture with IL-7, nearly all the WT and DKO bone marrow cells expressed CD19 and CD93. However, a lower percentage of the DKO progenitors expressed surface IgM as compared to wild-type B cell progenitors. Among the WT and DKO IgM-positive cells, IgD levels appeared similar. We found a slight reduction in ADAM10 expression on the day 0 DKO T0 and T1 cells. After 1 day of culture on the OP9-DL1 cells, ADAM10 expression increased on the WT transitional B cells, while we noted little to no increase on the DKO T0-T2 B cells. After 2 days of culture,   Figure 8C). Thus, the inability of the Gαi-deficient progenitors to efficiently upregulate ADAM10 expression likely explains the lack of MZ B cell development in these cultures.

DiscUssiOn
Whether or how Gαi proteins promote MZ B cell development is unknown. In this study, we tested three different hypotheses: an inhibitory role for Gαi proteins in BCR signaling in transitional B cells, an inability of Gαi-deficient transitional B cells to access Notch2 ligand expressing cells, and an intrinsic role for Gαi proteins in Notch2 processing or signaling. This report supports the third hypothesis. Suggesting a defect in Notch2 processing or transcriptional activity both WT and Gαi-deficient transitional B cells expressed similar Notch2 levels, yet the latter exhibited impaired Notch2 signaling. The Gαi-deficient transitional B cells had low levels of CD21 and a reduction in Hes1, Dtx1, and Id2 expression, known Notch2 target genes. Arguing that inadequate access to Notch2 ligands did not account for the MZ B cell development defect, plating Gαi-deficient B cell progenitors directly on a source of Notch ligands did not rescue MZ B cell development. Supporting a defect in Notch processing and/or translocation, the Gαi-deficient transitional B cells had reduced plasma membrane localized ADAM10 and either blocking Gαi nucleotide exchange or the lack of Gαi proteins inhibited progenitor B cells from upregulating ADAM10 expression. Together, these data indicate that Gαi signaling helps supports ADAM10 expression and Notch 2 signaling in transitional B cell.
Notch processing and subsequent transcriptional activity depends upon its cleavage by membrane localized ADAM10. Besides lacking MZ B cells, ADAM10-deficient transitional B cells express low levels of Notch target gene (14). An ADAM10 processing defect partially accounts for the reduced membrane ADAM10 expression noted in the Gαi-deficient transitional B cells as immunoblots revealed a threefold to fourfold reduction in the mature form of ADAM10. Supporting this observation, anti-IgM did not trigger ADAM10 to translocate to the plasma membrane in the Gαi-deficient T0 and T1 B cells. Subject to multiple layers of regulation, ADAM10 activity requires proprotein convertases within the secretory pathway to cleave the ADAM10 zymogen.
This releases the active enzyme and promotes its plasma membrane translocation. Removal of the prodomain depends upon a canonical site for a Furin-mediated cleavage located between the pro-and catalytic domains of ADAM10 (44). Previous reports have linked ADAM10 processing to GPCR signaling (49). Most of these studies focused on GPCR engagement triggering ADAM10mediated EGF receptor transactivation rather than Notch2 activation. For example, treatment of non-transformed N15C6 cells with CXCL12 enhanced ADAM10 prodomain removal and triggered ADAM10-dependent amphiregulin ectodomain shedding (50). The enhanced ADAM10 activity depended upon Src kinase activity. Both free Gβγ subunits and GTP bound Gαi have been implicated in Src kinase activation (51). Not only did the Gαideficient transitional B cells have reduced membrane ADAM10 expression but so did developing B cells in the bone marrow. Thus, the microenvironmental exposure to chemoattractants in the bone marrow and spleen likely supports the maturation of ADAM10 in developing B cells.
While a processing defect may explain the low ADAM10 membrane expression on the Gαi-deficient transitional B, Gαi signaling also promotes the rapid translocation of ADAM10 to the plasma membrane. Only 5-10% of the wild-type transitional B cells (B220 + CD93 + IgM + CD23 − ) have elevated ADAM10 expression. It is this population that contained MZ, but not FO B cell precursors, and its loss in the Taok3 −/− mice that accounted for the MZ B cell development defect (12). Like the Taok3 −/− B cells, the Gαi-deficient B had a similar reduction in this population. Since ADAM10 processing occurred normally in the Taok3 −/− transitional B cells, an ADAM10 translocation defect best explained their poor MZ B cell development. In addition, crosslinking surface Ig on wild-type B cells mobilized ADAM10 from intracellular stores to the plasma membrane as assessed by immunohistochemistry (12). Using flow cytometry, we confirmed that BCR crosslinking increased plasma membrane ADAM10 expression on transitional B cells. Although not as robust as surface IgM crosslinking, the exposure of transitional B cells to CXCL12 also led to increased ADAM10 surface expression. In line with these results, a brief exposure to either anti-Ig or CXCL12 increased the level of pTaok proteins in wild-type T0 and T1 cells.
However, bone marrow cell-derived B cell progenitors behaved differently when exposed to CXCL12 or anti-Ig. A 2-day culture on OP9-DL1 cells upregulated ADAM10 and CD23 expression. Perhaps because the OP9-DL1 cells secrete CXCL12 (52) and likely other GPCR ligands, the addition of exogenous CXCL12 to the OP9-DL1 cultures had no effect. However, pretreating the B cell progenitors with PTX strongly inhibited the induction of ADAM10, reduced CD81 expression, decreased the appearance of T2 B cells, and decreased MZ B cell development. Surprisingly, the addition of anti-IgM also inhibited CD23 upregulation and totally blocked the appearance of T2 cells. While a BTK inhibitor also prevented the appearance of T2 transitional cells, it did not affect ADAM10 or CD81 expression. Based on these studies, both antigen receptor crosslinking and Gi signaling can promote the membrane expression of ADAM10, a necessary step in splenic MZ B cell development. However, the timing and the context of the antigen receptor signal may be important in determining whether newly arrived bone marrow B cells arrest, or further differentiate toward either a FO or MZ B cell.
Signaling pathways triggered by Gαi nucleotide exchange or downstream of BCR engagement cause the translocation of ADAM10 to cell membrane in transitional B cells. They may share or use divergent intermediates to trigger the translocation of ADAM10. Both BCR signaling and CXCR4 engagement enhanced the phosphorylation of Taok proteins, arguing that BCR and Gαi signaling may converge on Taok3 activation, which either directly or indirectly controls the translocation of ADAM10. An intriguing target of Taok3 kinase activity is Mst1 (53). Taok3 functions in the Hippo pathway both in parallel and upstream of Mst1/2 to activate LATS1/2. In addition, Mst1 is also a known effector triggered by chemokine receptor engagement, whose activation depends upon Gαi signaling. Interestingly, the mst1 −/− mice also have a severe defect in MZ B cell development (54). It will be of interest to check whether the mst1 −/− transitional B cells also have a reduction in ADAM10 membrane expression.
The eventual target of these signaling pathway must be ADAM10 or an ADAM10 associated protein. Intriguingly, a subgroup of tetraspanins (Tspan5, 10, 14, 15, 17, and 33) that have eight cysteines, referred to as the TspanC8 directly interact with ADAM10 (55). They affect the exit of ADAM10 from the endoplasmic endothelium, its translocation to the plasma membrane, and even its substrate specificity. Based on their mRNA expression (http://www.immgen.org/databrowser/index.html), the best candidate among the TspanC8 group to control ADAM10 expression in murine transitional B cells is Tspan14. Unfortunately, Tspan14 antibodies are not available for flow cytometry. However, since tetraspanin proteins often interact with other family members to form a tetraspanin web, we checked whether the expression of other family members might correlate with ADAM10 expression on transitional B cells. This led to the findings that the CD81 high transitional B cells also expressed membrane ADAM10 and that the Gαi-deficient transitional B cells, not only expressed less ADAM10, but also less CD81. In agreement with these results, a previous study showed that CD81 can regulate ADAM10mediated cleavage of TNF-α and epidermal growth factor (56).
To our knowledge, GPCR or chemoattractant receptor signaling has not been linked to ADAM10 function and Notch signaling in B lymphocytes. Here, we showed that Gαi-deficient transitional B cells have decreased ADAM10 expression and reduced Notch2 target gene expression. Explaining the reduced ADAM10 expression on transitional B cells, we identified both an ADAM10 processing and a translocation defect. We suspect that these may be controlled by separate signaling pathways, likely downstream of different effectors. Reduced or absent Gαi protein expression in developing B cells results in a prolonged absence of input from chemoattractant receptors, which likely supports both ADAM10 expression and processing in developing B cells. By contrast, PTX treatment acutely inhibits Gi signaling by prevents chemoattractant triggered Gαi nucleotide exchange. Normal developing transitional B cells treated with PTX fail to mobilize ADAM10, while stimulation with CXCL12 rapidly mobilized ADAM10 to the cell membrane on a subset of T0/T1 cells. Both chemoattractant and BCR signaling triggered the translocation of ADAM10 from intercellular stores to the plasma membrane perhaps by targeting tetraspanins. Future studies will focus on unraveling the signaling pathways that control the expression, processing, and translocation of ADAM10 in transitional B cells.

