Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3–6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.


Hypoxia induces cell proliferation in normal and IPF human pulmonary fibroblasts.
To determine the effects of hypoxia on the proliferation of human lung fibroblasts, microscopic observations, cell counting with trypan blue staining and bromodeoxyuridine (BrdU) assays were performed. Using normal human pulmonary fibroblasts (HPFs) cultured in Fibroblast Growth Medium 2 containing 2% fetal bovine serum (FBS), 1 ng/ml basic fibroblast growth factor, and 5 µg/ml insulin provided by the Manufacturer (PromoCell), an increased number of cells under hypoxic conditions were observed compared to normoxic conditions at culture day 2 and day 3 (Fig. 1A). This increase was confirmed by direct cell counting (Fig. 1B). Cell proliferation, determined by BrdU incorporation assay, was increased in cells grown in hypoxic conditions (5% and 1% O 2 ) for 3 and 6 days, and there were no significant differences in fold-increases between day 3 and day 6 ( Fig. 1C). Similar results were observed with IPF fibroblasts (LL29) cultured in F12K medium containing 10% fetal bovine serum. However, fold-increases in IPF fibroblasts induced by hypoxia were smaller than those in normal HPFs (Fig. 1D). Cell viability was not affected by hypoxic conditions in both cell types, as assessed by MTT assay (Fig. 1E,F).
To determine whether the culture medium has any effects on hypoxia-induced cell proliferation, we cultured HPFs in F12K medium containing 10% FBS and performed BrdU assays. Our results showed that hypoxia still increased the cell proliferation, although the fold-change in the F12K medium was smaller compared to that in the manufacturer's medium (Fig. 1G). Furthermore, we tested 7 additional fibroblast lines: four normal human pulmonary fibroblasts (CCD-13Lu, CCD-19Lu, HLF153 and HPF154) and three IPF fibroblasts (LL97A, IPF12 and IPF14). These fibroblasts were cultured in F12K medium containing 10% FBS. Cell proliferation of all fibroblast lines was increased by 3-day hypoxic exposure (Fig. 1G). In all of the studies reported in this paper except Fig. 1G, HPF cells were cultured in in Fibroblast Growth Medium 2 containing 2% fetal bovine serum (FBS), 1 ng/ml basic fibroblast growth factor, and 5 µg/ml insulin as recommended by the manufacturer. In Fig. 1G, the proliferative response was measured in HPF cells cultured in F12K media to ensure that the responses could be compared across all control and IPF fibroblast cultures, all of which were maintained in F12K media.

Hypoxia promotes G1/S transition and alters cell cycle protein expression in HPFs.
To determine which phase of the cell cycle is affected, fluorescence-activated cell sorting (FACS) cell cycle analysis was performed on HPFs exposed to hypoxia for 3 days using a Cell Cycle Phase Determination kit. The results showed that the number of cells with propidium iodide incorporation in the S phase of the cell cycle was higher in HPFs exposed to hypoxic conditions compared to normoxic conditions ( Fig. 2A). These results suggest that hypoxia increases the G1/S phase transition. To determine which cell cycle proteins are affected by hypoxic treatment, levels of cyclins and cyclin-dependent kinases (CDKs) were assessed by Western blot analysis. The results showed that cyclin D1, which is involved in G1 phase, was downregulated (Fig. 2B,C). Cyclin E1, the prominent cyclin in the G1/S phase transition, was upregulated. Cyclin A2, which promotes the transition of the G2/M phase, was downregulated in HPFs exposed to hypoxia. CDK2, CDK4 and CDK6 protein levels were not affected by hypoxia.
ScienTific RepoRts | (2018) 8:2709 | DOI:10.1038/s41598-018-21073-x affect cell viability at concentrations up to 10 μM (Fig. 3C,D). These results were consistent with previous HIF knockdown studies in IPF fibroblasts 11 . Hypoxia activates NFATc2 signaling. Several studies have demonstrated that NFAT signaling is involved in the cell proliferation of various cell types, including PAMSCs 17,25-27 , neural stem cells 28 , β cells, cancer 29 and lymphocytes 30 . We have recently shown that NFATc2 is required for Wnt5a-mediated fibroblast proliferation 31 . We hypothesized that hypoxia-mediated fibroblast proliferation may be due to the activation of NFATc2 signaling by hypoxia. To test this hypothesis, we determined whether hypoxia could activate NFAT signaling via NFATc2 nuclear translocation. Immunofluorescence staining of HPFs exposed to hypoxia was performed to evaluate the cytoplasmic and nuclear location of NFATc2. Ionomycin, a known activator of NFAT signaling 32 , was used as a positive control. Under normoxic conditions, only 5 ± 2% of NFATc2 was located in the nuclei (Fig. 4A-C). Ionomycin treatment increased the number of cells with NFATc2 nuclear localization to 48 ± 1%. Hypoxia exposure resulted in a significantly higher number of NFATc2 nuclear translocated cells (50 ± 3%) compared to normoxia.
