extfieldsofstudy: Medicine Chemistry

text:
ROS-mediated activation of AMPK plays a critical role in sulforaphane-induced apoptosis and mitotic arrest in AGS human gastric cancer cells.

In this study, we investigated the role of AMP-activated protein kinase (AMPK) and the importance of reactive oxygen species (ROS) in apoptosis induction associated with cell cycle arrest induced by sulforaphane in AGS human gastric cancer cells. Our results demonstrated that sulforaphane inhibited proliferation of AGS cells by promoting apoptosis and accumulating the cellular portion of the G2/M phase via the buildup of cyclin B1 and cyclin-dependent kinase p21 (WAF1/CIP1). Moreover, the phosphorylation of histone H3 was markedly increased following treatment with sulforaphane, indicating that sulforaphane stimulated mitotic arrest. Sulforaphane concurrently induced phosphorylation of AMPK; however, compound C, an AMPK inhibitor, significantly blocked sulforaphane-induced apoptosis, suggesting that sulforaphane induces apoptosis of AGS cells through the AMPK-dependent pathway. Sulforaphane also activated the mitochondrial apoptotic signaling pathway with a decrease in mitochondrial membrane potential and the nuclear translocation of cytochrome c. Furthermore, sulforaphane provoked the generation of intracellular ROS; especially when ROS production was blocked by antioxidant N-acetylcysteine, both AMPK activation and growth inhibition by sulforaphane were completely abolished. Collectively, these findings suggest that sulforaphane inhibited growth of AGS cells, which was mediated by a complex interplay between cellular mechanisms governing redox homeostasis, apoptosis, and cell cycle arrest through an ROS/AMPK-dependent pathway.

Introduction
Cell cycle deregulation, which leads to uncontrolled cell proliferation, is one of the most frequent changes occurring during tumor development. Thus, inducing arrest of cell cycle progression is an effective way to regulate abnormal proliferation of cancer cells. Induction of apoptosis, a programmed cell death, with cell cycle control has also been evaluated as a major mechanism for the removal of cancer cells (Diaz-Moralli et al. 2013;Koff et al. 2015). Therefore, de-veloping appropriate methods to overcome these behaviors of cancer cells without affecting normal cells is recognized as the most basic process of cancer treatment (Visconti et al. 2016;Westhoff et al. 2016). Furthermore, cancer cells often exhibit several types of mitochondrial dysfunction, including mitochondrial DNA mutations, changes in energy metabolism, increased reactive oxygen species (ROS) production, and increased mitochondrial membrane potential (MMP), which may be key targets for cancer cell proliferation blocking. Especially, the loss of MMP damages mitochondrial function and induces ROS production; therefore, natural products that are likely to affect intracellular energy and metabolism have recently become the subject of investigation as promising anticancer agents (Rehman et al. 2014;Redza-Dutordoir and Averill-Bates 2016).
Many recent epidemiological data have shown that intake of cruciferous vegetables that are rich in dietary isothiocyanates is positively correlated with the incidence of diverse tumors (Tang et al. 2010;Liu et al. 2013). Sulforaphane is a natural isothiocyanate derived from cruciferous vegetables like broccoli and Brussels sprouts (Myzak and Dashwood 2006;Fimognari et al. 2008). This phytochemical has a strong antioxidant potential and has proven to be an important cancer-preventive candidate, exhibiting high activity that inhibits the proliferation of cancer cells and induces apoptosis in many cancers (Gupta et al. 2014;Sestili and Fimognari 2015). Although inhibition of the proliferation of cancer cells by sulforaphane in some cell lines has been reported to be associated with G1 arrest in the cell cycle (Shen et al. 2006;Bryant et al. 2010;Wang et al. 2012), many studies have shown that it induces G2/M arrest associated with apoptosis in multiple cancer types (Singh et al. 2004;Matsui et al. 2007;Chu et al. 2009;Suppipat et al. 2012;Wang et al. 2015;Byun et al. 2016;Shang et al. 2017). The effects of sulforaphane on cell cycle progression have also been reported to be specific for mitotic arrest, but not the G2 phase, which is associated with destruction of microtubule polymerization (Jackson and Singletary 2004;Mi et al. 2008;Xiao et al. 2012). In addition, it has been shown in various previous studies that ROS act as key regulators in the induction of apoptosis by sulforaphane (Singh et al. 2004;Wang et al. 2015;Byun et al. 2016;Shang et al. 2017). Further, recent results from Mondal et al. (2016) demonstrate that induction of apoptosis and inhibition of migration of human gastric cancer cells by sulforaphane are associated with increased ROS production and blocking of several intracellular signaling pathways. We have also previously reported that the production of ROS is a critical factor in inducing apoptosis of various cancer cells by sulforaphane, and mitotic arrest is also dependent on ROS production (Choi et al. 2008;Jo et al. 2014;Park et al. 2014).
AMP-activated protein kinase (AMPK), a serine/threonine kinase, is a metabolic-sensing protein kinase that plays an essential role in regulating cellular energy homeostasis during cell proliferation (Hardie et al. 2006;Kim and He 2013;Rehman et al. 2014). AMPK is activated in response to the phosphorylation of the critical amino acid residue Thr172 due to an increase in the ratio of intracellular AMP to ATP and phosphorylation by several upstream AMPKs (Hawley et al. 1996;Woods et al. 2003;Shaw et al. 2004). Several studies have shown that AMPK plays an important role in the induction of cell cycle arrest and the proapoptotic pathway in many cancer cell types, which is frequently accompanied by an increase in the generation of intracellular ROS (Duong et al. 2012;Sook et al. 2014;Li et al. 2016). Therefore, AMPK signaling has been considered as an attractive therapeutic target for cancer treatment in recent years. Recently, it has been shown that sulforaphane promotes lipolysis through hormone-sensitive lipase acti-vation and inhibits lipogenesis by reducing the activation of AMPK signaling (Lee et al. 2012;Choi et al. 2014). In addition, sulforaphane has been shown to protect prionmediated neurotoxicity induced by the AMPK signaling pathway in human neurons, and it has been reported that lipid toxicity and cardiomyopathy can be prevented through the attenuation of AMPK inactivation by oxidative stress Zhang et al. 2014). However, the correlation between ROS generation and AMPK activation in sulforaphane-induced cell cycle arrest and apoptosis has not yet been identified. Therefore, in this study, we performed experiments to investigate the roles of ROS and AMPK in sulforaphane-induced growth inhibition using AGS human gastric adenocarcinoma cells.

Materials and Methods
Cell culture and sulforaphane treatment AGS human gastric cancer cells were obtained from the American Type Culture Collection (Manassas, MD, USA). The cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (WelGENE Inc, Daegu, Republic of Korea) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) and maintained as a monolayer culture at sub-confluence in a 95% air and 5% CO 2 humidified atmosphere at 37°C. Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)-butane) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA); it was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemical Co.) and adjusted to final concentrations using complete culture medium. At the end of incubation, cells from each well were also photographed under a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany).

Cell viability assay
To evaluate the cytotoxic ability of sulforaphane, the cells were seeded in 96-well microtiter plates at a density of 1×10 3 cells per well. After incubation for 24 h, the cells were treated with different concentrations of sulforaphane in the presence or absence of other agents for 48 h. At the end of the incubation period, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Chemical Co.) was added to each well at 0.5 mg/ml, followed by incubation at 37°C in dark conditions. After incubation for 3 h, the MTT solution was removed and 200 μl of 5% DMSO was added to dissolve the crystals. The viable cells were detected by reading the absorbance of formazan at 540 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Dynatech Laboratories, Chantilly, VA, USA). The optical density of the formazan formed in control (untreated) cells was used to represent 100% viability (Hong et al. 2016).

Cell cycle distribution analysis
To analyze the cell cycle progression, the cells were removed from plates via trypsinization and pooled with cell culture supernatant containing nonadherent cells. The cells were washed twice with phosphate-buffered saline (PBS), followed by fixation with 70% ethanol, and stored at -20°C overnight. After washing with PBS, the cells were stained with 50 μg/ml of propidium iodide (PI, Sigma-Aldrich Chemical Co.) solution containing 50 μg/ ml of RNase A and 0.1% Triton X-100 for 10 min in the dark at room temperature. Then, the cell cycle distribution was determined using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA), and the percentage of cells at different cell cycle phases was analyzed using ModFitLT V3.0 software (Becton Dickinson).

Detection of nuclear morphological changes
Apoptotic cells containing chromatin condensation and nuclear fragmentation in the nuclei were detected by 4' ,6-diamidino-2-phenylindole (DAPI) staining. Briefly, the cells were harvested, washed with PBS twice, and fixed with 3.7% paraformaldehyde (Sigma-Aldrich Chemical Co.) in PBS for 10 min at 25°C. The fixed cells were washed with PBS and stained with 1 mg/ml of DAPI solution (Sigma-Aldrich Chemical Co.) for 10 min. The cells were then washed twice with PBS, and the morphology changes in the nucleus were observed using a fluorescence microscope (Carl Zeiss).

Western blot analysis
As described previously (Lee et al. 2016a), the cells were collected and lysed with cell lysis buffer; following this, protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). In a parallel experiment, the mitochondrial and cytosolic fractions were isolated using a mitochondrial fractionation kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. For Western blotting, equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH, USA) at 350 mA for 1 h at room temperature. Subsequently, the membranes were blocked with 5% nonfat dry milk/Tris-buffered saline containing 0.1% Triton X-100 (TBST) for 1 h; the membranes were then probed with specific primary antibodies at 4°C overnight. After washing the primary antibodies with TBST, the membranes were incu-bated with the appropriate horseradish peroxidase (HRP)conjugated secondary antibodies (Amersham Biosciences, Westborough, MA, USA) for 2 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences) according to the manufacturer's instructions.

Apoptosis analysis using flow cytometry
The apoptosis rate was determined using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer's instructions. Briefly, the cells were harvested, washed twice with binding buffer and stained with annexin V and PI for 20 minutes in the dark. The mixture was then analyzed using a flow cytometer. The degree of apoptosis was quantified as a percentage of the annexin V-positive and PI-negative (annexin V + /PI -) cells (Lee et al. 2016b).