eThics sTaTeMenT
This study was carried out in accordance with the recommendations of the NIH Animal Research Advisory Committee (ARAC) guidelines, NIAID Animal Care and Use Committee. The protocol was approved by the NIAID Animal Care and Use Committee.
aUThOr cOnTriBUTiOns I-YH, CB, and JK designed and analyzed the experiments. CB, I-YH, and KH did the experimental work; CB and JK wrote and edited the manuscript.

acKnOWleDgMenTs
The authors would like to thank Dr. Anthony Fauci for his continued support. This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.


extfieldsofstudy: Medicine Biology

text:
Tamoxifen mechanically deactivates hepatic stellate cells via the G protein-coupled estrogen receptor

Tamoxifen has been used for many years to target estrogen receptor signalling in breast cancer cells. Tamoxifen is also an agonist of the G protein-coupled estrogen receptor (GPER), a GPCR ubiquitously expressed in tissues that mediates the acute response to estrogens. Here we report that tamoxifen promotes mechanical quiescence in hepatic stellate cells (HSCs), stromal fibroblast-like cells whose activation triggers and perpetuates liver fibrosis in hepatocellular carcinomas. This mechanical deactivation is mediated by the GPER/RhoA/myosin axis and induces YAP deactivation. We report that tamoxifen decreases the levels of hypoxia-inducible factor-1 alpha (HIF-1α) and the synthesis of extracellular matrix proteins through a mechanical mechanism that involves actomyosin-dependent contractility and mechanosensing of tissue stiffness. Our results implicate GPER-mediated estrogen signalling in the mechanosensory-driven activation of HSCs and put forward estrogenic signalling as an option for mechanical reprogramming of myofibroblast-like cells in the tumour microenvironment. Tamoxifen, with half a century of safe clinical use, might lead this strategy of drug repositioning.

Introduction
The G protein-coupled estrogen receptor (GPER) is a seven transmembrane G protein-coupled receptor (GPCR) that mediates the acute response to extracellular estrogens [1,2]. Agonists for GPER include endogenous estrogens such as 17β-estradiol, as well as synthetic compounds, such as tamoxifen and fulvestrant. Tamoxifen is a GPER agonist that has been used in clinics for more than 50 years as hormonal therapy for breast cancer based on the classical genomic estrogen receptor (ER) signalling pathway, unrelated to GPER. Interestingly, tamoxifen has also been used in women at risk of developing breast cancer and has been observed to reduce mammographic density and fibrosis [3,4]. Due to its established pharmacology and non-toxicity, tamoxifen is well positioned to lead our efforts in exploring novel modes of action for this drug and investigating the possible clinical benefits of GPER-mediated estrogen signalling.
Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and, regardless of aetiology, occurs predominantly in patients with cirrhosis, which is characterised by excessive extracellular matrix (ECM) deposition that presents the ideal environment for promoting tumour formation. In this environment, hepatocyte necrosis, inflammation, oxidative stress and hypoxia are responsible for genetic alterations and deregulation of signalling pathways that promote HCC development [5][6][7]. It is also known that inflammation in the stroma in HCC can be modulated by estrogens [8].
Hepatic stellate cells (HSCs) are stroma resident mesenchymal myofibroblast-like cells [9] which initiate and modulate liver fibrosis by regulating the chemical [10] and mechanical [11] composition of the ECM. HSCs, like other myofibroblast-like cells [12], are highly responsive to mechanical cues, requiring a stiff microenvironment to become activated and therefore initiate and perpetuate fibrosis. They achieve this by (i) activating their contractile apparatus to apply endogenous forces to the ECM, and (ii) mechanosensing the rigidity from their surroundings [13,14]. Both processes, cell contractility and mechanosensing, rely on activation of the small GTPase RhoA [15,16]. RhoA is essential in maintaining the activated phenotype of HSCs, by ensuring cell contractility through regulation of ROCK and other modulators of actomyosin [17].
Here we report that tamoxifen induces the mechanical deactivation of HSCs via a previously unidentified mechanism that involves the GPER/RhoA/myosin axis. This inhibits activation of YAP (Yes-associated protein) and durotaxis in HSCs. We also show that cell contractility and ECM rigidity regulate the levels of hypoxia-inducible factor 1 alpha (HIF-1α) and lysyl oxidase (LOX) in HSCs, and that tamoxifen suppresses force-mediated regulation of both HIF-1α and LOX. HIF-1α is fundamental for cell survival in hypoxic conditions [18], as the LOX family regulates collagen crosslinking and ECM architecture and is therefore required for hypoxia-induced metastasis.