We further examined the effects of hypoxia on NFAT expression. We exposed HPF cells to normoxia and hypoxia (5% or 1% O 2 , respectively) for 3 and 6 days and determined mRNA expression levels of NFAT isoforms. Hypoxia had no effect on the mRNA levels of all NFAT isoforms (NFATc1-4 and NFAT5) at day 3 (Fig. 5A). However, hypoxia increased the mRNA expression levels of NFATc2 and NFATc4 by day 6. The induction of mRNA expression of NFATc2 by hypoxia was greater than that of NFATc4. NFATc2 protein expression was also significantly increased by hypoxia at day 6 in HPFs ( Fig. 5B,C).
Furthermore, we determined whether HIF-1α and HIF-2α could increase NFAT transcriptional activity. HEK 293 T cells were co-transfected with the NFAT reporter and either a HIF-1α or HIF-2α expression vector. Overexpression of HIF-2α significantly increased NFAT reporter activity, whereas HIF-1α overexpression had no effects on the NFAT reporter activity (Fig. 6H). Taken together, these results indicate that HIF-2α mediates the hypoxia-induced activation of NFATc2 signaling.

NFAT signaling is involved in hypoxia-induced fibroblast proliferation.
To determine whether hypoxia-induced cell proliferation in lung fibroblasts occurs through the NFAT signaling pathway, NFATc2 was knocked down in HPF cells using a shRNA lentiviral vector expressing GFP. A high efficiency of lentiviral infection was observed (Fig. 7A). Western blot analysis showed that the NFATc2 protein level was reduced by 44 ± 7% in the NFATc2 shRNA-treated HPFs (Fig. 7B,C). NFATc2 knockdown significantly inhibited hypoxia-induced HPF proliferation (Fig. 7D). A significant decrease in HPF proliferation was also observed in NFATc2-knocked-down cells under normoxic conditions, likely due to the basal activity level of NFATc2. These results indicate that NFATc2 is a key regulator of cell proliferation in both normoxic and hypoxic conditions. Hypoxia-induced proliferation does not occur through upstream components of NFAT signaling. Hypoxia activates voltage-gated Ca 2+ channels (VGCCs) 14,33 . With the exception of NFAT5, all the other isoforms of the NFAT family are activated through calcium sensing. An increased intracellular calcium influx activates calcineurin, which dephosphorylates cytoplasmic NFAT and results in NFAT nuclear translocation, leading to the transcription of genes involved in cell proliferation 18,20 . To determine which step of NFAT signaling is involved in hypoxia-induced fibroblast proliferation, we inhibited NFAT signaling at various steps, including Ca 2+ channels, intracellular Ca 2+ and calcineurin, using specific inhibitors and determined cell proliferation. Verapamil is an L-type voltage-gated Ca 2+ channel blocker that reduces Ca 2+ influx 34,35 . BAPTA-AM is a specific Ca 2+ chelator 36,37 . Cyclosporine A (CsA) is a calcineurin inhibitor that inhibits the enzymatic activity of calcineurin 38,39 . HPF cells were treated with different concentrations of these inhibitors and exposed to normoxia or hypoxia for 3 days. HPF cells treated with the Ca 2+ chelator BAPTA-AM and the calcineurin inhibitor CsA did not show any differential effects in HPF proliferation between normoxic and hypoxic conditions ( Fig. 8A-C). These results suggest that the upstream molecules of NFAT signaling prior to NFAT nuclear translocation are not involved in hypoxia-induced pulmonary fibroblast proliferation. However, treatment with the Ca 2+ channel blocker verapamil chloride showed differential effects on cell proliferation between normoxic and hypoxic conditions at higher concentrations (Fig. 8C). This could be due to its direct effects on proteins involved in cell cycle and proliferation 40 . The inhibitors did not affect cell viability, except for BAPTA-AM, which was associated with cell death at concentrations greater than 10 µM ( Fig. 8D-F).