Determination of intracellular ROS
To monitor the intracellular ROS production, we utilized cell-permeable, oxidation-sensitive fluorescent 5,6-carboxy-2'7'-dichlorofluorescein diacetate (DCF-DA) probes (Molecular Probes Inc, Leiden, Netherlands), as previously described (Kim 2016). In brief, samples of 2 × 10 4 cells were placed on six-well plates and preincubated with 10 μM DCF-DA for 30 min at 37°C in the dark. The cells were harvested and washed with PBS twice; they were then immediately analyzed using a flow cytometer. To confirm the involvement of elevated ROS in sulforaphane-induced cytotoxicity, the cells were preincubated with N-acetylcysteine (NAC, Sigma-Aldrich Chemical Co.), an established antioxidant, for 2 h before treatment with sulforaphane.

Statistical analysis
All numerical data are expressed as the mean ± standard deviation (SD). Significant differences between mean values were analyzed using the Student's t-test. All statistical analyses were conducted using the Statistical Package for the Social Sciences version 17.0 software (SPSS Inc, Chicago, IL, USA). Values of p < 0.05 were considered to indicate statistical significance.

Sulforaphane inhibits cell viability in AGS cells
Using MTT assay, the effect of sulforaphane on the growth of AGS human gastric cancer cells was evaluated. As shown in Fig. 1A, the AGS cell viability was not significantly decreased with a concentration of sulforaphane lower than 2.5 μM for 48 h. However, concentrations of sulforaphane ranging from 5 μM to 20 μM all exerted concentrationdependent cytotoxic effects on AGS cell growth, and treatment with 20 μM sulforaphane resulted in almost 60% inhibition.

Sulforaphane induces mitotic arrest in AGS cells
In many cases, inhibition of proliferation of cancer cells by chemotherapeutic agents is associated with cell cycle arrest. Therefore, flow cytometry analysis was performed to investigate the relationship between inhibition of proliferation and cell cycle progression of AGS cells by sulforaphane. As shown in Fig. 1B, exposure to sulforaphane led to an increase in the number of G2/M phase cells as well as a simultaneous decrease in G1 phase cells in a concentration-dependent manner. For example, the populations of cells in G2/M phase elevated 59.46% in 20 μM sulforaphane-treated cells compared with 17.25% in the control group. The results suggested that sulforaphane can induce G2/M arrest to decelerate cell cycle progression and prevent the cells from entering the G1 phase and proliferating. To understand the mechanism underlying sulforaphane-induced G2/M arrest, we examined the effects of sulforaphane on the expression level of key regulators responsible for G2/M-phase checkpoints using Western blot analysis. In comparison to the control cells, there was a marked increase in the expression of cyclin B1 in AGS cells when treated with sulforaphane even though the expression of cyclin-dependent kinase 1 (Cdk1) and Cdk2 was not changed. In addition, the expression of tumor suppressor p53 and Cdk inhibitor p21 (WAF1/CIP1), a member of the Cip/Kip family proteins, was concentration-dependently increased when cells were treated with sulforaphane ( Fig. 2).
Since it is difficult to distinguish between G2 and M arrest through cytometric analysis alone, we investigated whether sulforaphane induces phosphorylation of histone H3 protein, which is exclusively phosphorylated at serine 10 during mitosis by aurora kinase (Murnion et al. 2001). As consistent with our previous observation in human bladder cancer , the immunoblotting results showed that sulforaphane treatment markedly enhanced the phosphorylation level of histone H3 compared with the untreated control group (Fig. 2); this suggesting that inhibition of AGS cell proliferation by sulforaphane treatment is closely associated with mitotic arrest in the cell cycle.

Sulforaphane enhances apoptosis in AGS cells
We next investigated whether proliferation inhibition and mitotic arrest induced by sulforaphane were associated with apoptosis induction. The results in Fig. 3A showed that analysis by an inverted microscopy revealed distinct morphological changes in sulforaphane-treated cells. Sulforaphane-treated cells were variable in size, with extensive cytosolic vacuolization, the appearance of irregular cell membrane buds, membrane shrinkage, and cell rounding up. In addition, the morphological changes associated with apoptosis, such as chromatin condensation and nuclear fragmentation, were evident in AGS cells after sulforaphane treatment. Moreover, the cleavages of poly-ADP-ribose polymerase (PARP), a characteristic of apoptosis, were determined to further assess apoptosis. As shown in Fig. 3B, when challenged with sulforaphane, cleaved PARP increased as the concentration levels rose, as illustrated by the generation of an 85-kDa band. To quantitatively assess the degree of apoptosis induced by sulforaphane treatment, annexin V staining was attempted; this detects phosphatidylserine in the outer membrane. The flow cytometry results indicated that AGS cells treated with sulforaphane led to a markedly increased apoptotic ratio compared with controls (Fig. 3C), and the effect was concentration-dependent. These observations suggested that the proliferation inhibition and mitotic arrest effects of sulforaphane in AGS cells are related to apoptosis induction. A. The morphological changes in the cells were imaged using an inverted microscope (original magnification, ×200; upper panels). The cells were fixed and stained with DAPI solution. Stained nuclei were then observed with a fluorescence microscope (original magnification, ×400; lower panels). B. The cellular proteins were separated by SDS-PAGE and transferred to membranes. The membranes were probed with anti-PARP antibody. Proteins were visualized using an ECL detection system. Actin was used as an internal control. C. The cells were stained with annexin V and PI, and the percentages of apoptotic cells were analyzed using flow cytometric analysis. Each point represents the mean of two independent experiments.

Figure 4. Activation of AMPK by sulforaphane and the effects of AMPK inhibitors on sulforaphane-induced apoptosis in AGS cells.
A. Cells were treated with the indicated concentrations of sulforaphane for 48 h. The cells were lysed and cellular proteins were separated by SDS-PAGE and transferred to membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Equal loading was confirmed using actin as an internal control. B., C. The cells were pretreated with 10 μM compound C, an AMPK inhibitor, for 1 h and then with 20 μM sulforaphane for an additional 48 h. B. Nuclei were stained with a DAPI solution and observed with a fluorescence microscope (original magnification, ×400). C. The percentage of apoptotic cells was analyzed using flow cytometry. Each point represents the means of two independent experiments. D. Cell viability was analyzed using an MTT assay. The data are expressed as means ± SD of triplicate samples. The significance was determined by the Student t-test (* p < 0.05 vs. untreated control; # p < 0.05 vs. sulforaphane-treated cells).

Activation of AMPK is involved in sulforaphane-induced apoptosis in AGS cells
Because the AMPK signaling pathway plays an important role in apoptosis induction (Kim and He 2013;Rehman et al. 2014), we investigated whether this pathway is involved in apoptosis associated with sulforaphane. As shown in Fig. 4A, sulforaphane treatment led to an increase in the phosphorylated AMPK level (Thr 172) but did not cause changes in the total AMPK level. In addition, the amount of phosphorylated acetyl-CoA carboxylase (ACC, Ser 79), a major target kinase of AMPK (Hopkins et al. 2003), was augmented in a concentration-dependent manner. To examine whether sulforaphane-induced apoptosis is related to AMPK activation, we treated the cells with an AMPK inhibitor, compound C, prior to the addition of sulforaphane. The DAPI staining and flow cytometry results shown in Fig.  4B and C demonstrated that compound C could abolish sulforaphane-induced apoptosis in AGS cells. Conversely, the blocking of AMPK signaling significantly inhibited sulforaphane-mediated cell viability loss (Fig. 4D). Collectively, our results suggested that activation of AMPK signaling is important for sulforaphane-induced cytotoxicity in AGS cells.

ROS generation is associated with sulforaphane-induced apoptosis in AGS cells
ROS generation is usually associated with apoptosis related to impaired mitochondrial function ). Therefore, we used an oxidation-sensitive fluorescent dye, DCF-DA, to test the level of ROS generation in sulforaphanetreated AGS cells. As can be seen in Fig. 5A, intracellular ROS levels increased rapidly after treatment with sulforaphane and decreased again after 2 h. MMP values were also measured using JC-1 to investigate whether the increased production of ROS by sulforaphane treatment was associated with mitochondrial function impairment. The results in Fig. 5B show that sulforaphane caused a concentration-dependent loss of MMP in comparison to the untreated control. Since the breakdown of the MMP is amongst the sequences of events occurring during the mitochondrial-mediated apoptotic pathway, this study examined the effects of sulforaphane on the levels of cytochrome c. As indicated in Fig. 5C, Western blot analyses revealed a progressive decrease in mitochondrial cytochrome c and a concurrent increase in cytosolic cytochrome c in response to sulforaphane treatment. These results further showed that sulforaphane induces the loss of MMP, thereby resulting in mitochondrial dysfunction, release of cytochrome c from the mitochondria to the cytosol, and apoptosis induction.
To evaluate the role of ROS in sulforaphane-induced apoptosis, AGS cells were pretreated with an antioxidant NAC before sulforaphane administration. As shown in Fig.  6A and B, the sulforaphane-induced ROS generation and loss of MMP were markedly blocked by pretreatment with NAC, revealing that ROS contributed to the sulforaphane-induced dissipation in MMP. Furthermore, the degradation of PARP and translocation of cytochrome c were both completely reversed when cells were pretreated with NAC before applying sulforaphane (Fig. 6D). Moreover, the flow cytometry analysis and MTT assay also demonstrated that the blocking of ROS generation with NAC significantly reduced the apoptotic ratio and cell growth inhibition, respectively ( Fig. 6C and E). Together, these data confirmed that ROS generation plays a critical role in the mitochondria-mediated apoptosisinducing activity of sulforaphane in AGS cells.

ROS generation are involved in sulforaphane-induced AMPK activation in AGS cells
Recent studies have shown that ROS generation is one of the most important signaling networks involved in the activation of AMPK (Kim and He 2013;Rehman et al. 2014). Therefore, we postulated that sulforaphane may induce mitotic arrest, consequently leading to apoptosis through activating ROS-mediated AMPK signaling. To study the role of ROS as upstream modulating factors for AMPK activation in sulforaphane-induced mitotic arrest of AGS cells, we investigated the phosphorylation levels of AMPK and histone H3 after treatment with sulforaphane in the presence or absence of NAC. As shown in Fig.  7A, NAC pretreatment perfectly protected phosphorylation of AMPK and histone H3 in the sulforaphane-exposed cells without a change in their total protein levels. Importantly, our results revealed that blocking the ROS generation also markedly inhibited the induction of cyclin B1 and p21 in sulforaphanestimulated AGS cells (Fig. 7A). Consistent with these studies, the flow cytometry results in Fig. 7B show that NAC pretreatment almost blocked the sulforaphane-induced increase in the number of G2/M phase cells. Therefore, our results strongly revealed that the generation of ROS plays a pivotal role in sulforaphane-triggered mitotic arrest and eventually apoptosis through the activation of AMPK signaling.