Results
Tamoxifen treatment reduces myosin activation in HSCs via GPER/RhoA signalling Actomyosin contractility is a key characteristic of activated HSCs, allowing mechanotransduction and force generation. This behaviour is adaptive in wound healing but promotes fibrosis in HCC [11]. Regulation of actomyosin contractility is achieved through phosphorylation of the regulatory protein myosin light chain 2 (MLC-2). Tamoxifen is a 17βestradiol mimetic that activates GPER, and has been well characterised in its ability to selectively modulate estrogen receptors [19]. We used immunofluorescence staining to confirm the presence of GPER and the canonical estrogen receptors alpha and beta (ER-α and ER-β) in HSCs (Supplementary Fig S1). We also confirmed the levels of expression of GPER in HSCs using immunoblotting/ immunofluorescence and GPER knockdown/overexpression ( Supplementary Fig S2-S4). To assess actomyosin contractility, we determined the levels of active phosphorylated MLC-2 (pMLC-2), as well as the total MLC-2, in response to 10-day treatment with tamoxifen. We also included conditions with antagonists against ER and GPER to explore which receptor tamoxifen acted through. We used the selective ER antagonist (ICI182780) [20] and the specific GPER antagonist G15 [21]. For this experiment and the subsequent ones, ICI182780 and G15 were used simultaneously with tamoxifen treatment.
Across all four conditions (control, tamoxifen, tamoxifen + ER antagonist, tamoxifen + GPER antagonist), the staining intensity for MLC-2 remained constant, indicating that protein expression was unchanged following treatment ( Fig.  1a, b). Levels of pMLC-2 were significantly reduced in the tamoxifen and tamoxifen + ER antagonist conditions compared to control, indicating that tamoxifen greatly reduces MLC-2 phosphorylation, but does not act through the nuclear estrogen receptors. Conversely, tamoxifen + GPER antagonist showed pMLC-2 staining intensity comparable to the control condition, suggesting that tamoxifen achieves inhibition of MLC-2 activation through GPER (Fig. 1a, b).
To further confirm the specific role of GPER, we performed experiments with siRNA to knockdown GPER expression, in combination with tamoxifen or the estrogen 17β-estradiol (E2). Treatment was performed for 72 h since GPER knockdown with siRNA is only stable for this amount of time. We observed that 72-h treatment of HSCs with either tamoxifen or E2 led to a decrease in pMLC-2 without affecting MLC-2 abundance, similar to 10-day tamoxifen treatment. Knockdown of GPER with siRNA GPER showed abundance of pMLC-2 comparable to the control condition, even in the presence of tamoxifen or E2 (Supplementary Fig S5). This demonstrates that estrogenic signalling, instigated by either tamoxifen or E2, acts through GPER to reduce MLC-2 phosphorylation. Likewise, we assessed the effect of tamoxifen on the activation of MLC-2 after 24-h treatment and observed that while the total levels of MLC-2 were kept constant, pMLC-2 were significantly decreased in the tamoxifen group and this effect was mediated by GPER ( Supplementary Fig S6).
RhoA lies upstream of MLC-2 and controls MLC-2 activation [22]. We quantified the levels of total RhoA and active RhoA levels in HSCs under tamoxifen treatment. Both control and 10 day tamoxifen treated conditions showed similar levels of total RhoA. Control cells exhibited active RhoA levels of around 50% of total RhoA, significantly higher than in the tamoxifen treated cells where active RhoA levels were around 20% (Fig. 1c). Taken together, these data suggest that tamoxifen reduces myosin activation via the GPER/RhoA/MLC-2 axis.

GPER activation in HSCs impairs force generation and increases cell compliance
To further assess the effects of tamoxifen on cell contractility, we used elastic pillars as a form of traction force microscopy. This technique assesses the individual force applied to fibronectin-coated polydimethylsiloxane pillars during cell spreading. Using the deflection of each pillar in contact with the cell and the known Young's modulus of each pillar in the array, quantitative analysis of force generation was achieved. We report the value of mean maximum force, calculated from the mean value of the maximum force experienced by each pillar with cellular contact during the time of analysis. Control HSCs generated a mean maximum force of around 3.2 nN, and following 10-day tamoxifen treatment, this mean maximum force was significantly reduced to around 1.2 nN. When a GPER antagonist was present alongside tamoxifen, the mean maximum force returned to a value comparable to control and significantly higher than tamoxifen alone (Fig. 2a, b). Both 72-h tamoxifen treatment and E2 treatment also significantly reduced traction forces in HSCs, but force generation was rescued in the presence of siRNA against GPER. Additionally, G1, a specifically designed GPER agonist [23], reduced traction forces, but did not with GPER knockdown (Supplementary Fig S7). These results indicate that GPER regulates cell traction forces, with tamoxifen, E2 and G1 all acting as GPER agonists.
The ability of cells to generate force is also dependent on their rheological properties such as cell stiffness [24,25]. Drugs that disrupt the cytoskeleton are known to inhibit the activated phenotype of HSCs [26] and these types of drugs have also been shown to decrease cell stiffness [27]. We used atomic force microscopy (AFM) indentation of HSCs seeded on fibronectin-coated fluorodishes to determine cell elasticity. By indenting the cells at points between the nucleus and the cell edge, we ensured that our analysis would accurately assess the contribution of the cytoskeleton to cell elasticity, and would be unaffected by the nucleus or the underlying substrate.
We observed that control HSCs had a Young's modulus around 4.1 kPa, and this was significantly reduced to around 1.7 kPa with 10-day tamoxifen treatment. However, with a GPER antagonist, tamoxifen was unable to reduce the Young's modulus, and cells had an average Young's modulus similar to the control condition (Fig. 2c). 72-h treatment with either tamoxifen, E2 or G1 reduced cell stiffness, but not in the presence of siRNA against GPER (Supplementary Fig S7).