Since we used dimethyl sulfoxide (DMSO) to dissolve TC7009, KC7F2 (Fig. 3), BAPTA, and CsA, and ethanol (EtOH) to dissolve verapamil chloride (Fig. 8), we examined whether these solvents had any effects on cell proliferation and cell viability. HPFs were treated with 1% DMSO and 0.1% ethanol, the maximum concentrations used in this study, and exposed to 1% O 2 or normoxia for 3 days. These solvents at the concentrations used did not affect cell proliferation and viability (Fig. 8G,H).

Discussion
Hypoxia regulates many cellular events, including growth, proliferation and apoptosis, in different cell types. In this study, we demonstrated that 1) hypoxia promotes normal and IPF human pulmonary fibroblast proliferation, 2) hypoxia promotes the G1/S phase transition, 3) hypoxia activates NFAT signaling via HIF-2α, and 4) NFATc2 is required for hypoxia-induced cell proliferation. Based on these results, we propose the following model (Fig. 9). Hypoxia activates NFATc2 signaling by promoting NFATc2 nuclear translocation and/or transcriptional activity via HIF-2α, which in turn induces gene expression related to cell proliferation.
Our FACS analysis indicated that hypoxia (1% O 2 ) promoted the G1/S phase transition of pulmonary fibroblasts. This is consistent with several previous studies that have shown that moderate hypoxia (1-2% O 2 ) accelerates the G1/S phase transition in Jurkats (human T lymphocytes), SUP-T1s (human T lymphoblasts) 41 , human mesenchymal stem cells 42 , keloid fibroblasts 43 and normal human lung fibroblasts 13 . However, severe hypoxia (0.1-0.5% O 2 ) has been shown to arrest the G1/S phase transition in adult human lung fibroblasts 13 , as well as rat and mouse embryonic fibroblasts 44 .
The cell cycle is regulated by cyclins and cyclin-dependent kinases (CDKs). Cyclin E promotes the G1/S phase transition and its expression increases at the G1/S checkpoint. Cyclin A promotes the G2/M phase transition 45,46 . Our Western blot analysis showed that hypoxia increased cyclin E1 and downregulated cyclin A2 protein levels, which is consistent with our finding that hypoxia increased S phase transition in human pulmonary fibroblasts. Upregulation of cyclin E1 levels by hypoxia has been observed in prostate cancer cells 47 . Cyclin E has also been shown to overcome G1 arrest in rat embryonic fibroblasts 48 .
Cyclin D1 expression is high in the G1 phase, and its expression is reduced when cells enter the S phase 45,49-51 . Cyclin D1 promotes cancer cell growth [52][53][54] . Unexpectedly, we observed downregulation of cyclin D1 levels in pulmonary fibroblasts with hypoxia. However, minimal nuclear-localized cyclin has been observed when BrdU incorporation is high in proliferating human lung fibroblasts 49,51,55 . An inverse correlation between BrdU incorporation and nuclear cyclin D1 levels has also been reported 51 .
CDK2 is mainly involved in regulating S and G2 phase, while CDK4 and 6 are primarily involved in the G1 phase 45 . We did not observe any changes in CDK2, 4 and 6 levels with hypoxia. This is consistent with the observation that CDK protein expression persists stably throughout the cell cycle, whereas cyclin expression varies 46 . However, CDK2 expression is reduced by severe hypoxia (0.1%-0.5% O 2 ) 44 in healthy lung fibroblasts, but increased by hypoxia in prostate cancer cells 49 . Moderate hypoxia (2% O 2 ) has also been shown to inhibit CDK inhibitor p21 13 .
