Extracellular Calcium-sensing Receptor Activation Induces Vitamin D Receptor Levels in Proximal Kidney HK-2G Cells by a Mechanism That Requires Phosphorylation of p38α MAPK*

In hypocalcaemia, elevated parathyroid hormone transitorily down-regulates the kidney vitamin D receptor, which returns to normal levels with the rise in serum extracellular calcium [Ca2+]e. In this study, we investigated the mechanism that underlies VDR increase in kidney in association with elevated [Ca2+]e. Examination of MAP kinase signals in a proximal tubule human kidney (HK-2G) epithelial cell line showed that treatment of [Ca2+]e in the culture medium elevated phosphorylation of both ERK and p38 MAPKs. Blockade of p38 phosphorylation with SB203580 or SB202190 in turn abolished [Ca2+]e-mediated VDR protein increase, while treatment with PD98059 and U0126, specifically blocked ERK phosphorylation, but had no effect on VDR stimulation by [Ca2+]e. Furthermore, SB203580 treatment potently repressed [Ca2+]e-mediated activation of VDR promoter. We also demonstrate that si-RNA knock down of p38α completely diminished high [Ca2+]e-mediated VDR induction. Direct CaSR involvement was demonstrated by using an si-RNA of CaSR that impeded [Ca2+]e-mediated induction of VDR. In conclusion, a high extracellular [Ca2+]e concentration in the physiological range is capable of directly increasing renal proximal VDR expression, and the induction mechanism requires activation of the CaSR and signal mediation by the p38α MAP kinase pathway.

In hypocalcaemia, elevated parathyroid hormone transitorily down-regulates the kidney vitamin D receptor, which returns to normal levels with the rise in serum extracellular calcium [Ca 2؉ ] e . In this study, we investigated the mechanism that underlies VDR increase in kidney in association with elevated [Ca 2؉ ] e . Examination of MAP kinase signals in a proximal tubule human kidney (HK-2G) epithelial cell line showed that treatment of [Ca 2؉ ] e in the culture medium elevated phosphorylation of both ERK and p38 MAPKs. Blockade of p38 phosphorylation with SB203580 or SB202190 in turn abolished [Ca 2؉ ] e -mediated VDR protein increase, while treatment with PD98059 and U0126, specifically blocked ERK phosphorylation, but had no effect on VDR stimulation by [Ca 2؉ ] e . Furthermore, SB203580 treatment potently repressed [Ca 2؉ ] e -mediated activation of VDR promoter. We also demonstrate that si-RNA knock down of p38␣ completely diminished high [Ca 2؉ ] e -mediated VDR induction. Direct CaSR involvement was demonstrated by using an si-RNA of CaSR that impeded [Ca 2؉ ] e -mediated induction of VDR. In conclusion, a high extracellular [Ca 2؉ ] e concentration in the physiological range is capable of directly increasing renal proximal VDR expression, and the induction mechanism requires activation of the CaSR and signal mediation by the p38␣ MAP kinase pathway.
Extracellular ionized calcium [Ca 2ϩ ] e is a critical mediator of cell signaling for the storage and release of both parathyroid hormone (PTH) 2 and calcitonin. Homeostasis of [Ca 2ϩ ] e is important to proper neuromuscular contractions, cellular integrity, and the deposition of mineral in skeletal structures (1). A multi-organ system coordinates and maintains [Ca 2ϩ ] e homeostasis within a narrow physiological range (2). Activation of the an extracellular calcium sensing receptor (CaSR) when [Ca 2ϩ ] e concentrations are high maintains the storage form of PTH in the parathyroid gland (3,4). Release of PTH from the parathyroid gland is a direct result of a decrease in serum [Ca 2ϩ ] e (5,6). PTH is a potent stimulus of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) biosynthesis in the kidney proximal convoluted tubule by its induction of 1␣-hydoxylation (7). Resultant increase in [Ca 2ϩ ] e by 1,25(OH) 2 D 3 regulates PTH at two levels; where CaSR signaling shuts down PTH secretion (8,9) and 1,25(OH) 2 D 3 through transactivation of its nuclear receptor (VDR) inhibits prepro-PTH transcription (10).