GPER activation reduces HSC mechanosensing and YAP activation
We used magnetic tweezers microrheology to assess the ability of HSCs to respond to external mechanical forces, as would be experienced surrounded by a rigid stroma. Fibronectin-coated magnetic beads were attached to cells, and 12 consecutive pulses of equal force were applied with magnetic tweezers. Cells that displayed mechanosensitivity showed a reduction in bead displacement as the cytoskeleton reinforced following force application (Fig. 3a). We observed that control HSCs showed mechanosensitivity, significantly reducing the displacement of the bead on the 12th pulse to 71% of the displacement measured on the 1st pulse. With 10 day tamoxifen treatment, the 12th pulse had 85% of the displacement measured on the 1st pulse, a value not significantly different from its first pulse displacement, indicating a reduction in mechanotransduction. Using the GPER antagonist G15, mechanotransduction was restored to control levels, with the 12th pulse at 68% compared to the 1st pulse (Fig. 3b).
The transcriptional regulator YAP is a key cellular mechanotransducer, converting external mechanical signals into changes in gene expression through its translocation to the nucleus [28]. YAP has further been shown to be essential in the mechanosensitive phenomenon of durotaxis in HSCs [29]. We stained control and 10-day tamoxifentreated HSCs for YAP and assessed the intensity of staining in both the nucleus and cytoplasm. The ratio between these intensity values represents the level of YAP translocation to the nucleus and therefore activation. Control HSCs showed increased YAP nuclear localisation compared to 10-day tamoxifen-treated HSCs, suggesting that tamoxifen reduces levels of YAP mediated mechanotransduction (Fig. 3c, d).

Control
Tam Tam + GPER  Mann-Whitney test for significance, ***P < 0.001. All histogram bars represent mean ± sem, **P < 0.01,***P < 0.001. Three experimental replicates in all panels Seventy-two-hour treatment of HSCs with either tamoxifen or E2 also led to decreased YAP nuclear localisation, with GPER knockdown rescuing localisation to that of control HSCs ( Supplementary Fig S8), indicating the specific role of GPER in estrogen-mediated YAP deactivation. The expression of the downstream YAP target genes CTGF and ANKRD1 were reduced in 10 day tamoxifen treated HSCs, in concurrence with the immunofluorescence data ( Fig. 3e). We then performed correlation analysis for the expression profiles of the genes GPER, YAP and CTGF using the TCGA (Cancer Genome Atlas) database, and found that GPER expression in HCC patients negatively correlates with the expression of YAP and CTGF (Fig. 3f).

Tamoxifen treatment induces HSC deactivation
Force generation and mechanotransduction are the two pillars required for maintenance of the activated phenotype of HSCs, similar to other myofibroblast-like cells [13,30].
Since we observed tamoxifen to inhibit these mechanical properties, we assessed whether tamoxifen could promote HSC deactivation. We used immunofluorescence and qPCR to assess levels of α-SMA and vimentin, both markers of the activated phenotype. We observed a significant decrease in both α-SMA and vimentin with tamoxifen treatment at both the protein (Supplementary Fig S9) and mRNA (Supplementary Fig S10) levels.
With GPER knocked down with siRNA for 72 h, we observed no effect of tamoxifen in reducing the levels of both markers of quiescence in HSCs (α-SMA and vimentin), though 72 h treatment by tamoxifen did decrease these levels. Likewise, treating HSCs with 17 βestradiol also downregulated the expression of α-SMA and vimentin to levels comparable to those observed in the tamoxifen group (Supplementary Fig S9). These results support the notion that tamoxifen promotes mechanical deactivation in HSCs through GPER/RhoA signalling.

Tamoxifen treatment suppresses ECM protein production
A key role of activated HSCs in promoting further fibrosis and disease development is their ability to produce high levels of ECM proteins for secretion into their microenvironment. Collagen-I and fibronectin are abundant proteins within the ECM, playing critical roles in the organisation and structural integrity of the environment, and when overexpressed, can contribute to a pro-tumour microenvironment [31]. We used immunofluorescence to determine both the intracellular expression, and the extracellular secretion, of both collagen-I and fibronectin. We observed that 10-day tamoxifen treatment significantly reduced the intracellular and extracellular levels of both collagen-I (Fig. 4a, b, e) and fibronectin (Fig. 4c, d, f). 72-h tamoxifen treatment also reduced intracellular collagen-I and fibronectin levels, but could be rescued with GPER knockdown. E2 treatment for 72-h also showed the same trend (Supplementary Figure S11). Due to the highly contractile phenotype of activated HSCs, and their role in ECM protein production, we assessed how enhancing contractile ability influenced collagen-I and fibronectin expression. We transfected control HSCs with constitutively active myosin-2 (pMLC-2) to increase cell contractility, and we observed significant increases in expression of both collagen-I and fibronectin (Fig. 4g), suggesting a mechanical basis to transcriptional regulation of both ECM proteins by HSCs.
We also assessed how changes in matrix rigidity, achieved through fabrication of different rigidity polyacrylamide (PAA) gels for cell culture, could change the production of collagen-I and fibronectin. A 1 kPa gel, which approximates the rigidity of healthy liver [32], was used as a soft substrate and we observed that increasing this rigidity to 25 kPa gave significant increases in collagen-I and fibronectin mRNAs. We further observed that 10-day tamoxifen treated HSCs on 25 kPa gels showed mRNA levels of collagen-I and fibronectin comparable to the 1 kPa condition, i.e. significantly lower than the 25 kPa condition (Fig. 4h). This indicates that tamoxifen inhibits the mechanical signalling pathway that connects external rigidity and increased ECM deposition. Collectively these results show that increased contractility and ECM stiffness trigger a transcriptional increase in both collagen-I and fibronectin in HSCs, and that tamoxifen inhibits this forcemediated activation.
Tamoxifen treatment mechanically inhibits the HIF-1α/LOX and HIF-1α/LOX-L2 axes Liver fibrosis in HCC, along with excess consumption of oxygen by hepatocytes, leads to tissue hypoxia, and the survival of cells becomes dependent on expression of HIF-1α [33]. Hypoxia, through HIF-1α, can regulate expression of ECM protein genes, such as fibronectin [34] and collagen-I [35]. HIF-1α has many other downstream targets, including members of the lysyl oxidase (LOX) family. Lysyl oxidases are copper-dependent enzymes that have fundamental roles in ECM organization in cancer. For instance, LOX is essential in hypoxia driven metastasis [36] and LOX-L2 is involved in ECM remodelling in fibrosis [37].
Mechanical induction of HIF-1α has also been observed in endothelial cells exposed to low shear stress [38], and in the myocardium in response to mechanical stress [39]. While hypoxia is the most common method of HIF-1α activation, upregulation of HIF-1α expression has also been seen in the presence of oxygen, with GPCRs on the cell surface responding to microenvironmental cues [40].
We observed that levels of HIF-1α are reduced in HSCs following 10-day tamoxifen treatment (Fig. 5a, b). Furthermore, levels of LOX and LOX-L2 are also reduced with tamoxifen ( Fig. 5c-f), suggesting that the ability of HSCs to cross-link collagen fibres in the ECM may be affected by tamoxifen treatment. Similarly, 72-h treatment by tamoxifen or E2 reduced levels of HIF-1α, LOX and LOX-L2, but no reduction was seen with GPER knockdown (Supplementary Figure S12).
Notably, we observed that the levels of HIF-1α, LOX and LOX-L2 are responsive to mechanical cues independent of tamoxifen-mediated signalling. The mRNA expression of these proteins is increased following transfection of HSCs with active myosin-2 (Fig. 5g). Similarly, the culturing of HSCs on polyacrylamide gels of differing rigidities also affected mRNA production. Compared to a 1 kPa substrate, HSCs cultured on a 25 kPa substrate showed a significantly increased expression of HIF-1α, LOX and LOX-L2 (Fig. 5h). This suggests that mechanotransduction alone can drive processes that promote survival under hypoxic conditions. When tamoxifen was added to HSCs cultured on the stiff 25 kPa substrate for 10 days, levels of HIF-1α, LOX and LOX-L2 were reduced, becoming equivalent to the levels of these species on the soft 1 kPa substrate. To gain mechanistic insight into tamoxifen-induced downregulation of LOX, LOX-L2 and fibronectin, we used HIF-1α siRNA to knockdown HIF-1α expression. We observed that the mRNA levels of LOX, LOX-L2 and fibronectin, when treated with HIF-1α siRNA, were equivalent to the mRNA levels seen with tamoxifen treatment (Fig. 5i and Supplementary Fig S13). When taken together these results suggest that tamoxifen decreases LOX, LOX-L2 and fibronectin expression via HIF-1α, and that the effect of tamoxifen on HIF-1α levels is mechanically regulated by reducing myosin-2 dependent HSCs contractility and tissue stiffness.