The involvement of HIF isoforms varies in different cellular mechanisms associated with proliferative responses. Our inhibitory study demonstrated that a HIF-2α inhibitor, but not a HIF-1α inhibitor, decreased the hypoxia-mediated proliferation in HPFs, suggesting that hypoxia-mediated pulmonary fibroblast proliferation is HIF-2α-dependent. This finding was supported by knockdown experiments in which the knockdown of HIF-2α in IPF fibroblasts reduced cell proliferation under hypoxic conditions (3% O 2 ) 11 . Furthermore, pulmonary arterial fibroblast proliferation is decreased by the knockdown of HIF-2α, but not HIF-1α, under moderate hypoxic conditions (1% O 2 ) 56 . Interestingly, HIF-2α promotes cell proliferation in renal-cell adenocarcinoma cells (WT8, 786 -O RCC), whereas HIF-1α inhibits the proliferation of HCT116 colon carcinoma cells at 0.5% O 2 9 . The mechanisms underlying the increase in fibroblast proliferation in response to hypoxia may be complex and involve several pathways. It has been reported that hypoxia increases IPF fibroblast proliferation by inducing miR-210 expression, which in turn reduces the c-myc inhibitor MNT 11 . Hypoxia-induced increases in miR-210 level is in a HIF-2α-dependent fashion and appears early and persists through day 6. In the current study, we Figure 5. Effect of hypoxia on NFAT mRNA and protein expression. HPFs were exposed to normoxia (21% O 2 ) or hypoxia (5% or 1% O 2 ) for 3 and 6 days. (A) mRNA expression levels of NFAT isoforms were determined by real-time PCR and normalized to β-actin. (B,C) NFATc2 protein expression levels were determined by Western blot, analyzed by ImageJ and normalized to the protein loading control β-actin. The resulted data were then normalized to normoxia group and represented as a percentage change relative to normoxia. Data represents means ± SE. *p < 0.05, *p < 0.01, ***p < 0.001 vs. normoxic group. n = 3 independent experiments. propose that hypoxia increases fibroblast proliferation by activating NFATc2 signaling via HIF-2α, which in turn regulates cell cycle protein expression. The activation of NFATc2 signaling by hypoxia is supported by our observations that hypoxia promotes NFATc2 nuclear translocation at an early time point (day 3) and increases NFATc2 mRNA levels at a late time point (day 6). It is conceivable that effects of HIF-2 on fibroblast proliferation may be mediated by several pathways -an early pathway through miR-210 and NFATc2 nuclear translocation and later effects involving NFATc2 transcriptional activity.
The nuclear translocation of NFAT isoforms caused by hypoxia has been previously reported in other cells. Increased NFATc3 nuclear translocation has been observed in PASMCs of mice that have been subjected to chronic hypoxic conditions 21 . In another study, exposure of human PASMCs from PAH to hypoxia for 96 hrs resulted in increased NFATc2 nuclear signal compared to normoxic conditions 17 . Neural stem cells that are exposed to hypoxia showed increased NFATc4 nuclear localization 28 . In an ischemia and reperfusion kidney model, rat IMCD (inner renal medulla Sprague-Dawley) cells exposed to anoxia (O 2 replaced by N 2 ) for 2 hrs had higher NFAT5 nuclear signals compared to cells exposed to normoxia 57 . U373MG human glioblastoma astrocytoma cells treated with the hypoxia-mimicking agent CoCl 2 had increased NFAT nuclear localization 58 . PASMCs isolated from mice exposed to chronic 21-day hypoxia showed higher NFAT activity compared to those from normoxic mice 59 .
Hypoxia-mediated activation of NFATc2 occurs through HIF-2α, not HIF-1α, as knock-down or chemical inhibition of HIF-2α, not HIF-1α, inhibited hypoxia-induced NFATc2 nuclear translocation, and overexpression of HIF-2α, not HIF-1α, increased NFATc2 transcriptional activity. There are two possibilities by which HIF-2α may activate NFATc2 signaling. One possibility is that HIF-2α induces the expression of positive components in NFAT signaling or the genes that upregulate NFAT signaling. Indeed, our study showed that hypoxia increases NFATc2 expression in HPFs. It has also been shown that the activation of HIF-2α increases NFATc2 and c3 mRNA expression levels 60,61 . Another possibility by which HIF-2α may activate NFAT signaling is that HIF-2α directly interacts with NFATc2.