Discussion
Mounting evidence has shown that sulforaphane suppresses most cancer cell proliferation and induces apoptosis in Figure 5. Increase in ROS generation, loss of MMP and translocation of cytochrome c into the cytosol by treatment of AGS cells with sulforaphane. Cells were treated with 20 μM sulforaphane for the indicated times (A) or the indicated concentrations of sulforaphane for 48 h (B and C). A. The medium was discarded and cells were incubated at 37°C in the dark for 20 min with new culture medium containing 10 μM DCF-DA. ROS generation was measured using a flow cytometer. B. The cells were collected and incubated with 10 μM JC-1 for 20 min at 37°C in the dark. The cells were then washed with PBS and the values of MMP were analyzed using a flow cytometer. C. The mitochondrial and cytosolic proteins were extracted and separated by SDS-PAGE, followed by Western blot analysis using anti-cytochrome c antibody and an ECL detection system. Cytochrome oxidase IV (COX IV) and actin were used as internal controls for the mitochondrial and cytosolic fractions, respectively.
conditions where there is no significant effect on normal cells. The anticancer efficacy of sulforaphane was found to be associated with mitosis-specific arrest during cell cycle progression and ROS production Redza-Dutordoir and Averill-Bates, 2016). Recently, it has been reported that inhibition of cancer cell proliferation by natural products with anticancer activity is related to the activity of the AMPK pathway (Sook et al. 2014), and sulforaphane has been reported to activate AMPK in some experimental systems (Lee et al. 2012;Choi et al. 2014;Lee et al. 2014;Zhang et al. 2014); however, the underlying mechanism related to anticancer activity is not well known. In the current study, we investigated the relationship between ROS and the AMPK pathway in the induction of apoptosis associated with cell cycle arrest by sulforaphane in AGS gastric cancer cells. Our results revealed for the first time that sulforaphane induces mitotic arrest and apoptosis of AGS cells via a ROSdependent activation of AMPK signaling.
It has been found that excessive production of ROS that cannot be attenuated by intracellular redox systems in many cancer cell lines can not only arrest cell cycle but also induce apoptosis Redza-Dutordoir and Averill-Bates 2016). This means that generation of ROS can act as a secondary messenger device to regulate the intracellular signaling pathways for antitumoral function, suggesting that excessive ROS production is an important factor in determining the fate of cancer cells. Our flow cytometry study showed that sulforaphane-induced inhibition of AGS cell proliferation was associated with G2/M arrest, which is consistent with some previous results (Singh et al. 2004;Matsui et al. 2007;Chu et al. 2009;Suppipat et al. 2012;Wang et al. 2015;Byun et al. 2016;Shang et al. 2017). In this process, expression of p21 was increased with the increase of p53 expression, which was also in good agreement with previous results observed in some cancer cells (Herman-Antosiewicz et al. 2007). Interestingly, the expression of cyclin B1 was increased in the absence of changes in the expression of Cdks, such as Cdk1 and Cdk2, and the enhanced phosphorylation of histone H3 was observed with sulforaphane treatment. Although, it has been reported that cyclin B1 expression is Figure 6. Induction of ROS-dependent apoptosis by sulforaphane in AGS cells. Cells were pretreated with 10 mM NAC for 2 h prior to 20 μM sulforaphane treatment. A. After 2 h of incubation, ROS generation was detected using DCF-DA. B. The loss of MMP was investigated using JC-1 following 48 h treatment with 20 μM sulforaphane. C. After 48 h of incubation, the cells were stained with annexin V, and the percentages of apoptotic cells were then analyzed using flow cytometry. The results are expressed as the mean of two independent experiments (A-C). D. The proteins were separated by SDS-PAGE and then transferred to membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. E. Cell viability was analyzed using an MTT assay. The data are expressed as means ± SD of triplicate samples. The significance was determined by Student's t-test (* p < 0.05 vs. untreated control). inhibited in cervical cancer cells during mitosis arrest by sulforaphane treatment (Cheng et al. 2016), its expression is increased in the mitotic arrest process by sulforaphane treatment in many cancer cells, including bladder, prostate, and breast cancer cell lines (Jackson and Singletary 2004;Herman-Antosiewicz et al. 2007;Park et al. 2014), as shown in this study. Thus, it can be inferred that cell cycle arrest by sulforaphane occurs specifically at the mitosis stage, considering that histone H3 phosphorylation, accompanied by increased cyclin B1 expression, is a hallmark of mitotic stasis (Murnion et al. 2001).
We also investigated whether cell cycle arrest of AGS cells by sulforaphane is associated with apoptosis induction of by morphological nuclear changes, degradation of PARP protein, and flow cytometry through annexin V staining. The apoptosis-induction pathway known to date is divided into an extrinsic pathway involving the death receptor and an intrinsic pathway emerging as the center of the mitochondria. Both pathways depend on caspase activation, and the intrinsic apoptosis pathway is accompanied by the loss of MMP and cytochrome c release in the cytoplasm (Koff et al. 2015;Westhoff et al. 2016). In the present study, sulforaphane-triggered apoptosis was accompanied by increased MMP loss, generation of ROS, and translocation of cytochrome c from the mitochondria to the cytoplasm, which was similar to previous study by Mondal et al. (2016).
Although some prior studies have reported that the extrinsic and intrinsic pathways are simultaneously activated in apoptosis induction by sulforaphane, our results give good support for several previous studies suggesting that sulforaphane-induced apoptosis is caspase mediated in relation to mitochondrial dysfunction (Singh et al. 2005;Rudolf et al. 2009;Xiao et al. 2009;Negrette-Guzmán et al. 2013;Mondal et al. 2016). In particular, the increased expression of pro-apoptotic Bax protein by sulforaphane is associated with increased ROS production and activation of intrinsic apoptosis pathway associated with cytosolic release of cytochrome c and loss of MMP (Jo et al. 2014;Mondal et al. 2016;Shang et al. 2017). However, when the production of ROS was artificially blocked, induction of apoptosis by sulforaphane was completely blocked, indicating that induction of apoptosis by sulforaphane in AGS cells is due to ROS production dependent mitochondrial dysfunction.
According to the results of many recent results, the activation of AMPK signaling pathway is a key factor in inducing apoptosis of cancer cells, which is reported to be associated with energy metabolism disturbance (Hopkins et al. 2003;Hardie et al. 2006). Since mitochondria are the key organ of energy production in cells and mitochondrial dysfunction in mitochondria-mediated apoptosis processes disturbs intracellular energy metabolism, AMPK is recognized as a new target to control cancer cell proliferation (Kim and He 2013;Rehman et al. 2014). Nevertheless, the role of AMPK in the inhibition of cancer cell proliferation by sulforaphane has not been studied to date. According to the results of our current study, the phosphorylation of ACC, its downstream target of AMPK, and AMPK, was apparently dependent on the treatment concentration of sulforaphane, indicating that the AMPK pathway was activated in sulforaphane-stimulated AGS cells. However, when compound C, an inhibitor of AMPK, was treated with sulforaphane at the same time, apoptosis induction by sulforaphane was significantly blocked, and the decreased cell viability returned to almost normal level; this indicated that activation of AMPK was required for sulforaphane-induced cytotoxicity in AGS cells. Interestingly, when ROS production was blocked, activation of AMPK, which was increased in sulforaphane-treated AGS cells, was also completely blocked. In addition, expressions of cyclin B1 and p21 were inhibited simultaneously, as well as the phosphorylation of histone H3, representing mitotic arrest. Moreover, the increased frequency of the cells belonging to the G2/M phase by sulforaphane treatment was restored to normal level. These results strongly suggest that mitotic arrest and induction of apoptosis by sulforaphane are mediated by ROS-dependent AMPK pathway activation.
In conclusion, the results of this study revealed that inhibition of AGS cell proliferation by sulforaphane is mediated by apoptosis induction associated with mitotic arrest, and that ROS production and increased AMPK activation are involved in this process. It was also found that the generation of ROS acts as a potential upstream factor for sulforaphaneinduced AMPK activation. Although further studies on the role of AMPK in energy homeostasis following mitochondrial disturbance are needed, the results of this study are the first reports to show that ROS-dependent AMPK activity is involved in the inhibition of human tumor cell proliferation by sulforaphane.

Conflict of interest.
The author has no conflict of interest to declare.


s2fieldsofstudy: Biology Medicine

text:
Pseudomonas aeruginosa Protease IV Exacerbates Pneumococcal Pneumonia and Systemic Disease

S. pneumoniae remains the leading cause of bacterial pneumonia despite widespread use of pneumococcal vaccines, forcing the necessity for appropriate treatment to control pneumococcal infections. Coinfections involving S. pneumoniae with other bacterial pathogens threaten antibiotic treatment strategies and disease outcomes. Currently, there is not an effective treatment for alveolar-capillary barrier dysfunction that precedes bacteremia. An understanding of the dynamics of host-pathogen interactions during single and mixed pulmonary infections could elucidate proper treatment strategies needed to prevent or reduce invasive disease. Antibiotic treatment decreases bacterial burden in the lung but also increases acute pathology due to cytotoxins released via antibiotic-induced bacterial lysis. Therefore, targeted therapeutics that inhibit or counteract the effects of bacterial proteases and toxins are needed in order to limit pathology and disease progression. This study identifies the cooperative effect of PIV and Ply, products of separate lung pathogens that additively alter the lung environment and facilitate invasive disease.