[Ca 2ϩ ] e is also a potent mediator of the balance between cellular proliferation and differentiation while VDR mediates biological functions of 1,25(OH) 2 D 3 and is expressed in many different cell types (11). Past work has focused on the role of VDR in mineral homeostasis, with VDR activators used mainly to treat hyperparathyroidism secondary to chronic kidney disease (12). A more recent perspective includes the use of vitamin D analogs in combination with calcimimetics and phosphate binders in the management of hyperparathyroid disorders (13). It is therefore important to identify the mechanisms whereby CaSR signaling and VDR activation coincide in the tissues that co-express these two receptors. We previously reported for the first time that [Ca 2ϩ ] e is a direct regulator of VDR in proximal human kidney HK-2G cells (14). The present study was designed to better explore the mechanism by which [Ca 2ϩ ] e influences VDR increase in these cells.
PTH exerts several actions in the renal proximal tubule that include control of phosphate transport and induction of 1,25(OH) 2 D 3 -1␣-hydroxylase (CYP27B1) mRNA (15)(16)(17). The receptor for PTH is a another member of the GPCR family capable of coordinating bidirectional signaling through pathways involving adenylate cyclase or activation of protein kinase C depending on the concentration of PTH and the specific cell type (18,19). In proximal tubule epithelial cells, PTH stimulates 1␣-hydroxylation, mediates 1,25(OH) 2 D 3 -24-hydroxylase (CYP24) down-regulation and represses 1,25(OH) 2 D 3 -receptor (VDR) transcription all by increasing cAMP and activating the cAMP-dependent response element-binding protein (CREB) (20 -22). However, the set of molecular mechanisms for transmitting and reversing these PTH effects is still unclear.
In addition to its more well studied effects in the distal tubule, reports demonstrate that CaSR also is expressed in the proximal tubule suggesting that [Ca 2ϩ ] e plays an as yet undefined role in proximal kidney physiology (23,24). Studies of inherited disorders of calcium sensing have established the central role of CaSR in calcium homeostasis (25). Dysregulation of CaSR may cause an irregular pattern of PTH secretion and excessive tubular calcium reabsorption (26). Because PTH indirectly influences [Ca 2ϩ ] e homeostasis largely through regulated synthesis of 1,25(OH) 2 D 3 in the kidney proximal tubule (27,28), we asked the question of whether [Ca 2ϩ ] e plays a direct role in the compensatory down-regulation of the vitamin D system in the proximal tubule.
The effect of [Ca 2ϩ ] e on renal vitamin metabolism has been addressed both in vitro (29 -31) and in vivo (32) by studying the regulation patterns of CYP27B1 or CYP24. The direct role of [Ca 2ϩ ] e in cell culture is minimal, but a correlation has been seen between the rise in serum [Ca 2ϩ ] e and the suppression of CYP27B1 (32). The CaSR has been shown to be involved in multiple mechanisms that include mitogen activated protein kinase (MAPK) intermediates and release of stored intracellular Ca [Ca 2ϩ ] i (33). These pathways are known for their roles in mediating increases in VDR (34,35) and renal VDR is a key determinant for the reciprocal regulation of CYP27B1 and CYP24 by PTH (36). It is therefore plausible to hypothesize that [Ca 2ϩ ] e is counter-regulatory to the effects of PTH in the proximal tubule by inducing VDR up-regulation (14).
In the present study, the mechanism regarding high [Ca 2ϩ ] emediated VDR up-regulation in HK-2G cells was explored. We found this effect to be associated with rapid activation of p38 MAPK and more specifically phosphorylation of p38␣ MAPK, but not the ERK MAPK signaling pathways. Thus, specific intracellular targets of [Ca 2ϩ ] e in proximal tubule epithelial cells were confirmed, establishing [Ca 2ϩ ] e as a potential trigger for the counter-regulatory effects by interaction with its CaSR.