Tamoxifen impairs directed migration via GPER signalling
HSCs have been observed to migrate up a stiffness gradient, a process also known as durotaxis and this has been suggested to be a further step in the perpetuation of fibrosis in the liver [29]. Since this process was shown to be highly dependent on mechanotransduction, and our results here have shown tamoxifen to inhibit mechanotransduction through GPER, we investigated the ability of tamoxifen to inhibit HSC durotaxis.  FN in HSCs, normalized to RPLP0 (60 S acidic ribosomal protein P0) and relative to control. h qPCR levels of collagen-I and FN in HSCs, normalized to RPLP0 and relative to 1 kPa, t-test for b, d and g; and Anova and Tukey's test for (h). g, h three biological samples analysed in three different experiments. All histogram bars represent mean ± sem, *P < 0.05, **P < 0.01,***P < 0.001. Three experimental replicates in all cases

Control
We prepared PAA gels of dual rigidity to assess HSC durotaxis in vitro following a protocol previously described [29]. On these gels, control HSCs moved an average distance in the X-axis (up the stiffness gradient) of around 70 µm over 5 ½ h, with an average speed of 0.99 µm/min along their individual paths. Ten-day tamoxifen treated cells and tamoxifen combined with an ER antagonist treated cells both showed no durotaxis, with significantly reduced cell movement speeds of 0.20 µm/min and 0.15 µm/min, respectively. When tamoxifen was combined with a GPER antagonist, durotaxis was rescued (average distance in Xaxis = 61 µm over 5 ½ h) and cell speed became similar to the control condition (0.98 µm/min) ( Fig. 6 and videos 1-4). Seventy-two-hour treatment of HSCs with tamoxifen or G1 also abrogated durotaxis behaviour, though GPER knockdown was able to rescue the tamoxifen treated cells, with   Figure S14 and videos 5-8). These combined results show that tamoxifen inhibits the ability of HSCs to migrate to stiffer substrates via GPER signalling.

Discussion
Estrogens regulate a manifold of physiological and pathological processes and although endogenous estrogen is mainly derived from the ovaries in premenopausal women and mostly regarded as a female hormone [41], estrogen is also produced in other tissues, such as adipose tissues and arteries in both men and women [19,42]. Until recently, the field of estrogens was dominated by studies that explored their transcriptional effects via nuclear estrogen receptors. However, the last decade has witnessed an explosion of interest in GPER-mediated estrogen signalling.
From our results, GPER comes to light as a comprehensive and effective mechanoregulator that targets the activation of fundamental proteins in cell mechanics, such as RhoA [22,43] and MLC-2 to control force generation, mechanosensing and durotaxis in hepatic stellate cells. Increased levels of MLC-2 are required for the ability of stromal cells to remodel the ECM [44] to perpetuate fibrosis [45]. Likewise, high levels of active YAP, a mechanoresponsive transcriptional regulator [28], are indispensable for the activation of tumour-associated myofibroblasts in the stroma [46], and we show that YAP is downregulated in tamoxifen treated HSCs. Due to the similarity of activated HSCs to myofibroblasts, we posit that GPER is therefore likely to influence the mechanical properties of other stromal cells (Fig. 7).
The activation of HIF-1α is required for cell survival under hypoxic conditions. The dense stroma that surrounds solid tumours limits the accessibility of nutrients and Results are expressed as mean ± sem. Anova and Tukey's post hoc tests were used for the analysis oxygen to cells, promoting HIF-1α stabilisation [47]. The rapid growth of cancer cells within HCC also leads to high consumption of oxygen, further generating a hypoxic environment [48]. Our data suggest that HIF-1α is the unifying factor through which tamoxifen subsequently reduces the levels of LOX, LOX-L2 and fibronectin in HSCs. We suggest that HIF-1α may act as a converging point to mechanically regulate the adaptive response of HCC to hypoxia and the overall architecture of the tumour microenvironment. We propose that this mechanical regulation of HIF-1α by tamoxifen in an oxygen-independent manner may result in an effective reduction of cell fitness to cope with hypoxic condition in HSCs, and potentially in cancer cells as well, leading to decreasing fibrosis. Development of fibrosis relies on positive feedback pathways, including mechanotransduction and ECM protein deposition [49] and durotaxis [29]. The directed migration of cells to stiffer fibrotic areas leads to further activation by mechanotransduction, leading to an increase in ECM protein production, which in turn, promotes a stiffer microenvironment [13] that might lead to increased chemoresistance in cancer cells [50]. Durotaxis can also play a role in facilitating cross-talk between cancer cells and activated stromal cells such as activated HSCs [51], and therefore the inhibition of directed migration by tamoxifen, combined with its ability to induce HSC quiescence, indicates the multiple ways in which tamoxifen could mechanically influence the tumor-stroma cross-talk.
Within the liver tissue, HSCs reside within the ECM, a mixture of scaffolding proteins secreted by HSCs, among other stromal cells. Interactions between cells are mostly through paracrine signalling, as well as interactions with the ECM proteins, rather than direct cell-cell interactions [9]. For our in vitro studies, we used culture-activated HSCs seeded on fibronectin-coated glass, a widely employed model, which recapitulates the activated phenotype in vivo with good approximation [10]. However, the behaviour we observe in vitro may well differ from that in vivo, where HSCs are influenced by factors secreted by cancer cells and other stromal cells, as well as the complex architecture of the ECM [9], which are not present in our in vitro setup. Further studies with HSCs are therefore necessary for a full comprehension of the role of GPER in vivo.
Our work lays the foundations for further studies that could directly influence therapeutic development. Tamoxifen is a widely used drug in clinics, with well-established pharmacodynamics [52] and safety [53], and due to the pleiotropic effects of estrogens and the commonality of GPCR signaling pathways, it is possible that tamoxifen regulates many genes involved in the function of myofibroblast-like cells such as activated HSCs or cancer associated fibroblasts. This could lead to development of stromal reprogramming strategies in which GPER agonists could modulate the fibrovascular stroma of HCC to increase vascular density and perfusion by reducing overall solid stress, achieved through inhibiting expression of collagen and fibronectin. This would increase intratumoral drug perfusion, while concurrently impeding the adaptive fitness of tumour and stromal cells to survive under hypoxic conditions (via HIF-1α) and thus promote widespread hypoxic necrosis.