Our recent studies showed that NFATc2 is required for Wnt5a-mediated cell proliferation 31 . Our current studies indicate that the silencing of NFATc2 reduced the fibroblast proliferation in hypoxic conditions, suggesting that NFATc2 signaling is also involved in hypoxia-mediated fibroblast proliferation. Different isoforms of NFAT have been reported to be involved in the regulation of cell proliferation in different cell types [25][26][27][28]62 . In pulmonary arterial hypertension (PAH), PASMCs show an increased proliferation and resistance to apoptosis. A chemical treatment (Plumbagin), which inhibits NFATc2 gene expression and NFATc2 nuclear localization, resulted in decreased cell proliferation of PASMCs derived from PAH patients, suggesting a positive relationship between NFATc2 signaling and cell proliferation of PASMCs in PAH 27 . Serotonin treatment in PASMCs increases cell proliferation and activates calcineurin and NFAT signaling. The inhibition of calcineurin by CsA and silencing of NFATc2 by siRNA decreases DNA synthesis in PASMCs treated with serotonin, indicating that NFATc2 is involved in proliferation 25 . In mice, conditional expression of NFATc1 in β-cells promoted β-cell proliferation 62 . In another study, silencing of NFATc4 in mouse brains through in utero electroporation of siRNA reduced the number of cells in the S phase of the cell cycle of neural progenitor cells and the hypoxia-mediated neurosphere progenitor forming capacity in neural stem cells, indicating the involvement of NFAT signaling in cell proliferation 28 .
Hypoxia activates voltage-gated Ca 2+ channels (VGCC) 14,33 , which in turn may activate NFAT signaling and thus cell proliferation. However, in our studies, inhibiting the upstream intracellular Ca 2+ and calcineurin molecules in NFAT signaling did not differentially affect cell proliferation under normoxic and hypoxic conditions, although treatment with a Ca 2+ channel blocker did inhibit cell proliferation more efficiently under hypoxic conditions than normoxic conditions. A direct interaction of calcium channels and cell cycle proteins and kinases has previously been reported 40 . Thus, the observed effects of the Ca 2+ channel blocker on fibroblast proliferation may be due to the direct interaction of cell cycle proteins and kinases with the Ca 2+ channels.
In summary, we conclude that hypoxia-induced cell proliferation occurs via the activation of NFAT signaling through HIF-2α.

Methods
Cell culture. Primary human pulmonary fibroblasts (HPFs) isolated from a 74-year-old male Caucasian subject were purchased from PromoCell (Heidelberg, Germany, Cat. No: C-12361). They were cultured in Fibroblast Basal Medium (PromoCell, Cat. No: C-23220) with its recommended supplements (PromoCell, Cat. No: C-39320), including 0.02 ml/ml fetal calf serum, 1 ng/ml basic fibroblast growth factor and 5 µg/ml insulin. IPF fibroblasts LL29 and LL97A were isolated from a 26-year-old female Caucasian patient and a 48-year-old male Caucasian patient, respectively, and were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human normal lung fibroblasts CCD-13Lu and CCD-19Lu were isolated from a 71-year-old male Black patient with carcinoma and a 20-year-old Caucasian female, respectively, and were also purchased from ATCC. Human normal lung fibroblasts HLF153 and HLF154 and IPF fibroblasts IPF12 and IPF14 were isolated as previously described 11 . All of these cells were maintained in F12K medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Human embryonic kidney (HEK) epithelial 293 T cells were purchased from ATCC and maintained in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin.
ScienTific RepoRts | (2018) 8:2709 | DOI:10.1038/s41598-018-21073-x Hypoxia exposure. Cells were exposed to normoxia (21% O 2 ) in a normal CO 2 cell culture incubator or hypoxia (5% or 1% O 2 ) in a hypoxia chamber (Billups-rothenburg, Del Mar, CA). Sterile water was placed inside the chamber to maintain moist conditions. Hypoxic conditions were created by filling the chamber with 5% or 1% O 2 , 5% CO 2 and balanced N 2 . The gas was passed through the chamber at 1-2 psi for 3 min, and the chamber was sealed and placed in a 37 °C cell culture incubator. The oxygen concentration in the chamber was monitored using an oxygen sensor (OxyCheq, Marianna, FL). Every 2 days, media were replaced with fresh media that were degassed using a SpeedVac Concentrator system (Thermo Fisher Scientific, Waltham, MA).