Cell Culture-The human proximal kidney cell type (HK-2) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). This cell line was originally developed for the purpose of a well differentiated proximal kidney cell line derived from human adult kidney (37). To establish a human renal proximal cell model to study the mechanism by which PTH and high [Ca 2ϩ ] e influences VDR gene expression, HK-2 cells were further stably transfected with human PTHR1 and selected in G418 and made a stable clone (HK-2G) to study VDR and CYP27B1 expression in response to PTH (14). Cells were maintained in DMEM/F12 medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and grown at 37°C in a humidified 5% CO 2 atmosphere. Cells were passaged every 4 -5 days with 0.05% Versene and used for experimentation within 10 passages. Experiments were started by switching the medium to non-supplemented synthetic Ketatinocyte serum-free medium (Invitrogen) containing a minimal calcium concentration (0.02 mM), and antibiotics. In this medium, the cells could be manipulated with changes in calcium concentration. No effect on cell growth or viability during the experimental periods used were observed at concentrations of [Ca 2ϩ ] e at or below 3 mM. Treatments were as described in figure legends using calcium chloride as the active agent for [Ca 2ϩ ] e delivery. In addition to HK-2G cells, the study used the human proximal cell line HKC-8 obtained from Dr. Loraine Racusen, murine proximal MPCT and distal DKC-9 cells provided by Dr. Peter Friedman and human MG-63 osteosarcoma cells purchased from the ATCC.
Transfection and Luciferase Assay-For transient transfection experiments, HK-2G cells were seeded in 6-well dishes in DMEM/F12 plus 10% fetal bovine serum (1.5 ϫ 10 5 cells/well, ϳ80% confluent) and incubated overnight. The next day cells were transfected with 1 g of hVDR promoter fused to luciferase (from Dr. Sylvia Christakos (38) and 500 ng of ␤-galactosidase and 4 l of Lipofectamine (Invitrogen) in 1 ml of serum deficient DMEM/F12 per well. At 3 h post-transfection, media containing 10% fetal bovine serum replaced the transfection medium for the next 24 h. The following day, cells were serumstarved in DMEM/F12 overnight and pretreated for an hour with the MEK inhibitor, PD98059 (10 M) or the p38 MAPK inhibitor, SB203580 or SB202190 (10 M). Following incubations with the inhibitors, cells were treated with or without [Ca 2ϩ ] e . Lysates for luciferase assays were extracted 6 h after Ca treatment. The cells were washed in phosphate-buffered saline and lysed in 150 l of lysis buffer (1% Triton X-100, 15 mM MgSO 4 , 4 mM EGTA, 1 mM dithiothreitol, 25 mM glycyl-glycine) on ice. Extracted samples were subjected to a luciferase assay system (Promega, Madison, WI) in triplicate and measured using a luminometer (Applied Biosystem TR17, Foster City, CA). The ␤-galactosidase measurements were performed on a Spectramax plus 384 (Molecular Devices, Sunnyvale, CA). Luciferase activity was normalized to ␤-galactosidase activity.
For siRNA experiments, HK-2G cells were seeded in complete medium without antibiotics the day before the experiment in 6-well plates at a density of 1.5 ϫ 10 5 cells per well on the day of the experiment. Cells were transfected with 100 nM SMARTpool PLUS siRNA, specifically targeting either CaSR or p38␣, along with scrambled SMARTpool PLUS siControl (Dharmacon Inc., Lafayette, CA) using DharmaFECT 1 (Dharmacon Inc.) diluted in Opti-MEM I (Invitrogen) for 24 h. Transfectants were then incubated with fresh complete medium for a total time period of 48 or 72 h before protein extraction. For other experiments, siRNA transfected HK-2G cells were grown in complete medium for 48 h and serumstarved overnight, stimulated with or without [Ca 2ϩ ] e for 12 h.
Immunoprecipitation-Cells were lysed in 1ϫ lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , and 10 g/ml protease inhibitor mixture as described previously (39) with slight modification. Briefly, aliquots of cleared lysates containing 1 mg total proteins were pre-cleared with protein A/Gagarose beads and incubated with 2 g of p38, p38␣, p38␤, phospho-p38 antibodies overnight with gentle rocking at 4°C. Immune complexes were precipitated with protein A/G-agarose beads. For other experiments, protein lysates were incubated with immobilized phospho-p38 MAPK (Thr 180 /Tyr 182 ) mouse mAb beads overnight, and immune complexes containing beads were isolated by centrifugation. Washed and precipitated protein/bead complexes were boiled with 2ϫ SDS sample buffer and spun down the beads. The sample was loaded without beads on a 10% SDS-PAGE for Western blot analysis.