P neumonia is the leading cause of childhood mortality worldwide and is also a primary reason for hospitalizations associated with high morbidity and financial burden in the United States (1,2). The causative agent in cases of pneumonia has classically been diagnosed and treated as a single infecting microorganism, and the principal etiological agents identified during episodes of bacterial pneumonia include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Pseudomonas aeruginosa (3). However, increases in concurrent lung infections have been reported during both nosocomial and community-acquired pneumonia (CAP), and these mixed bacterial infections have resulted in more severe disease presentations than single infections (4)(5)(6)(7)(8). Specifically, S. pneumoniae is the most common causative agent of CAP and is also the most prevalent bacterium during mixed CAP infections (5)(6)(7).
In healthy individuals, the environment of the lower respiratory system is nutrient poor and unfavorable to invading pathogens (9). During acute lung injury or chronic lung disease, the alveolar environment favors bacterial growth through nutrient-rich fluid accumulation, temperature gradients, development of oxygen pockets, and a host inflammatory response that selectively favors potential pathogens (10)(11)(12)(13). Contrary to the previous assumption that the lung is a sterile environment, a heterogenous microbiome has been identified in the lungs of healthy individuals (9). Furthermore, the lung microbiome shapes the ecological forces that influence the colonization or elimination of potential pathogens in the lower airways (10). Lung microbiome dysbiosis occurs during acute lung injury such as viral infection and favors the growth of P. aeruginosa and S. pneumoniae (10). Moreover, lung coinfections linking P. aeruginosa and S. pneumoniae have been reported (7,(14)(15)(16). This coexistence implies that these two pathogens are cooperatively interacting during pneumonia. These interactions may be a result of secreted virulence factors that alter the lung environment, select for outgrowth of these pathogens, and thus enhance persistence of both pathogens.
Here, we investigated the ability of a secreted Pseudomonas protease, protease IV (PIV), to augment an active lung infection caused by S. pneumoniae EF3030, a strain of pneumococcus with limited virulence in a murine model (17). PIV has been shown to alter the lung mucosal defense by proteolytically degrading host immune effectors, such as interleukin 22 (IL-22) and surfactant proteins (18,19). IL-22 is a critical facilitator of mucosal innate immunity whose effects maintain mucosal barrier integrity through specific targeting of mucosal epithelial cells to induce tissue repair and secretion of antimicrobial peptides (20). Surfactant proteins are innate immune effectors that bind, aggregate, and increase phagocytic uptake of bacterial pathogens in the lung (21). Furthermore, IL-22 and surfactant proteins have been shown to be essential for limiting pneumococcal pneumonia, and EF3030 is a pneumococcal strain that does not readily induce bacteremia (22,23). Thus, a lung environment altered by PIV activity may favor the outgrowth of pneumococci, leading to enhanced lung pathology driven by pneumococcal virulence factors. Pneumolysin (Ply) is a cytotoxin and prominent virulence factor of pneumococci that has been shown to be essential during pulmonary infection and dissemination into the bloodstream (24)(25)(26)(27). Based on these prior studies, we hypothesize that depletion of IL-22 by PIV in the localized lung environment exacerbates pneumococcal pneumonia and permits invasive disease mediated by Ply.

RESULTS
Pulmonary coinfection involving S. pneumoniae and P. aeruginosa expressing PIV results in pneumococcal bacteremia. EF3030 is a pneumococcal strain with limited virulence that does not readily induce bacteremia during pneumonia in a murine model of infection. We performed a coinfection of S. pneumoniae EF3030 with either the P. aeruginosa 103-29 parent strain (designated PA103-29) or a piv deletion mutant of PA103-29 to determine if secreted virulence factors of P. aeruginosa could stimulate pneumococcal bacteremia. In mice coinfected with EF3030 and PA103-29, there was no significant difference in pneumococcal densities in the lungs compared to mice infected with EF3030 alone (Fig. 1). However, pneumococci were isolated from the blood of all mice coinfected with EF3030 and PA103-29. A deletion of piv in PA103-29 significantly reduced (P Ͻ 0.001) pneumococcal loads in murine lungs and abrogated pneumococcal bacteremia (Fig. 1).
Protease IV is active in murine lungs during infection. To further examine PIV-specific effects on pneumococcal pulmonary infections, we measured protease activity in lung homogenates of mice intratracheally administered phosphate-buffered saline (PBS), PA103-29, or PIV alone to determine a physiologically relevant concentration of PIV to administer (Fig. 2). When evaluating the secretome of P. aeruginosa, the Chromozym PL assay has been previously shown to be specific for measuring PIV activity (28). Compared to mock-treated mice, significantly higher (P Ͻ 0.05) proteolytic activity was measured in the lungs of mice during PA103-29 infection. Administration of 10 g PIV resulted in a nonsignificant 0.25-fold decrease in proteolytic activity compared to proteolytic activity in the lungs of mice during active infection (Fig. 2).
PIV exacerbates pneumococcal pneumonia that develops into highly fatal bacteremia in a murine model of infection. We investigated whether the proteolytic effects of PIV could augment the virulence of EF3030 in a C57BL/6 murine pneumonia model. LasB is a Pseudomonas protease that cleaves a number of host proteins, including surfactants, but this specific protease does not cleave IL-22 (18). Therefore, LasB was used as a negative control for IL-22 cleavage. EF3030 combined with PIV FIG 1 P. aeruginosa expressing protease IV (PIV) during murine lung coinfection with S. pneumoniae EF3030 stimulates pneumococcal bacteremia. C57BL/6 mice were intratracheally inoculated with 10 3 CFU of P. aeruginosa 103-29 (PA103-29) or PA103-29 piv deletion mutant followed by intratracheal inoculation of 10 6 CFU of S. pneumoniae EF3030 at 24 h after Pseudomonas infection. Pneumococcal burden in the lungs and blood was enumerated by plating on selective blood agar 48 h after pneumococcal infection. Deletion of piv in PA103-29 significantly reduced pneumococcal load in the lung and abrogated pneumococcal bacteremia. Each symbol represents the value for a single mouse, and each bar represents the mean for that group of mice. Data are representative of two independent studies. *, P Ͻ 0.05; **, P Ͻ 0.01.

FIG 2
Protease activity in murine lungs at 24 h after intratracheal administration. C57BL/6 mice were intratracheally inoculated with phosphate-buffered saline (PBS; control), 10 6 CFU of P. aeruginosa 103-29 (PA103-29), or 10 g protease IV (PIV). At 24 h after intratracheal administration, protease activity in murine lungs was measured on a spectrophotometer using the chromogenic substrate Chromozym PL. Optical density readings at 405 nm were converted into proteolytic units per milliliter as described by the manufacturer. Protease activity significantly increased during PA103-29 infection, and there was no significant difference between protease activity measured during active infection and that measured during administration of PIV alone. Data represent two independent studies performed in triplicate. Error bars represent standard errors of the means. *, P Ͻ 0.05; ns, no significant difference. significantly increased (P Ͻ 0.01) bacterial burden during pneumococcal pneumonia ( Fig. 3A) and allowed the infection to advance to significant bacteremia (P Ͻ 0.05) compared to EF3030 alone (Fig. 3B). Heat-inactivated PIV or LasB did not significantly increase bacterial burden during pneumococcal pneumonia compared to EF3030 alone (Fig. 3A). However, there was a strong trend (P ϭ 0.0625) toward increased bacteremia in mice inoculated with LasB compared to EF3030 alone (Fig. 3B). PIV and EF3030 combined inoculation resulted in 100% mortality, while all other inoculations caused very low or no mortality by 2 days postinfection (see Fig. 5).
PIV increases host susceptibility to Ply. Pneumolysin (Ply) is a virulence factor of pneumococcus that compromises alveolar-capillary barrier function, leading to permeability and flooding of alveoli (29). Since PIV enhanced pneumococcal pneumonia in a murine model (Fig. 3), we examined whether this increase in pathology was a result of additive Ply activity. Whether PIV was administered or not, there was no significant difference in bacterial load in the lungs (Fig. 4A) or blood (Fig. 4B) of mice during experimental pneumonia when infected with the Ply-deficient strain JLB12. Higher CFU were recovered from mice infected with JLB12 alone (Fig. 4A) than from mice infected with EF3030 alone (Fig. 3A), with some mice infected with JLB12 alone becoming bacteremic (Fig. 4B). Nonetheless, all mice infected with either EF3030 or JLB12 alone survived through 2 days postinfection (Fig. 5). Moreover, PIV administered with EF3030 resulted in higher bacterial burdens in murine lungs and blood (Fig. 3) as well as significantly increased mortality (Fig. 5) compared to combined PIV and JLB12-infected mice ( Fig. 4 and 5).
PIV does not impact pneumococcal adherence to human pulmonary epithelial cells. Pneumococcal adherence to human pulmonary epithelial cells was examined to determine if PIV alters S. pneumoniae EF3030 interactions with cells relevant to bacterial pneumonia. PIV coadministered with bacterial cells did not impact adherence to epithelial cells in either EF3030 or JLB12, a ply mutant strain of EF3030 (Fig. 6). However, a deletion of ply significantly increased (P Ͻ 0.05) pneumococcal adherence to pulmonary epithelial cells (Fig. 6). EF3030 and JLB12 were not able to invade epithelial cells with or without the presence of PIV (data not shown).

PIV and Ply induce neutrophil (polymorphonuclear leukocyte [PMN]) recruitment and lung damage during active pneumonia.
We used histology to visualize relative lung pathology during active pneumonia. Intratracheal administration of EF3030 and PIV led to massive infiltration of immune cells and shrinkage of alveolar spaces with evidence of necrosis and abscess formation (Fig. 7, panels 3 and 4). No indication of pneumonia was observed when mice were inoculated with PBS or with EF3030 combined with heat-inactivated PIV (Fig. 7, panels 1 and 2 and panels 5 and 6, respectively). Mice inoculated with a combination of active PIV and the Ply-deficient strain JLB12 displayed evidence of focal, mild pneumonia characterized by restricted infiltration of immune cells and attenuated shrinking of alveolar air spaces (Fig. 7, panels 7 and 8).
PIV depletes IL-22 levels in vivo during active pneumococcal pneumonia. IL-22 is required for host control of pneumococcal pneumonia, and IL-22 is cleaved by Pseudomonas PIV but not LasB (18,22). To assess if IL-22 cleavage was occurring in vivo, we measured IL-22 levels in murine lungs during infection. IL-22 levels were signifi-  cantly reduced (P Ͻ 0.05) when PIV was administered in combination with either EF3030 (Fig. 8A) or JLB12 (Fig. 8B) compared to IL-22 levels in mice infected with EF3030 alone or JLB12 alone, respectively. When mice were inoculated with heat-inactivated PIV or LasB, there was no significant difference in IL-22 levels compared to inoculation with EF3030 alone (Fig. 8A). IL-22 levels were also higher in mice infected with EF3030 ( Fig. 8A) than in mice infected with the Ply-deficient strain JLB12 (Fig. 8B).