Cell proliferation assay. Cell proliferation was determined using a BrdU assay (EMD Millipore, St
Charles, MO), which measures the incorporation of bromodeoxyuridine (BrdU), a thymidine analog of DNA. Fibroblasts were seeded at a density of 2,000 cells/well in 96-well plates. After 24 hrs, the medium was replaced with fresh medium and incubated under normoxic or hypoxic conditions for 3 or 6 days. Then, 3-12 hrs prior to the endpoint, BrdU was added and cells were fixed with fixing solution provided by the BrdU assay kit manufacturer. A BrdU detection antibody was added and incubated for 1 hr at room temperature. After washing, peroxidase-labeled goat anti-mouse IgG was added and incubated for 30 min at room temperature. After a final wash, substrate was added and incubated for 30 min in room temperature at dark. Then, stop solution was added, and the absorbance was measured at 450 nm using a Spectramax M2 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).
Cell viability. Cell viability was determined using an MTT assay kit (ATCC, Manassas, VA) or LDH assay (Thermo Scientific, Waltham, MA). Briefly, cells were cultured in 96-well plates at a density of 2,000 cells/well and exposed to normoxia or hypoxia for 3 or 6 days. For MTT assays, 10 µL of MTT reagent was added to the cells. After 4 hrs of incubation, 100 µL of detergent reagent was added. Plates were maintained at room temperature in the dark for 2 hrs, and the absorbance values were subsequently recorded at 570 nm. For LDH assay, 50 µL of the supernatant was transferred to new 96-well plates. Then, 50 µL of the reaction mixture (combination of substrate and assay buffer) was added to the supernatant, and plates were incubated at room temperature in the dark. After 30 min, 50 µL of stop solution was added, and the absorbance was measured at 490 nm and 680 nm. Absorbance values at 680 nm (background) were subtracted from absorbance values at 490 nm to obtain LDH activity. Briefly, HPF cells were seeded at a density of 100,000 cells/well in 6-well plates and serum-starved for 24 hrs. The following day, fresh medium with serum was added, and the cells were exposed to normoxic and hypoxic (1% O 2 ) conditions for 3 days. The cells were trypsinized, counted, fixed and analyzed according to the manufacturer's instructions.
Inhibitor studies. HPF cells were seeded at a density of 2,000 cells/well in 96-well plates. Then, 24 hrs after seeding, the medium was replaced with fresh medium with and without the following inhibitors at concentrations ranging from 0-100 µM: HIF-1α inhibitor KC7F2 (TOCRIS, Pittsburg, PA), HIF-2α inhibitor TC-S 7009 (TOCRIS, Pittsburg, PA), calcium chelator BAPTA-AM (Life Technologies, Carlsbad, CA), calcium channel blocker verapamil chloride (Sigma, St. Louis, MO), and calcineurin inhibitor cyclosporine A (CsA, R&D Systems, Minneapolis, MN). Cells were treated with these compounds and exposed to normoxic (21% O 2 ) or hypoxic conditions (1% O 2 ) for 3 days in the presence of the inhibitors for the entire 3 days. Separate sets of wells were maintained for media and solvent controls. Cell proliferation and cell viability were determined by BrdU assay (EMD Figure 8. Effects of NFAT signaling inhibitors on hypoxia-induced proliferation. HPFs were treated with a calcium chelator (BAPTA-AM) and calcineurin inhibitor (cyclosporine A, CsA) and a calcium channel blocker (verapamil hydrochloride) and exposed to normoxia (21% O 2 ) and hypoxia (1% O 2 ) for 3 days. (A,B and C) Cell proliferation as determined by BrdU assay. Cells were incubated with BrdU for 12 hrs. (D,E and F) Cell viability as determined by LDH assay. (G,H) HPFs were treated with the solvents used [1% DMSO and 0.1% Ethanol (EtOH)] and exposed to normoxia (21% O 2 ) and hypoxia (1% O 2 ) for 3 days. Cell proliferation and cell viability were determined by BrdU assay and LDH assay, respectively. Data were expressed as a percentage of the control (0 µM inhibitor) at each oxygen condition. The absorbance of control at normoxia for proliferation was 0.39 ± 0.06 (n = 3) and for hypoxia was 0.66 ± 0.16 (n = 3). Viability % was calculated as 100% -cytotoxicity %. Cytotoxicity % = [(LDH activity of sample -Spontaneous LDH activity)/(Maximum LDH activity -Spontaneous LDH activity)] × 100. The absorbance of control at normoxia for LDH activity was 0.17 (n = 2) and for hypoxia was 0.16 (n = 2). Maximum and spontaneous LDH activity values for normoxia were 0. 