Western Blot Analysis-For the determination of ERK1/2 and p38 phosphorylation and VDR protein regulation, monolayers of HK-2G cells were grown on 6-well plates up to 80 -90% confluent. Cells were incubated for 18 h in serum-free DMEM/F12 medium with normal (1.2 mM) calcium. This medium was removed and replaced with same medium supplemented with a variable Ca concentration or the 3 mM [Ca 2ϩ ] e treatment as outlined in each experiment. Inhibitors were treated an hour before [Ca 2ϩ ] e treatment. At the end of incubation period, media was removed and the cells were washed with ice-cold phosphate-buffered saline and then lysed in MPER lysis buffer (Pierce) containing 10 l/ml of protease inhibitor mixture (Pierce). Cell lysates were centrifuged at 14,000 ϫ g to separate cell protein from cell debris. The soluble protein content was measured by a Bradford assay (Bio-Rad) and was stored at Ϫ70°C. After thawing, the cell lysates were combined with an equal volume of Laemmli sample buffer (Bio-Rad) heated at 95°C, and separated using a 10% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred electrophoretically to nitrocellulose membranes (Bio-Rad). After transfer, the membranes were blocked with 5% powdered nonfat milk in TBST (20 mM Tris-HCl, 137 mM NaCl, and 0.05% Tween 20) pH 8.0 for 1 h at room temperature. ERK1/2 and p38 MAPK phosphorylation and VDR induction were detected by immunoblotting using an overnight incubation with 1:1,000 dilution of rabbit/mouse monoclonal/polyclonal antiserum specific for phospho-ERK1/2, phospho-p38 MAPK and VDR. Blots were then washed in TBST 4 times for 10 min each at room temperature and then incubated at 1:10,000 for 1 h with Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) diluted in 5% nonfat milk in TBST. The membranes were washed with TBST again four times for 10 min each. After washing the membranes, bands were visualized by chemiluminescence according to the manufacturer's protocol for SuperSignal West Pico detection (Pierce). The same membrane was used after 30 min stripping for determination of ␤-tubulin band as loading control.
Statistical Analysis-Results were expressed as the mean Ϯ S.E., and significance was determined by analysis with two tailed Student's t test. Values sharing the superscript are significant at p Ͻ 0.05.

HK-2G Cell Model-In HK-2G cells, VDR protein is increased by a 3 mM dose of [Ca 2ϩ
] e at 1 h post-treatment, then peak at 12 h and be sustained beyond 24 h (14). VDR induction at 12 h was selected for the remaining experiments. The regulation of VDR in response to elevated [Ca 2ϩ ] e was examined in two additional proximal cell models (HK-2G plus human HKC-8 and murine MPCT), a distal cell model (murine  and an osteoblast cell model (human MG-63). Interestingly, HK-2G cells had the greatest response among the proximal cell models, whereas the DKC-8 distal cells and MG-63 osteoblast cells both displayed VDR decreases following 3 mM [Ca 2ϩ ] e exposure ( Fig. 1).
High [Ca 2ϩ ] e Activates Phosphorylation of ERK1/2 MAPK in HK-2G Cells-It has been shown that high levels of [Ca 2ϩ ] e causes rapid protein tyrosine phosphorylation and activation of ERK1/2 in both primary cells of the parathyroid gland and HEK-293 cells transfected with CaSR (33). The MAPKs are pleiotropic regulators of numerous cellular activities. It was our interest to examine the involvement of MAPKs in high [Ca 2ϩ ] emediated VDR up-regulation in HK-2G cells, in which CaSR was found to be endogenously expressed. To identify the signal transduction pathways involved in VDR up-regulation, we first examined whether ERK1/2 is phosphorylated by high [Ca 2ϩ ] e in HK-2G cells. Fig. 2 showed that high [Ca 2ϩ ] e increased phosphorylation of ERK1/2 in a time ( Fig. 2A) and dose-dependent manner (Fig. 2C), as assayed by use of a phospho-ERK1/2 specific polyclonal antibody. Fig. 2  MC3T3-E1 cell line (40). Therefore, we next examined whether p38 MAPK was activated by high [Ca 2ϩ ] e in HK-2G cells. Fig. 3 showed that high [Ca 2ϩ ] e increased phosphorylation of p38 MAPK in a time (Fig. 3, A and B) and dose-dependent manner (Fig. 3, C and D), as assayed by use of a phospho-p38 MAPK specific polyclonal antibody. The results demonstrate that 3 mM [Ca 2ϩ ] e treatment caused a rapid and cyclical activation of p38 MAPK ϳ2-3-fold at 0.5 min that subsided at 1 min and rose again at 5 and 10 min time intervals (Fig. 3, A and B). Fig. 3 (Fig. 4C). These results clearly indicate the involvment of the p38 MAPK pathway in [Ca 2ϩ ] e -mediated VDR up-regulation in HK-2G cells. To check if this regulation also occurs at the level of transcription of VDR, transfection experiments were done with minus 1500 to plus 60 bp (Ϫ1500/ϩ60) of the hVDR promoter fused to a luciferase gene (38) and subjected to the same treatments. Luciferase assay results revealed that pretreatment of SB203580 had a significant inhibitory effect both alone and further on high [Ca 2ϩ ] e -mediated VDR gene expression (Fig. 4D). These data plainly demonstrate that high [Ca 2ϩ ] e activates multiple MAPKs, but only activation of p38 MAPK is involved in VDR up-regulation, which occurred at the level of VDR gene transcription in HK-2G cells.