DISCUSSION
In this study, we demonstrate that a Pseudomonas protease augments the virulence of an S. pneumoniae strain in a murine model of acute lung infection. Our findings reveal that PIV and Ply, virulence factors exclusively expressed by two separate pathogens, additively potentiate an active lung infection. The coadministration of active PIV and Ply-expressing strain EF3030 resulted in the highest bacterial loads and severe bacteremia (Fig. 3), as well as 100% mortality (Fig. 5) and enhanced immune cell infiltration leading to large abscess formation (Fig. 7, panel 4). This severe pathology must be attributed to a protease function that is not shared by PIV and LasB, as LasB coadministration with EF3030 did not result in significantly increased pulmonary bacterial loads, bacteremia, or mortality ( Fig. 3 and 5). Since PIV did not impact interactions of S. pneumoniae EF3030 with pulmonary epithelial cells (Fig. 6), it is likely that altered immune signaling events are responsible for increased pathology within our murine model of lung infection. Both PIV and LasB cleave host surfactant proteins, but PIV exclusively cleaves IL-22 (18). To our knowledge, the activity of exogenously administered PIV has not been previously investigated in the lung. Our findings were able to quantify PIV activity (Fig. 2) and confirm the depletion of IL-22 by PIV in vivo (Fig. 8). This observation during an active murine lung infection is consistent with previous findings of IL-22 degradation in tracheal aspirates of P. aeruginosa-infected patients (18). Therefore, it is likely that IL-22 depletion in the lung is allowing for enhanced pneumococcal burden. PIV is a very weak immunogen (30), so increased pathology was not an effect of PIV immunogenic properties perpetuating deleterious inflammation. Accordingly, specific activity of PIV was required, as heat inactivation of PIV attenuated pathological findings (Fig. 3, 5, and 7). Although IL-22 played a large role in severity of pneumonia in our studies, the role of surfactants cannot be overlooked. LasB coadministration with EF3030 did enhance bacteremia, albeit not to statistical significance (Fig. 3B). Since surfactants have been shown to be important in restricting pneumococcal pneumonia (23), it is likely that the compounded effects of both Nonetheless, increased pathology was not solely dependent on the activity of PIV. PIV coadministration with Ply-deficient strain JLB12 did not enhance pneumococcal pneumonia or bacteremia (Fig. 4) and resulted in restricted, focal evidence of pneumonia (Fig. 7). Surprisingly, we recovered higher pneumococcal CFU from the lungs of mice infected with JLB12 alone than from mice infected with EF3030 alone (Fig. 3A and  4A), and 50% of mice inoculated with JLB12 alone were bacteremic at 2 days postinfection (Fig. 4B). However, increased adherence to pulmonary cells (Fig. 6) and decreased induction of protective IL-22 (Fig. 8) afforded by JLB12 compared to EF3030 may have allowed for the increased pneumococcal burden in the lung and breakdown of epithelial barrier integrity. The major cellular sources of IL-22 during pneumococcal pneumonia are innate-like cells (ILCs) and thymocyte cells (T cells) expressing ␥␦ T-cell receptors, which are stimulated by IL-23 secreted from activated dendritic cells and macrophages in the lung (22,31). Ply has been shown to activate macrophages through Toll-like receptor 4 (TLR-4) signaling (32). Thus, decreased activation of macrophages responsible for producing proinflammatory cytokines is likely contributing to the decreased induction of IL-22 and enhanced bacteremia in JLB12-infected mice ( Fig. 4B and 8).
Overall, innate mucosal barriers to infection play an important role in preventing pneumococcal invasive disease (PID). Altered lung environments provided by comorbidities or coinfection that lead to microbiome dysbiosis and pathogen outgrowth result in intimate interactions between invading pathogens. Specifically, our study reveals that a secreted Pseudomonas virulence factor dampens the host immune response and intensifies disease severity during S. pneumoniae pulmonary infections. Our findings also show that Pseudomonas PIV and Streptococcus Ply production have a combinatorial effect on disease severity. Unfortunately, the effects of these combined virulence factors cannot be alleviated with antibiotic usage. Since Ply is not secreted and is released upon bacterial lysis (33,34), bactericidal antibiotics that cause a rapid release of Ply could result in major lung damage. In an altered lung such as the environment created by PIV, increased damage mediated by rapid Ply release could be catastrophic. Thus, studies examining targeted therapeutics that minimize damage created by proteases and cytotoxins are necessary in order to develop proper treatment strategies for patients with critical lung infections.

MATERIALS AND METHODS
Materials. Recombinant P. aeruginosa protease IV (PIV) was purified and assayed using Chromozym PL (Roche) as previously described (35). PIV was heat inactivated at 60°C for 1 h, and inactivity was verified by gelatinase zymography. P. aeruginosa protease LasB was purchased from Elastin Products  Bacterial strains and growth conditions. S. pneumoniae strains were grown at 37°C with 5% CO 2 in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) or on sheep blood agar (BA) containing 0.5 g/ml gentamicin. EF3030 is a serotype 19F S. pneumoniae strain (36). An EF3030 isogenic pneumolysin (ply) deletion mutant, JLB12, was created by allelic replacement of ply with an erythromycin resistance cassette amplified from ΔPLY2 as previously described (37). JLB12 was cultured using selective medium containing 3 g/ml erythromycin. P. aeruginosa strains were grown at 37°C on MacConkey agar (MA) or in Luria-Bertani (LB) broth with shaking at 180 rpm. P. aeruginosa 103-29 and the isogenic protease IV (piv) deletion mutant have been previously characterized (38). During murine pulmonary coinfections, lung and blood samples were plated on BA containing 200 g/ml polymyxin B to select for S. pneumoniae growth and MA without antibiotics to select for P. aeruginosa growth.
Murine pulmonary infections. Adult B6 mice (10 to 12 weeks old, 20 to 25 g) were anesthetized with an intraperitoneal injection of 500 l Avertin (6.25 mg tribromoethanol dissolved in 2.5% amylene hydrate solution) and intratracheally inoculated with 50 l PBS containing 10 6 CFU pneumococci or 10 g of PIV. For bacterial coinfections, mice were inoculated with 50 l containing 10 3 CFU of Pseudomonas followed by 50 l containing 10 6 CFU of pneumococci at 24 h after Pseudomonas infection. For mixed exogenous administration, mice were inoculated with 50 l containing 10 6 CFU of pneumococci and 10 g of either PIV, heat-inactivated PIV, or LasB. The Chromozym PL assay (Roche) was used according to the manufacturer's instructions to measure the proteolytic activity in fresh lung homogenates of mice administered PIV or infected with 10 6 CFU of P. aeruginosa 103-29. Mice were monitored for survival and euthanized at 2 days postinfection. Blood was collected by retroorbital bleed or from the heart. Lungs were removed and homogenized in 2 ml PBS. Blood and lung samples were plated on selective BA or MA to enumerate CFU per milliliter recovered from each mouse. Mouse studies were approved by the University of Mississippi Medical Center Institutional Animal Care and Use Committee in accordance with National Institutes of Health laboratory animal use guidelines.
Pneumococcal adherence to human pulmonary epithelial cells. Adhesion assays were performed as previously described (39). Briefly, human A549 pulmonary epithelial cells that had reached at least 90% confluence in 24-well plates were washed three times with PBS and incubated with approximately 10 6 CFU of pneumococci in each well. Pneumococci were allowed to adhere for 30 min before epithelial cells were washed three times with PBS, lifted with 0.25% trypsin-EDTA, suspended in PBS, and plated on BA to enumerate adherent pneumococcal CFU. Recovered pneumococcal CFU were adjusted by standardizing inocula to 10 6 CFU.
Histology of murine lungs. At 24 h postinfection, mice were euthanized and all five lobes were fixed in modified Davidson's fixative (20% ethanol, 6% acetic acid, 10% formaldehyde dissolved in water). Fixed tissues were processed and stained with hematoxylin and eosin (H&E) by Excalibur Pathology Inc. Stained slides were examined by a reviewer blind to the specimen source. Pneumonia was characterized by exudate of fibrin and neutrophils within the airspaces in order to determine inflammation loci (focal versus diffuse) and severity of pneumonia (severe versus mild).
IL-22 levels in murine lungs. Murine lungs were homogenized in PBS containing 1ϫ Halt protease inhibitor cocktail (Thermo Scientific). Lung homogenates were centrifuged at 1,500 ϫ g, and supernatants were collected for use in a sandwich enzyme-linked immunosorbent assay (ELISA). IL-22 levels in supernatants were quantified using the mouse IL-22 Ready-Set-Go! ELISA kit according to the manufacturer's instructions.
Statistical analysis. Results were analyzed using the InStat program (Prism 4 software; GraphPad, San Diego, CA). The Mann-Whitney test was used to test significant differences in the means of two groups. The Kruskal-Wallis test with Dunn's post hoc comparisons was used for analysis of differences in means for three or more groups. Survival curves were analyzed with a log rank test. A P value of Ͻ0.05 was considered statistically significant.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
The emerging roles of ribosomal histidyl hydroxylases in cell biology, physiology and disease

Hydroxylation is a novel protein modification catalyzed by a family of oxygenases that depend on fundamental nutrients and metabolites for activity. Protein hydroxylases have been implicated in a variety of key cellular processes that play important roles in both normal homeostasis and pathogenesis. Here, in this review, we summarize the current literature on a highly conserved sub-family of oxygenases that catalyze protein histidyl hydroxylation. We discuss the evidence supporting the biochemical assignment of these emerging enzymes as ribosomal protein hydroxylases, and provide an overview of their role in immunology, bone development, and cancer.