Immunofluorescence staining
Cell immunofluorescence staining was done on coverslips coated with 10 μg ml −1 fibronectin (Gibco, phe0023). Following pertinent treatment cells were fixed with 4% paraformaldehyde (Sigma, P6148) in D-PBS (Sigma, D8537) for 10 min, and then blocked and permeabilized with 0.2% BSA-0.1%Triton (Sigma, T8787) in PBS for 30 min. After blocking, cells were incubated with primary antibodies prepared in blocking solution for 1 h at room temperature in a humidified chamber. Then, cells were washed in D-PBS and incubated with Alexa Fluor 488-conjugated secondary antibodies and Phalloidin (Invitrogen, A22283, 1/1,000 dilution) prepared in PBS for 30 min at room temperature. Finally, coverslips were washed in PBS and mounted in mounting reagent with 4,6-diamidino-2-phenylindole (Invitrogen, P36931). Immunofluorescent images were taken with Nikon Ti-e inverted microscope (Nikon, Kingston-upon-Thames, United Kingdom) with NIS elements software.
The procedures for the analysis of gene expression using TCGA data, traction forces using elastic pillars, cell mechanosensing, durotaxis, atomic force microscopy, GLISA and the statistical analysis can be found in supplementary methods.

Data availability
All relevant data are available from the authors. the support of The Drug Discovery and Chemical Biology − Biocenter Finland.
Author contributions EC and ADRH conceived the idea and designed research. EC, DL, AR, BR and GY performed experimental research and analyzed data under the supervision of ADRH. SDT performed immunoblotting assays under the supervision of DAL. LG and KR analyzed mRNA data from the TCGA. EC, AR, and ADRH wrote the paper.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry

text:
Thymoquinone inhibits the metastasis of renal cell cancer cells by inducing autophagy via AMPK/mTOR signaling pathway

Abstract Thymoquinone (TQ, 2‐methyl‐5‐isopropyl‐1,4‐benzoquinone), a bioactive constituent extracted from the seeds of Nigella sativa, has been proved to exert anti‐tumor efficiency in various cancers. Autophagy is a self‐digestion phenomenon, and its role in tumor formation and progression remains controversial. In the present study, we investigated the effects of TQ on renal cell cancer (RCC) cell lines (786‐O and ACHN) using wound healing assay, transwell assay and western blot analysis. We found that TQ effectively inhibited the metastatic capacity of RCC cells in vitro, which was also verified in a xenograft model. Meanwhile, we observed LC3 puncta and detected the expression of LC3 in TQ‐treated RCC cells, and then found that autophagy was induced by TQ in 786‐O and ACHN cell lines. In addition, TQ inhibited the migration and invasion as well as the EMT in RCC cells in an autophagy‐dependent manner. To further explore the underlying mechanism, we detected the AMPK/mTOR signaling pathway. The results indicated that TQ inhibited the metastasis of RCC cells by inducing autophagy via AMPK/mTOR signaling pathway. In conclusion, our findings provide a novel therapeutic strategy that aims at TQ‐induced autophagy in RCC treatment.

has been proved to exert wide-ranging pharmacological activities, with anti-tumor, anti-inflammatory, anti-oxidant, anti-microbial, anti-convulsant and anti-diabetic properties. [6][7][8][9] Therefore, TQ has been regarded as a promising agent for numerous diseases. Previous studies have revealed that TQ may exhibit high anti-tumor efficiency against various cancers, such as breast cancer, gastric cancer, cervical cancer, colon cancer, central nervous system cancer, ovarian cancer, prostate cancer, bladder cancer and renal cell cancer. [10][11][12][13][14][15][16][17][18] It is well known that invasion and metastasis play vital roles in the progression of tumors. Recent research has shown that TQ can inhibit metastasis and EMT in prostate cancer via the transforming growth factor-beta/Smad2/3 signaling pathway. 19 Jun Li and his team also indicated that TQ inhibited the migration and invasion abilities in cervical cancer cells, which might be regulated via the expression of Twist1, Zeb1 and E-cadherin directly. 12 However, there are few studies that consider the effect of TQ on metastasis in renal cell cancer.
Autophagy is a self-digestion phenomenon, first identified by Christian de Duve in 1963, which is evolutionarily conserved from yeast to mammals. During the autophagy process, a doublemembrane vesicle, named autophagosome or autophagic vacuole, sequesters cytoplasmic proteins and organelles and then delivers them to lysosome, where they are degraded. 20 The role of autophagy in tumors constantly changes with the progression of tumors. In the early stages of tumor formation, autophagy suppresses cancer initiation through eliminating inflammation, genome instability and tissue damage. Alternatively, as a tumor grows, autophagy promotes tumor growth through providing substrates for metabolism. 21,22 Emerging observations indicate that there is an intricate relationship between autophagy and metastasis in cancer. On the one hand, autophagy helps to support viability for the potentially metastatic cancer cells. On the other hand, autophagy tends to inhibit early metastasis and to reverse of epithelial-mesenchymal transition (EMT) in cancer cells. 23,24 Hence, in the present study, we aim to explore the effect of TQ on metastasis in RCC and the role of autophagy in this process. We also elucidate the underlying molecular mechanisms. at 37°C in a humidified incubator with 5% CO 2. Invitrogen (Carlsbad, CA, USA). Protease inhibitor and phosphatase inhibitor were obtained from Roche (Basel, Switzerland).