Phosphorylation of MKK3/6 and ATF2 by High [Ca 2ϩ ] e in HK-2G Cells-To demonstrate the involvement of p38 MAPK pathway in high [Ca 2ϩ ] e mediated up-regulation of VDR in HK-2G cells, we investigated additional upstream and downstream signals to p38 MAPK in this experiment. We tested whether the effect of high [Ca 2ϩ ] e involves phosphorylation of MAP kinase kinase-3/6 (MKK3/6) and activation of the transcription factor ATF2. Fig. 5, A and B demonstrate that MKK3/6 was phosphorylated quickly at 30 s, reached its maximum at 1 min and declined at 5 min, activated again at 10 min ] e and ERK1/2 phosphorylation (P-ERK1/2) was measured by Western blot using the phospho-ERK1/2-specific antiserum. Anti-ERK2 was used as the loading control. B, blots were scanned and P-ERK1/2 bands were normalized with loading control (ERK2) and plotted as fold increase with the control level set at 1 (n ϭ 1 blot). C and D, dose response of [Ca 2ϩ ] e (0 -4 mM) for 10 min on the activation P-ERK1/2 in HK-2G cells (n ϭ 2 blots). Equal amounts of protein from the whole cell lysates were analyzed in Western blots with anti P-ERK1/2. ERK2 was used as the loading control. ] e and p38 MAPK phosphorylation (P-p38) was measured by Western blot with the phospho-p38 MAPK-specific antiserum. Anti-ERK2 was used as the loading control. B, blots were scanned and P-p38 MAPK bands were normalized with loading control and plotted as fold increase with the control level set at 1 (*, p Յ 0.05; n ϭ 3 blots). ERK2 was used as the loading control. C and D, dose response of [Ca 2ϩ ] e (0 -4 mM) for 0.5 min on the activation P-p38 MAPK in HK-2G cells (***, p Յ 0.001; n ϭ 3 blots). ERK2 was used as the loading control. and declined at 30 min following high [Ca 2ϩ ] e treatment. Results also show that ATF2, a downstream effector molecule of p38 MAPK activation, had detectable phosphorylation at 0.5 min and sustained this elevated state of phosphorylation out to 10 min (Fig. 5, C and D).

CaSR Is Involved in High [Ca 2ϩ ] e -mediated Up-regulation of VDR in HK-2G Cells-In a previous study it was observed that
HK-2G cells express CaSR endogenously, and we showed that VDR could be increased in these cells by treatment of 20 mM gadolinium (Gd 3ϩ ) (14). Western blot results confirm that the CaSR agonist, Gd 3ϩ , dose-dependently activates VDR in HK-2G cells (Fig. 6A).