Introduction
Post-translational modification (PTM) is a major level of protein control that plays critical roles in all fundamental cellular processes. Our understanding of the proteomic landscape of PTMs and the discovery of new protein modifications has been partly fuelled by the development of mass-based detection, sequencing and quantification technologies. Mass spectrometry has revolutionized the ability to research small and difficult-to-detect PTMs. One such PTM is 'hydroxylation', the enzymatic incorporation of a single oxygen atom to create an alcohol, or hydroxyl, group. Although hydroxylation was once considered to be a very rare PTM [1], it is now appreciated to be an important modification that deserves more scrutiny for its roles in biology and for its potential medical applications [2].
Protein hydroxylation is generally catalyzed by a family of '2-Oxoglutarate (2OG)-dependent oxygenases' (2OGoxygenases) (see below). These enzymes utilize fundamental nutrients to catalyze oxidative modifications to a variety of biological molecules in addition to protein including DNA, RNA and lipid [3,4]. They target their substrates with similar levels of specificity as other signaling enzymes: Protein hydroxylases generally modify a single residue in a limited number of substrates. Thus far, the types of amino acids targeted for hydroxylation include prolyl, lysyl, asparaginyl, arginyl, aspartyl, and histidyl residues [5][6][7][8][9][10][11][12]. Whilst our understanding of prolyl, lysyl and asparaginyl hydroxylation has been driven by extensive research into their roles in collagen synthesis and hypoxia signaling [7,10], the biological roles of the other types of protein hydroxylation are less well understood. That being said, it is clear that even poorly characterized hydroxylases are involved in diverse aspects of physiology and in important disease processes [13,14]. In this article, we focus on summarizing our current understanding of protein histidyl hydroxylation, the main enzymes responsible for catalyzing this novel PTM, and their emerging roles in cell biology, physiology and disease.
Histidyl hydroxylation was first characterized in 2011, with the discovery that factor-inhibiting HIF (FIH), the 1 3 primary activity of which is asparaginyl hydroxylation, can also catalyze site-specific histidyl hydroxylation in the ankyrin repeat domain of over-expressed Tankyrase-2 [12]. Whether this modification occurs in endogenous Tankyrase-2, and whether it has a physiological role in regulating Tankyrase-2 function, remains unclear. More recently, two closely related 2OG-oxygenases, Myc-induced nuclear antigen (MINA) and nucleolar protein 66 (NO66), were reported to hydroxylate specific histidyl residues in endogenous ribosomal proteins [6]. Therefore, we focus our attention here on MINA and NO66. First, we provide a general background to the wider family of 2OG-oxygenases.

2OG-oxygenases
FIH, MINA and NO66 belong to a gene family of approximately sixty 2OG-oxygenases. These enzymes catalyze various oxidative modifications, including hydroxylation and demethylation (via a hydroxylation reaction) (Fig. 1a), through the action of a common catalytic domain known as a 'double-stranded beta helix' (DSBH) [15,16]. This domain consists of eight anti-parallel beta strands which together form a barrel-like or 'cupin' fold. This structure ensures the integration of essential co-factors including Fe-(II), the Krebs cycle intermediate 2OG, molecular oxygen, and the substrate (Fig. 1b) [15,16]. A conserved '2-His/1-Carboxylate' motif mediates Fe-(II)-binding, whilst the 2OG-binding residues vary across different sub-families.
Catalysis by 2OG-oxygenases initially proceeds via formation of a highly reactive iron (IV)-oxo intermediate (by the oxidative decarboxylation of 2OG), which oxidizes the target substrate (Fig. 1b) [15,16]. Where a 2OG-oxygenase has a demethylase function, the product formed is an unstable hydroxy-methyl group, which spontaneously fragments producing formaldehyde and the demethylated substrate ( Fig. 1a) [13,17]. The requirement for fundamental nutrients and metabolites means that the activity of these enzymes has the potential to be regulated by changes in the intracellular and extracellular micro-environment. Low tissue oxygen tensions and dysregulation of the Krebs' cycle have been implicated as important regulators of 2OG-oxygenase function in physiological and disease processes [18,19]. For example, increased concentrations of the metabolites succinate and fumarate have been shown to competitively inhibit some 2OG-oxygenases [19,20], and oxygen limitation inhibits hydroxylases that control the proteasomal degradation of the hypoxia-inducible transcription factor HIF [7,18,21]. The catalysis of hydroxylation and demethylation by 2OGoxygenases is described in depth in a number of excellent reviews [2-4, 13, 15-17].
2OG-oxygenases have been implicated in several fundamental cellular processes [4,14]. Thus, it is perhaps unsurprising that 2OG-oxygenases have been linked with a variety of diseases. These include obesity, cardiovascular and pulmonary disease, neurological abnormalities and cancer [14,22]. This in turn has stimulated efforts to understand the biochemical activities and cellular functions of 2OGoxygenases. Indeed, histidyl hydroxylases were initially characterized in disease models, sparking efforts to identify and characterize their substrates and biological functions.

Initial discovery and characterization of histidyl hydroxylases
MINA and NO66 belong to a subgroup of 2OG-oxygenases whose DSBH has homology to that of 'Jumonji', a protein originally named after the cruciform phenotype observed in the corresponding knockout mouse [23]. These DSBH domains, now termed Jumonji-C (JmjC) are present in 2OGoxygenases that (generally) catalyze protein hydroxylation. The largest phylogenetic group of JmjC hydroxylases catalyze lysyl demethylation of histones and have attracted significant interest for their roles in epigenetics [17]. In addition to the JmjC domain, these proteins also contain functional motifs involved in chromatin biology including ARID, Tudor and zinc finger domains [13].
MINA was first described in 2002 as a c-myc target gene following gene expression profiling in HL60 cells that conditionally expressed c-myc [24]. Although MINA appears to be ubiquitous [25], its expression does not always correlate with myc levels [26], indicating that other factors also likely contribute. Indeed, SMAD and Sp1 transcription factors have also been identified as regulators of MINA transcription [27]. Consistent with its regulation by growth-responsive transcription factors, MINA levels are induced by mitogens [24], and it is highly expressed in rapidly proliferating tissues such as the testes [28]. Interestingly, MINA levels may also be under the control of stress signaling pathways: Independent gene expression profiling experiments discovered MINA as a gene whose transcription was induced by silica treatment in a Jun kinase-dependent manner (resulting in the label 'mineral dust-induced gene', or Mdig) [29,30].
Proteomic analyses of nucleoli purified from Xenopus laevis oocytes also led to the discovery of MINA as a novel nucleolar protein [26]. Eilbracht et al. reported that MINA was localized to the granular component of the nucleoli, a sub-compartment normally associated with late stages of ribosomal biogenesis. Indeed, mass spectrometry analysis of MINA complexes supported its interaction with preribosomal particles [6]. This led to the proposal that MINA may play a role in ribosome biogenesis. Although MINA is enriched in the nucleolus, it is also partially localized to the nucleoplasm [26,31]. Whether its differential localization reflects multiple cellular functions is unclear, but could be consistent with proposed roles in chromatin biology and transcription (see below).
Interestingly, the biochemical exploration of the nucleolus that discovered MINA also identified a related JmjC-only protein called NO66 [32]. Like MINA, NO66 is also highly conserved through evolution and is ubiquitously expressed [32]. Furthermore, sucrose-gradient experiments demonstrated that NO66 co-fractionates with pre-ribosomal particles, again suggestive of a role in ribosome biogenesis. However, additional localization in nucleoplasmic dots that colocalize with Ki-67, HP1α and PCNA, suggested that NO66 may also have functions in late replicating chromatin [32]. Similar to MINA, these observations may be consistent with independent reports of NO66 controlling chromatin modifications and transcriptional regulation (see below). Indeed, NO66 was independently identified as a member of the myc transcriptional complex and named 'MAPJD': Suzuki et al. suggested that transactivation of myc by NO66 could lead to upregulation of genes involved in oncogene signaling and the TGFβ pathway [33].
Overall, the studies outlined above suggested that MINA and NO66 are ubiquitously expressed proteins that have functions in ribosome biogenesis and/or protein translation, and possibly extra-nucleolar functions in genome biology. However, the molecular mechanisms involved in these processes remained unclear, partly because of a lack of understanding regarding the biochemical activities of these enzymes.