26  Quantitative real-time PCR. Fibroblasts were seeded at a density of 35,000 cells/well in 6-well plates and incubated at 37 °C overnight. The following day, after replacing the media with fresh media, cells were exposed to normoxia and hypoxia for 3 and 6 days. Total RNA was extracted using TRIzol Reagent (Molecular Research Center, Cincinnati, OH). One microgram of DNase-treated RNA was used for first-strand cDNA synthesis using random primers and Moloney Murine Leukemia Virus reverse transcriptase (Thermo Fisher Scientific, Waltham, MA). cDNA was diluted 100 times and then used for quantitative real-time PCR analysis using SYBR green (Eurogentec, Fremont, CA). β-actin was used as the reference gene. Forward and reverse primers for human NFTAc1-c4, NFAT5, and β-actin are listed in Table 1  Western blot. To analyze NFATc2 levels, HPF cells were cultured in 6-well plates at a density of 35,000 cells/ well and exposed to normoxia and hypoxia for 6 days. Proteins were extracted with RIPA buffer (Cell Signaling, Beverly, MA) containing 1X phosphatase and protease inhibitors (Thermo Fisher Scientific, Waltham, MA). Cell debris was removed by centrifugation (20,000 × g for 15 min) and supernatants were collected. Protein concentration was determined using a Bio-Rad (Hercules, CA) protein assay kit. Fifty-five µg of proteins were separated on 8% SDS PAGE gels for detecting NFTAc2 expression.
After being transferred to the membranes, the blots were blocked with 5% non-fat milk in Tris-Buffered Saline with Tween ® 20 (TBST) buffer. The following antibodies were added, and membranes were incubated at 4 °C over- green fluorescent protein (GFP)-positive cells were counted at 20 × magnification under a fluorescence microscope in 10-15 fields per well. For titer calculation, the average number of GFP-positive cells was multiplied by 594 (which is the number of fields in a well of a 12-well plate at 20X magnification) and divided by the volume of the medium in the well and the virus dilution.
Effect of NFATc2 silencing on hypoxia-mediated cell proliferation. HPF cells were seeded at a density of 2,000 cells/well in 96-well plates. Cells were infected with a lentivirus containing either vector control or NFATc2 shRNA at a multiplicity of infection (MOI) of 100 using a fibroblast growth medium containing polybrene (8 µg/mL, EMD Millipore, St Charles, MO). After 24 hrs, cells were examined under a fluorescence microscope for GFP to determine the infection efficiency. Then, the virus was removed and fresh media was added. The infected cells were exposed to normoxic and hypoxic conditions (1% O 2 ) for 3 and 6 days. Cell proliferation was measured using BrdU assay (EMD Millipore, St Charles, MO).
Immunofluorescence staining. HPF cells were cultured at 2,000 cells/well in 8-well glass chamber slides (EMD Millipore, St Charles, MO) and exposed to normoxia (21% O 2 ) and hypoxia (1% O 2 ) at 37 °C for 3 days. For positive controls, cells were treated with ionomycin (Sigma-Aldrich, St. Louis, MO,) at a concentration of 1 μg/mL for 24 hrs. For silencing HIF-1α and HIF-2α, the cells were infected with a shRNA lentivirus at an MOI of 100 for 24 hrs. After adding fresh media, cells were exposed to normoxia and hypoxia for 3 days. After incubation, cells were fixed with 10% formalin and permeabilized with 0.1% Trition-X-100. Rabbit polyclonal anti-NFATc2 antibodies (Santa Cruz biotechnology, Santa Cruz, CA) were added at a dilution of 1:50 and incubated at 37 °C for 1 hr. Alexa Flour 488-or 546-conjugated goat anti-rabbit secondary antibody (Life technologies, Carlsbad, CA) was added at a 1:300 dilution and incubated for 1 hr at 37 °C Nuclei were stained with Hoechst 33342 (Molecular probes, Waltham, MA) at a final concentration of 5 µg/mL. Images were captured by a digital camera and MetaVue software (Universal Imaging Co., Downingtown, PA) under a fluorescence microscope (Nikon, Melville, NY). Fields were randomly selected, and five images were taken for each condition. Nuclear NFATc2-positive cells and the total number of cells were counted in each field. NFATc2 nuclear-translocated cells were calculated as the percentage of nuclear NFATc2-positive cells relative to the total number of cells.