In several cell types, activation of MAPK via G-protein-coupled receptors has been reported to be sensitive to pertussis toxin (PTX) (41). We therefore examined the effects of PTX on high [Ca 2ϩ ] e induced VDR activation in HK-2G cells. Serumstarved cells were preincubated with PTX for 2 h both in presence and absence of [Ca 2ϩ ] e. Fig. 6B demonstrates that PTX did not inhibit VDR induction. This supports the involvement of the G q/11 coupling system in CaSR activated MAPK cascade in HK-2G cells. To further characterize the signaling mechanism that underlies CaSR-mediated VDR activation, we treated HK-2G cells with the phospholipase C (PLC) inhibitor, U73122. Pretreatment with 1 M of U73122 disrupted the catalytic activity of PLC (data not shown) and blocked the induction of VDR both in the presence and absence of high [Ca 2ϩ ] e (Fig. 6C).
siCaSR Accordingly, as it was observed that gadolinium potently induced VDR up-regulation via CaSR (Fig. 6A), it was confirmed that the gadolinium-mediated VDR up-regulation could also be diminished by knocking down CaSR using siRNA targeting CaSR. Fig. 7C clearly demonstrates that VDR induction was profoundly inhibited when endogenous expression of CaSR in HK-2G cells was knocked-down compared with a scrambled siControl treatment. Surprisingly, down-regulation of endogenous expression of CaSR caused an increase in phosphorylation of ERK1/2, that we were not able to explain.

Activation of p38␣ Not p38␤ MAPK Associated with High [Ca 2ϩ ] e -mediated VDR Up-regulation in HK-2G Cells-To further confirm that high [Ca 2ϩ
] e -mediated VDR up-regulation in HK-2G cells is associated with activation of specific isoform of p38 MAPK, we used a selective p38␣ and p38␤ MAPK specific pharmacological agent, SB202190 (42). Results demonstrated that pretreatment of this agent completely abolished [Ca 2ϩ ] emediated VDR up-regulation in HK-2G cells (Fig. 8A), indicating that activation of one or both p38␣ In C, serum-deprived HK-2G cells were pretreated 1 h with or without the p38 MAPK inhibitor, SB203580 (10 M). In each experiment, the cells also were treated in the presence and absence of 3 mM [Ca 2ϩ ] e for 12 h, or as a combination of calcium treatment and the respective inhibitor. Equal amounts of protein from the whole cell lysates were analyzed in Western blots with anti VDR, anti P-p38. Anti-␤-tubulin was used as the loading control (n ϭ 3 blots). In D, a VDR promoter-luciferase construct was transfected along with ␤-galactosidase plasmid in HK-2G cells for 24 h and followed by overnight serum deprivation. The serum-depleted cells were pretreated 1 h with or without the p38 MAPK inhibitor, SB203580 (10 M) and treated in combination with or without 3 mM [Ca 2ϩ ] e for 6 h. VDR promoter levels were measured as a ratio of luciferase/␤-galactosidase and plotted as relative light units (RLU) normalized to ␤-galactosidase (**, p Յ 0.01 between Cnt and Ca-treatment; whereas † equals p Յ 0.05 between SB203580 & SB203580 plus Ca; n ϭ 4 replicates). and ATF2 phosphorylation (P-ATF2) was detected using anti-ATF2 antibodies (C). Anti-␤-tubulin antibody was used as loading control. Blots were scanned and P-MKK3/6 (B) and P-ATF2 (D) bands were normalized with loading control (␤-tubulin) and plotted as fold increase with the control level set at 1 (*, p Յ 0.05; **, p Յ 0.01; n ϭ 3 blots for each experiment). and p38␤ MAPK isoforms are involved in [Ca 2ϩ ] e -mediated VDR up-regulation. Additional experiments were done to determine if a specific single isoform of p38 MAPK is activated in response to high [Ca 2ϩ ] e in HK-2G cells. Total p38 MAPK was first immunoprecipitated with a pan p38 antibody, and immunoblotted with antibodies specific for p38␣ and p38␤ (Fig. 8B). This showed that HK-2G cells expressed both isoforms of p38 and [Ca 2ϩ ] e treatment did not affect the total level of either p38␣ or p38␤. The increase in p38␣ phosphorylation in high [Ca 2ϩ ] e -activated cells was confirmed by immunoprecipitation with specific antibodies against p38␣ or p38␤ and subsequent immunoblot analysis with phospho-p38 antibody (Fig. 8C). We further confirmed that high [Ca 2ϩ ] e treatment can induce the specific activation of p38␣ MAPK in HK-2G cells. In this experiment, total phospho-p38 was immunoprecipitated using immobilized phospho-p38 MAPK (Thr 180 / Tyr 182 ) mouse mAb followed by an immunoblot with antibodies specific for p38␣ and p38␤ (Fig. 8D). These data clearly demonstrated that high [Ca 2ϩ ] e treatment activates p38␣, but not p38␤ MAPK and that this activation of p38␣ is linked to up-regulation of VDR HK-2G cells.