Assigning the biochemical activity of MINA and NO66
Early studies on MINA and NO66 successfully identified the presence of the JmjC domain [24,26,32]. Although it was not recognized as catalyzing histone lysine demethylation at that time, the JmjC domain had been proposed to have a role in chromatin remodeling [34,35]. Subsequently, the assignment of histone lysine demethylase ('KDM') activity to some JmjC domains [17] led investigators to test whether MINA and NO66 regulate the methylation status of histone lysine marks. Candidate approaches screened the expression levels of these marks in cells overexpressing MINA or NO66, and led to the assignment of both as KDMs [36,37], as outlined below.
Lu et al. noted an inverse correlation between MINA and global H3K9me3 levels in lung cancers, and reported that manipulating MINA levels by overexpression or RNA interference caused the anticipated changes in H3K9me3 expression in bronchial epithelial cells [36]. However, a subsequent study by the same group noted marginal effects of MINA on H3K9me3 levels in A549 cells [38]. Interestingly, Chen and colleagues observed that MINA upregulates the expression of KDM4A (a JmjC H3K9me3 demethylase of the KDM family) which they proposed may contribute to the observed regulation of H3K9me3.
A proteomic analysis of Osterix, an osteoblast-specific transcription factor (see below), led to the identification of NO66 as an Osterix repressor [37]. Sinha et al. reported that NO66 regulates Osterix-specific target promoters and proposed that the molecular mechanism involved an unusual H3K4 and H3K36 demethylase activity. Chromatin immunoprecipitation experiments indicated that the chromatin localization of NO66 inversely correlates with H3K4me3 and H3K36me3 levels [39,40], suggesting that NO66 may regulate the abundance of these epigenetic marks.
Independent, unbiased proteomic screens subsequently identified a distinct biochemical activity for both MINA and NO66. Mass spectrometry analyses of the MINA and NO66 interactomes identified a large number of ribosomal and nucleolar proteins [6], consistent with the work of Eilbracht et al. [26,32]. Synthetic peptides spanning all these interacting proteins were tested for a +16 Da shift by mass spectrometry, indicative of hydroxylation. These biochemical assays led to the discovery that MINA efficiently targets His-39 of the large ribosomal subunit Rpl27a, and that NO66 is equally effective at modifying His-216 of Rpl8 (Fig. 1d, e) (Rpl27a and Rpl8 are respectively known as uL15 and uL2 under the current naming convention). A subsequent study by an independent group confirmed Rpl8 His-216 hydroxylation by NO66 in vitro [41]. NMR studies confirmed the biochemical assignment and demonstrated that these enzymes hydroxylate at the beta position of the side chain (Fig. 1f) [6]. Endogenous Rpl27a and Rpl8 hydroxylation were dependent on MINA and NO66, respectively, and (importantly), this occurred in a non-redundant fashion. Interestingly, the occupancy of histidyl hydroxylation was extremely high in all healthy cell lines and tissues tested (> 90%), which may suggest a positive role in ribosome biogenesis and/or translation. Structural analyses indicated that Rpl27a His-39 is proximal to the ribosomal E-site tRNA, and His-216 of Rpl8 is close to the peptidyl transferase center (PTC) (Fig. 1d) [42]. Subsequent chemical footprinting analyses of differentially hydroxylated Rpl8 indicated that the NO66 hydroxylation reaction likely serves to properly orientate the 28S rRNA in the vicinity of the PTC, thus ensuring appropriate organization of the PTC in the assembled ribosome [42]. The assignment of 'ribosomal oxygenase' activity has recently led to NO66 and MINA being officially named RIOX1 and RIOX2, respectively. Interestingly, the finding that MINA and NO66 are related to YcFD, an Escherichia coli JmjC 2OG-oxygenase that catalyzes arginyl hydroxylation of the ribosomal protein Rpl16 [6], suggests that ribosomal oxygenase activity is very highly conserved. The function of MINA, NO66 and YcfD in ribosomal protein hydroxylation is consistent with an emerging role for JmjC-only enzymes, and the wider 2OG-oxygenase family, in protein translation [43].
Are MINA and NO66 'bifunctional' enzymes that possess both ribosomal oxygenase and KDM activity? A comprehensive study began to address this question by screening the catalytic domains of 15 JmjC enzymes from across the 2OG-oxygenase family against a panel of histone H3 peptides that spanned each methylation status (me1-3) of four lysyl residues; H3K4, H3K9, H3K27 and H3K36 [44]. These sites included the proposed demethylation targets of both MINA and NO66. Under conditions in which JmjC enzymes of the KDM family catalyzed efficient demethylation of their reported substrates, MINA and NO66 were completely inactive. Importantly, however, under the same conditions, MINA and NO66 catalyzed efficient hydroxylation of Rpl27a and Rpl8 peptides, respectively. Similar findings were independently reported following a focussed study on NO66 [41]. These data would suggest that the primary activity of MINA and NO66 is histidyl hydroxylation.
The specific biochemical activity of an enzyme can also be inferred from detailed structural analysis of its active site. Indeed, structural analyses of JmjC KDMs and hydroxylases have helped to rationalize differences in their biochemical specificities (reviewed in [15]).

Structural analysis of histidyl hydroxylases
To investigate the relationship between JmjC ribosomal oxygenases and KDMs, Chowdhury et al. solved the crystal structures of MINA, NO66 and YcfD [45]. Consistent with predictions from primary sequence analyses, these enzymes lacked functional motifs commonly associated with KDMs. Interestingly, however, they all contained a conserved helical motif immediately C-terminal to the JmjC domain that was required for dimerization (Fig. 2). Similar to other JmjConly hydroxylases [9,46], oligomerization appears to be required for full RIOX activity [41,45]. Moreover, MINA, NO66 and YcfD also share another C-terminal fold termed the winged helix (WH) domain (Fig. 2) [41,45]. Because of the absence of this domain in other 2OG-oxygenases [13] it has been proposed that KDMs likely evolved from RIOXs following the loss of the WH domain [45]. Although its exact function is unclear, the restriction of the WH domain to RIOXs would suggest a highly conserved role in ribosomal protein hydroxylation. WH domains are generally implicated in protein-protein or protein-DNA interactions [47]. However, Chowdhury et al. suggest that the overall negative charge of this domain in MINA, NO66 and YcfD makes direct DNA/RNA-binding unlikely [45]. Therefore, it is possible that the WH domain mediates an interaction with another protein, perhaps a highly conserved factor that targets RIOXs to ribosome biogenesis pathways.
Further evidence supporting that MINA and NO66 are biochemically distinct from KDMs came from detailed structural analyses of their active sites and the binding mode of their substrates [45]. RIOXs bind their substrates in a conserved manner that is similar to related JmjC-only hydroxylases such as FIH, with a specific N-to C-directionality with respect to the catalytic machinery. Importantly, this differs significantly from that of most KDMs, including those considered to be phylogenetically more closely related to MINA and NO66 [45]. Furthermore, there are important differences in how deeply the target side chain binds in the catalytic pockets of RIOXs versus KDMs. Because JmjC-only protein hydroxylases target a carbon in the middle of the side chain, the target residue binds deep within the pocket [45]. In contrast, KDMs target the N Ɛ -methyl site at the end of the lysyl residue, so their target residues penetrate less deeply [15]. Finally, the JmjC domains of ribosomal oxygenases also lack two flexible loops within the DSBH that are highly conserved within the KDM subfamily and essential for lysyl demethylase activity. These loops form important interactions with the methylated lysyl side chain to facilitate its accommodation within the active site of the enzyme [15].
Overall, the combined phylogenetic, evolutionary, structural and biochemical data are consistent with the assignment of MINA and NO66 as protein histidyl hydroxylases.
Next, we summarize the current literature describing the emerging roles of these histidyl hydroxylases in diverse aspects of biology and disease.

NO66 and MINA in physiology and disease
The early studies that led to the discovery and biochemical characterization of MINA and NO66 involved diverse approaches, including enzyme-focussed screens (e.g., MINA/NO66 proteomics), function-specific proteomics (e.g., Osterix), and differential gene expression profiling experiments (e.g. c-myc, silica treatment, and lung cancer). This diversity has, in turn, contributed to an expanding literature on the role of these histidyl hydroxylases in mammalian physiology and disease, as reviewed below.

NO66
Previous research has identified two main roles for NO66 in physiology and disease, namely skeletal bone formation and tumorigenesis.

NO66 and skeletal development and ossification
As outlined above, NO66 was detected in a proteomic screen aimed at identifying the interactome of Osterix, an osteoblast-specific transcription factor that is required for osteoblast differentiation and bone formation [37]. Further characterization of this interaction identified that NO66 inhibits the ability of Osterix to drive osteoblast-specific gene transcription (Fig. 3a), through an interaction between the dimerization domain of NO66 and a defined 16 amino acid sequence within the transactivation domain of Osterix [37,48]. Overexpression of NO66 is sufficient to suppress the transactivation domain of Osterix using in vitro cell models [37], and mesenchymal-specific overexpression inhibits osteoblast proliferation and differentiation, skeletal growth and bone formation in mice [49]. Conversely, mesenchymalspecific ablation of NO66 increases the expression of genes that promote bone growth and development in mice, with a consequent increase in bone mass and density [50].
Importantly, overexpression experiments in HEK293T cells suggested that the ability of NO66 to repress the transactivation domain of Osterix is dependent on its enzymatic activity [37]. Although de Crombrugghe and colleagues suggest that NO66 regulates Osterix target genes via an intrinsic histone demethylase activity [37,40], this may require further consideration, for reasons outlined above [15,41,44,45]. Interestingly, other work suggests that NO66 exists in complexes containing important epigenetic modifiers including DNA methyltransferase 1A (DNMT1A), histone Reported crystal structures indicate a unique topology for JmjC ribosomal oxygenases that is conserved from Homo sapiens (Hs) to prokaryotes (including R. marinus, Rm). Note that YcfD catalyzes arginyl hydroxylation of Rpl16 in prokaryotes [6]. Critical catalytic residues are indicated. The dimerization domain is required for oligomerization and activity [45]. The function of the WH domain is unclear but, because of its overall negative charge, is thought unlikely to mediate an interaction with nucleic acid [45] deacetylase 1A (HDAC1A), HP1 and PRC2 (Fig. 3a) [39,40], raising the possibility that NO66 might regulate histone methylation status and target gene expression via an intermediary.
Whilst the molecular mechanism by which NO66 influences bone formation in vivo and osteoblast differentiation in vitro remains unclear (Fig. 3a), it is evident that NO66 is an important regulator of bone growth and mineralization (a mechanistic discussion of the roles of NO66 (and MINA) is presented at the end of this review). Considering the potential implications for diseases such as osteoporosis and osteopenia [51], further research in this area is warranted.