| Cell viability assay
786-O and ACHN cells were plated into 96-well culture dishes (Corning, NY, USA) at a density of 5 × 10 3 cells/well with complete culture medium. After being incubated overnight, the medium was removed and replaced with fresh medium containing different concentrations of TQ (0, 10, 20, 40, 60, 80 and 100 μmol/L) for 24 or 48 hours, respectively.
Then the medium was removed and 20 μL CCK8 was added into each well and incubated for 4 hours at 37°C. The optical density (OD) of each well was measured at 490 nm by microplate reader.

| Transwell migration and invasion assay
We used 24-well plates with chambers of 8μm pore size (Millipore, Germany) in the transwell analysis. In the migration assay, 1 × 10 4 RCC cells were seeded in the upper chamber. In the invasion assay, 2 × 10 4 cells were seeded in the upper chamber pretreated with Matrigel. Cells in the upper chamber were resuspended in 200 μL serum-free medium with different concentrations of TQ. The lower chamber was filled with 800 μL complete medium containing the same concentration of TQ as in the upper chamber. After incubation for 24 hours, the non-migrated or non-invaded cells in the upper chamber were removed by cotton swab, and the migrated or invaded cells were fixed with 4% paraformaldehyde for 30 minutes and then stained with .1% crystal violet for another 30 minutes. Then, the cells were counted under a microscope and 4 random fields for each chamber were selected for statistical analysis.

| Wound healing assay
Renal cell cancer cells were seeded into 6-well plates with complete medium. We used 200-μL pipet tips to scratch wounds when the cells had grown to 90%. The plates were washed 3 times to remove the floated cells and then filled with serum-free medium containing different concentrations of TQ for 24 hours. The cells were observed at different time points and the wound closure was determined by inverted microscope.

| Tandem fluorescence microscopy
Renal cell cancer cells were plated into 6-well dishes with complete medium. When cells grew to 70%, the medium was discarded and replaced with serum-free medium. Then, the cells were transfected with tandem fluorescent mRFP-EGFP-tagged LC3 expressing plasmids using Lipofectamine 2000. After 6 hours, the medium containing Lipofectamine 2000 was abandoned and new complete medium was added to the dishes to maintain cells for 18 hours. Next, cells were treated with TQ for 24 hours and then fixed with 4% paraformaldehyde. Then, the expression of enhanced green fluorescent protein (EGFP) and mRFP was observed by fluorescence microscopy (Olympus, Tokyo, Japan).

| Western blot
Renal cell cancer cells were lysed by RIPA buffer containing protease inhibitor or phosphatase inhibitor and PMSF for 10 minutes on ice. Then, the cell debris was collected and centrifuged at 12 400 g for 15 minutes. The supernatants were transferred into new tubes, and protein concentrations were detected by BCA Protein Assay Kit (Heart, Xi'an, China). The supernatants were mixed with 1/5 volume of 6X buffer and then boiled for 5 minutes. The whole proteins were subjected to 10%-15% SDS-PAGE and then transferred to PVDF membranes. The membranes were first sealed with 10% skim milk for 2 hours at room temperature and then incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were washed with TBST 3 times and incubated with secondary antibodies for 1 hour. The membranes were visualized and measured using an ECL system (Bio-Rad, CA, USA).

| In vivo experiments
All animal experiments were performed in accordance with standard ethical guidelines and approved by the Ethics Committee of Xi'an Jiaotong University. Ten male nude mice were included in the study.
All mice were 5 weeks old and each weighted 19-22 g. Harvested 786-O cells (1 × 10 6 cells) were suspended in 200 μL serum-free medium containing 100 μL Matrigel, and subcutaneously injected into the right flank of every mouse. When the tumors had grown to approximately 100-150 mm 3 in size, the mice were randomly divided into 2 groups (5 in each group) and intraperitoneal injected with DMSO (control group) or TQ 20 mg/kg, respectively, every 3 days.
During the treatment, the tumor volumes were calculated and the mice were weighted with the same frequency. After 30 days, tumors were harvested, weighted and analyzed. The volume was calculated using the following formula: tumor volume = (length × width 2 ) ×.5.
To establish the metastatic tumor model, luciferase-tagged 786-O cells were injected into mice via tail veins. Then, the mice were divided into 2 groups and received the same treatment as above. After 30 days, the mice were intraperitoneal injected with D-luciferin (150 mg/kg). Ten minutes later, mice were anesthetized with 10% chloral hydrate (.004 mL/g) and imaged using the IVIS Lumina II with Living Image Software.
The lung metastatic tumors were then harvested and stained with H&E.

| Immunohistochemical assay
Renal tumors were separated from xenograft mice and fixed with 10% formaldehyde for 24 hours. Then, they were embedded in paraffin and cut into 5μm-thick sections. After that, the tissue sections were subjected to deparaffinization, rehydration, endogenous peroxidase blocking and antigen retrieval. Next, the sections were blocked with 1% BSA for 10 minutes. Subsequently, they were incubated with primary antibodies overnight and appropriate secondary antibodies for 1 hour. The sections were then visualized using a DBA kit following the manufacturer's instructions.

| Statistical analysis
All data were presented as mean ± SD of 3 independent experiments and analyzed using GraphPad Prism 5.2 software. In all cases, differences were considered statistically significant when P-value <.05.

| Thymoquinone suppresses migration, invasion and epithelial-mesenchymal transition in renal cell cancer cells
To choose proper concentrations of TQ in the present study, first we  (Table S1). As shown in Figure 1A Figure 1D). In addition, we observed EMT-related markers (E-cadherin and vimentin) in TQ-treated ACHN by fluorescent immunostaining and found that the results were consistent with those for western blot analysis ( Figure S2). These results suggest that TQ effectively inhibits migration, invasion and the EMT process in RCC cells in vitro.  Figure 2D). Collectively, these data indicate that TQ can induce autophagy in RCC cells.

| Inhibition of autophagy attenuates thymoquinone-induced anti-tumor effects in renal cell cancer cells
Accumulated evidence has suggested that there is an association between autophagy and metastasis. We hypothesized that autophagy induced by TQ may affect the migration and invasion in RCC cells.

| AMPK/mTOR pathway is involved in anti-tumor effects of autophagy induction by thymoquinone in renal cell cancer cells
Numerous studies have indicated that autophagy can be mediated by various pathways, including PI3K/AKT/mTOR, AMPK/mTOR, MAPK/ERK1/2, P53/DRAM and HGF/MET. 25 Among these pathways, the AMPK/mTOR signaling pathway plays a vital role in the process of autophagy. It positively regulates autophagy through increasing AMPK phosphorylation and decreasing mTOR phosphorylation. To confirm whether the AMPK/mTOR signaling pathway is involved in the induction of autophagy by TQ in RCC cells, the protein levels of AMPK, p-AMPK (Thr172), mTOR, p-mTOR (Ser2448) and p-S6K (Thr389) were detected after treatment with different concentrations of TQ. As expected, the expression of p-AMPK was elevated by TQ, while expressions of p-mTOR and p-S6K were reduced, which indicated the activation of the AMPK/mTOR pathway ( Figure 4A). Then, we pretreated the RCC cells with AICAR (an activator of AMPK) or compound C (an inhibitor of AMPK) and detected the expression of p-AMPK, p-mTOR and LC3. As shown in Figure 4B, the effects of TQ on LC3 could be receded when the AMPK/mTOR signaling pathway was inhibited by compound C, which suggested the clinical performance of mTOR inhibitors in relation to avoiding drug resistance is still less than satisfactory at present. Therefore, drugs that target multiple components of pathways, such as autophagy/AMPK/mTOR, are urgently needed.

| Anti-tumor effects of thymoquinone on renal cell cancer cells in vivo
We further investigated the effects of TQ on RCC cell growth in a xenograft tumor model. As shown in Figure 5A, tumors in the group treated with TQ grew more slowly than those in the control group.  Figure S4). These data indicate that TQ inhibits RCC cell growth and metastasis in vivo.  EMT can transform the epithelial phenotype into mesenchymal phenotype, leading to an increase in cell motility and invasiveness. 29 Accumulating research has suggested that EMT has a close correlation with the migratory and invasive abilities of cancer cells.

| D ISCUSS I ON
Then, we detected EMT-related proteins in our study. The results showed that TQ could upregulate epithelial markers and downregulate mesenchymal markers, suggesting that TQ could inhibit EMT in RCC cells. To observe the effects of TQ on tumor growth in vivo, we calculated the volume of the tumors and found that tumors in the group treated with TQ grew more slowly when compared to those in the control group. Moreover, the tumors in the TQ-treated group showed higher expression of E-cadherin and lower expression of N-cadherin, which concurred with our findings in vitro. We further established a metastatic tumor model and the results indicated that TQ significantly inhibited metastasis compared with the control group, which was consistent with our expectations.
Autophagy is a catabolic cellular process in which the dysfunctional cytoplasmic constituents are removed to maintain metabolic balance. It occurs not only in normal cells but also in cancer cells. 30 It is widely accepted that autophagy plays a complex role in oncogenesis: it represses tumor growth in the early stages on the one hand but is required for progression to advanced or invasive tumors on the other hand. 24 During the past 5 years, autophagy induced by plantderived drugs has been widely studied in various tumors. In the present study, we first demonstrated that TQ-induced autophagy in a concentration-dependent manner in RCC cells, which was verified by inverted microscope, LC3 turnover assay and western blot.
There is an intricate connection between autophagy and metastasis. Autophagy is usually induced during the progression of various cancers to metastasis and it can promote tumor metastasis through numerous mechanisms, including promoting a stem cell phenotype, production of secreted factors and modulating the Rho signaling pathway. 24 In contrast, autophagic cells can act as a barrier against tumor progression through inhibiting the tumor transfer from primary sites to secondary sites. 31 A study has shown halofuginoneinduced autophagy suppresses breast cancer cell migration and invasion. 32 Meanwhile, in osteosarcoma cells, the activation of autophagy is associated with the suppression of EMT. 33 In the study, we used 3-MA to inhibit autophagy and observed the effects of TQ on migration, invasion and EMT in RCC cell lines 786-O and ACHN.
We, therefore, found that inhibition of autophagy could attenuate TQ-induced anti-metastasis effects in RCC cells.
The mTOR signaling pathway has been reported to be activated in nearly 100% of RCC, and inhibitors targeting the mTOR signaling seem to offer a promising therapy against RCC. In our supplementary experiments, we compared TQ with everolimus.
The results indicated that everolimus showed more anti-tumor effects as compared to TQ. However, the emergency of drug resistance has limited the application of mTOR inhibitors in clinical treatment and agents targeting multiple pathways have been widely studied. Because the mTOR signaling pathway plays a vital role in the process of autophagy, it is important to study the relationship between the mTOR pathway and autophagy. The mTOR includes 2 functionally and biochemically distinct complexes, mTORC1 and mTORC2. 34 Evidence has shown that mTORC1, but not mTORC2, plays a vital role in the regulation of autophagy.
The mTORC1 inhibition increases autophagy, while activation of mTORC1 represses this process. 34 AMPK is a cellular energy sensor that regulates homeostatic balance. It positively regulates autophagy by inhibiting the activity of mTORC1 through direct phosphorylation of its receptor, a regulatory-associated protein of mTOR. 25 To verify whether TQ induced autophagy through the AMPK/mTOR signaling pathway, we detected the main molecules in this pathway and found that the expression of p-AMPK was elevated by TQ, while expressions of p-mTOR and p-S6K were reduced, accompanied by an increase in LC3. Moreover, we observed an upregulation of LC3 in TQ-treated RCC cells when activating the AMPK/mTOR pathway, while a downregulation when inhibiting this pathway; taken together, we can conclude that TQ could induce autophagy via the AMPK/mTOR pathway. Then, the migration and invasion experiments showed that the AMPK inhibitor attenuated the anti-metastatic effects of autophagy induced by TQ, which suggested that TQ could inhibit the metastasis of RCC cells by inducing autophagy through the AMPK/mTOR pathway.
In conclusion, our study demonstrated that TQ exerted antimetastatic properties in RCC cell lines 786-O and ACHN. In addition, these characteristics, including inhibiting migration, invasion and EMT, were mainly dependent on TQ-induced autophagy.
Furthermore, we revealed that the mechanism by which TQ-induced autophagy inhibited the metastasis of RCC cells was regulated through the AMPK/mTOR pathway. These findings indicated the potential role of TQ-induced autophagy in cancer treatment and suggested that combining TQ with autophagy inhibitors might enhance its cancer suppression effects.

D I SCLOS U R E
The authors have no conflict of interests.