NO66 and cancer
2OG-oxygenases are attracting significant academic and pharmaceutical interest for their roles in tumor development and metastasis [14]. These roles are diverse and include, but are not limited to, cellular dedifferentiation via histone demethylation, hypoxia sensing in tumors, and regulation of inflammation and resistance to apoptosis [13,14,18]. In addition to aberrant expression and genetic alteration, 2OG-oxygenases may be deregulated by additional mechanisms in cancer, including competitive inhibition by 'onco-metabolites' (e.g., fumarate, see Introduction), and by low oxygen tensions in poorly vascularized areas of the tumor [13,[18][19][20]. Although it is currently unclear whether such mechanisms regulate MINA and NO66 activity in tumors, there is some evidence suggesting that ribosomal hydroxylation may be reduced in severe hypoxia. Ribosomal oxygenases are some of the only 2OG-oxygenases downregulated at the transcriptional level during hypoxia [52]. Indeed, reduced NO66 protein expression under severe hypoxia correlated with impaired Rpl8 hydroxylation [6]. Whether this effect was primarily due to reduced enzyme abundance, and/or a direct effect on enzyme activity remains unclear. Furthermore, it is not yet known whether MINA and NO66 'sense' oxygen Whether this involves a direct effect of an NO66 histone demethylase activity, or results from the recruitment of multiple epigenetic modifiers (e.g., PRC2, HP1, etc.), is currently unclear (signified by '?'). b NO66 is over-expressed in lung and colorectal cancers and promotes tumor cell growth and invasion in vitro. Whether the enzymatic activity of NO66 is involved is unclear ('-OH?'). The molecular mechanisms involved are also unknown, but could be related to roles in myc-driven transcriptional control (left) and/or ribosome biogenesis/ translation (right). HDAC1A histone deacetylase 1A; PRC2 polycomb repressor complex 2; HP1 heterochromatin protein 1; DNMT1A DNA methyltransferase 1A; Col1a1 collagen type I alpha 1 chain; Bsp bone sialoprotein; Oc osteocalcin; HAT histone acetyltransferase complex (TRRAP and TIP60) in the physiological range in a manner akin to the HIF hydroxylases, and whether such effects are relevant to tumor biology. That being said, there is mounting evidence supporting the role of both enzymes in cellular processes implicated in cancer.
The first indication that NO66 may have a role in cancer followed comparative gene expression profiling in normal versus non-small cell lung cancer (NSCLC) [33]. Suzuki et al. identified NO66 mRNA as frequently over-expressed in the majority of NSCLC samples and lung cancer cell lines tested. Overexpression of NO66 conferred increased growth potential upon NIH3T3 fibroblasts, whilst RNA interference in LC319 and A549 lung cancer cells reduced growth. Of interest with respect to the function of NO66 in transcriptional regulation, and the potential role of MINA downstream of Myc, Suzuki et al. also characterized transcriptional activation mediated by a complex containing NO66, Myc and histone acetyltransferases (Fig. 3b) [33].
In addition to the cell biology approaches outlined above, pathology analyses have investigated the expression of NO66 in human tumor samples. High levels of NO66 in colorectal cancer samples were associated with metastatic potential, venous invasion and lymph node metastasis: Patients whose tumors stained positively for NO66 had significantly shorter survival and disease-free survival [53]. Similar to the work in lung cancer cell lines described above, overexpression of NO66 in colorectal cells was also sufficient to promote their growth, and their invasion (Fig. 3b).
The mechanism(s) by which NO66 promotes tumor cell growth and invasion remains unclear. Further work is required to determine the relative contribution of Rpl8 hydroxylation versus other potential functions in gene expression control and epigenetics (discussed in more detail below).

MINA
MINA has generally been studied in more detail than NO66 and is implicated in a wider range of physiological and pathological processes, particularly those related to the immune system and cancer.
Recent work suggests that the role of MINA in cancer may be more complex than first thought, however. Specifically, MINA overexpression may not always correlate with poor patient prognosis. Although MINA overexpression was associated with worse survival in lung cancer patients who were staged 'lymph node negative' (N0) or 'possible proximal lymph node metastasis' (N1) (consistent with the cancer 'driver' work reviewed above), MINA expression was associated with improved prognosis in patients with distant lymph node invasion (N2) or 'distant metastasis likely' (M1) [31]. This observation might suggest that MINA has opposing roles at different stages of tumorigenesis, with a growthpromoting role in early cancer, and a tumor suppressor role in later stage disease, possibly via modulating invasion and/ or metastasis (Fig. 4a). Perhaps consistent with the latter, overexpression of MINA suppressed the migration and invasion of A549 and H226B lung cancer cells in vitro [31,69]. Conversely, MINA knockdown enhanced their invasion and migration [31,69], and in a manner that was associated with changes in markers of epithelial-mesenchymal transition [70].
Importantly, the complex association of MINA expression with prognosis may not be restricted to lung cancer. Breast cancer patients without lymph node involvement are reported to have worse survival if their tumors express higher levels of MINA, whereas MINA expression is associated with improved survival of patients that are lymph node positive [66]. This, together with the studies outlined above, might suggest that MINA can have a tumor suppressor function in some contexts, possibly via the control of invasion and migration. However, it remains unclear whether MINA is a bona fide tumor suppressor gene, and if so, what the molecular mechanisms involved might be (Fig. 4a) (further discussed below).

MINA and immunology
The first evidence implicating MINA as an immuno-modulator came from mouse studies in which it was identified as a genetic determinant of 'T-helper type 2 (T H 2) bias' [71], the tendency of naive CD4 + T cells to differentiate into IL4-producing T H 2 cells. T H 2 bias is an important immunological process that regulates susceptibility to autoimmune, allergic, and infectious disease [72]. Okamoto et al. reported that MINA modulates T H 2 bias by acting as a direct repressor of IL-4 transcription (Fig. 4b) [71].
An independent study subsequently implicated MINA in controlling the balance between additional T cell sub-types, specifically Th17 and Treg cells [73]. Th17 cells are proinflammatory IL17-producing T-cells that have important roles in protecting against extracellular pathogens, and in the onset of autoimmune diseases [74]. Yosef et al. undertook a temporal analysis to identify the dynamic regulatory gene expression network involved in Th17 differentiation, and studied the role of novel targets in these networks using nanowire-based siRNA delivery [73]. This approach identified that MINA mRNA is strongly induced during Th17 differentiation, and that MINA promotes the expression of Th17 factors such as ROR-γt, Batf, and Irf4, whilst suppressing the expression of the Foxp3 transcription factor, which plays a pivotal role Treg in biology (Fig. 4b).
The two studies outlined above suggested, for the first time, that MINA has potentially important roles in regulating T-cell differentiation, and that it might, therefore, be involved in the etiology of some autoimmune, allergic, and infectious diseases. Indeed, recent evidence implicates MINA in asthma and pulmonary fibrosis. A case-control study in a Han-Chinese population identified a singlenucleotide polymorphism (SNP) of the MINA gene that was associated with a significant increase in the risk of atopic asthma, and that patients heterozygous for this allele had elevated serum levels of IgE and IL-4 (the effect on MINA expression was not reported) [75]. Interestingly, a recent study using a mouse model of asthma induced by house dust mite aeroallergens found that MINA knockout significantly ameliorated airway hyper-responsiveness and decreased inflammation [76]. Counter to the role of MINA in repressing IL-4 in naive CD4 + T cells (as above), this MINA knockout phenotype was associated with decreased levels of IL4. The authors speculated that this may be mediated via an independent role for MINA in promoting T H 2 cytokine expression in antigen presenting cells. Others have proposed that the reduced airway disease in this model may be associated with a defective Th17 response [77]. If correct, this would suggest that the known role of MINA in Th17 differentiation (outlined above) may be dominant with respect to its role in T H 2 bias, at least in this particular disease. This hypothesis may be supported by a recent study in which heterozygous knockout of the MINA gene in mice was associated with reduced silica-induced pulmonary fibrosis, possibly due to an observed decrease in the Th17 response (and corresponding increase in the Treg response) [78].
In summary, the finding that MINA influences the balance between multiple T-cell subsets may be consistent with important roles in several T-cell-mediated responses and diseases, including perhaps other pathological contexts such as transplant rejection, autoimmunity and infection. The key now is to understand the determinants that influence the role of MINA in T-cell biases and specific immunological disease states. In addition, further studies are needed to shed light on the molecular mechanisms by which MINA exerts its complex effects on the immune system.

What are the molecular mechanisms under-pinning the role of MINA and NO66 in physiology and disease?
In the preceding sections of this review, we have summarized the current literature on the protein histidyl hydroxylases MINA and NO66, including reports of diverse roles in fundamental cellular pathways and important physiological and pathological processes. Although the biomedical importance of these enzymes is becoming increasingly clear, the molecular mechanisms involved, and how these might be deregulated in disease, have not yet been established. In the future it will be important to determine whether the enzymatic activity of these 2OG-oxygenases is required for any of the functions described. It is possible, for example, that non-enzymatic functions may be involved, perhaps mediated via the WH domain. If enzyme activity is required, however, it will be of interest to determine how hydroxylation regulates substrate function to elicit the corresponding biological outcome. It will be worthwhile considering whether Rpl27a and Rpl8 modification are likely to solely account for the diversity in biological functions reported. If histidyl hydroxylation functions in ribosome biogenesis to promote increased rates of bulk protein synthesis, it could perhaps explain the role of these enzymes in supporting cell growth, and the association of their expression with poor cancer prognosis (Figs. 3b, 4a). However, it is perhaps more difficult to conceptualize how increased ribosome biogenesis could elicit biological specificity in terms of transcriptional control (e.g., MINA and IL17/Foxp3, Fig. 4b) or physiology (e.g., NO66 and bone development, Fig. 3a). Such specificity might be more easily rationalized if the function of ribosomal protein hydroxylation was to control the translation of specific mRNAs, as opposed to bulk ribosome biogenesis. The former could, perhaps, be consistent with the localization of MINA-and NO66-catalyzed histidyl hydroxylations to functionally important sites of the ribosome (Fig. 1d). Of course, other explanations are also possible, and these may not necessarily be mutually exclusive. An intriguing possibility is that other, as yet unidentified substrates exist for these protein hydroxylases. Considering the biology reviewed above, one might predict that such novel targets could have functions in transcription and/or chromatin biology/epigenetics (Figs. 3, 4). Thus, further study of the role of MINA and NO66 histidyl hydroxylase activity is warranted. Future findings will be critical for not only providing mechanistic insight into how these enzymes regulate their respective physiological processes and disease, but also for determining whether the development of small molecule inhibitors is merited. Importantly, competitive inhibitors of 2OG-oxygenases are in clinical trials for other indications [79], suggesting that targeting MINA and/or NO66 is theoretically possible. Such inhibitors would likely benefit from stringent enzyme specificity considering the role of other 2OG-oxygenases in fundamental cellular processes [4,14,22]. Bearing in mind the paradoxical roles of MINA in cancer (Fig. 4b) [31,69], the contexts of their potential medical applications should also be very carefully considered.

Concluding remarks
Here, we have reviewed our current understanding of the role of the histidyl hydroxylases MINA and NO66 in cell biology, homeostasis and disease. We hope to have highlighted that these enigmatic enzymes have intriguing roles in fundamental cellular processes that warrant further investigation. In particular, this review emphasizes the need for future mechanistic studies that clarify the role of hydroxylase activity, and the identity of critical targets, in the cellular pathways and diseases discussed. Such work will be necessary to build a solid foundation from which to efficiently and successfully translate these 2OG-oxygenases into the clinic. Finally, it remains to be seen whether the proteome contains hydroxy-histidyl residues deposited by other protein hydroxylases. These could include enzymes from novel classes, or perhaps the biochemically unassigned members of the JmjC-only family (Fig. 1c). It is intriguing to consider the possibility that MINA and NO66 might represent the 'tip of the iceberg' with respect to the biology of histidyl hydroxylation.