si-p38␣ MAPK Abrogates High [Ca 2ϩ ] e -mediated Up-regulation VDR in HK-2G Cells-To elucidate the regulation of VDR signaling by p38␣ MAPK isoforms in HK-2G cells, we inhibited p38␣ expression by using siRNA. High [Ca 2ϩ ] e does not affect the level of total p38␣ in siControl cells, but expression of total p38␣ was abrogated in si-p38␣-treated cells (Fig. 8E, middle  panel). As expected, high [Ca 2ϩ ] e -mediated up-regulation of VDR was inhibited in si-p38␣-treated cells (Fig. 8E). These data illustrate that activation of p38␣, not p38␤ MAPK, is associated with high [Ca 2ϩ ] e -mediated VDR up-regulation in HK-2G cells.

DISCUSSION
In this study we sought to determine whether high [Ca 2ϩ ] e treatment could stimulate VDR in a kidney proximal epithelial cell model and to identify the receptor and signaling pathways mediating this effect. Here, the data suggest that VDR is a potential on/off switch for regulating CYP27B1. The proximal tubule is the main site of regulated CYP27B1 expression and synthesis of 1,25(OH) 2 D 3 (43,44). PTH regulates its effects on CYP27B1 by elevating cAMP and activation of CREB followed by additional transcriptional modification at the CYP27B1 promoter (20,45). Loss of VDR by PTH probably relieves repression of CYP27B1 and this loss would appear to be corrected by the eventual rise in [Ca 2ϩ ] e and CaSRmediated activation of p38 in renal proximal cells. It is also well established that 1,25(OH) 2 D 3 activates its own break down by induction of CYP24  and 72 h. Following siRNA treatment whole cells lysates were analyzed by Western blot. Membranes were probed with primary antibody against CaSR. Stripped blots were re-probed with ␤-tubulin as loading control. B, 48 h siRNA-transfected HK2G cells from A, were serum-deprived overnight, stimulated with or without [Ca 2ϩ ] e for 12 h. Western blot was done using VDR monoclonal antibody and ␤-tubulin was used as loading control. In C, knocking down endogenous expression of CaSR diminished gadolinium (Gd 3ϩ )-mediated VDR up-regulation. HK-2G cells were transfected with 100 nM SMARTpool Plus siRNA targeted to CaSR or scrambled siControl for 24 h. Following transfection cells were serum-deprived for 16 h and treated with 3 mM [Ca 2ϩ ] e or Gd 3ϩ (100 ng/ml) for 12 h. Whole cell lysates were prepared, and equal amounts of protein were used for Western blot analysis. Blots were probed with monoclonal antibodies raised against VDR, phospho ERK1/2, and phospho-p38. Equal loading was checked by re-probing the same blots with either ␤-tubulin or ERK2 antibodies, as indicated. expression (30) and simultaneously down-regulation of CYP27B1 expression (30,46). The molecular details of these events are just beginning to unfold, but both of these effects by 1,25(OH) 2 D 3 require VDR (47). In the case of CYP27B1, the regulation by 1,25(OH) 2 D 3 might be indirect because a classical VDRE is absent in the first 1500 bp of CYP27B1 promoter, while suppressive effects on this promoter segment still occur via ligand-mediated transrepression via a novel negative VDRE (48). The mechanism for the transrepressive regulation appears to involve recruitment of acetylated histones and methylation (49). The phosphaturic molecule, fibroblast growth factor-23 (FGF-23), is a secondary repressive mechanism on CYP27B1, where FGF-23 induction from bone tissue is under 1,25(OH) 2 D 3 control (50). Based on this finding and the data of this study, Ca/VDR/1,25(OH) 2 D 3 and FGF-23 represent distinct mechanisms for controlling CYP27B1. Whether or not [Ca 2ϩ ] e can directly repress CYP27B1 will require more data.
The HK-2G model used in this study was previously demonstrated to be responsive to PTH, which down-regulated VDR (14). This prior study suggested that high [Ca 2ϩ ] e treatment in HK-2G cells is counter-regulatory to the effects of PTH on VDR. These in vitro changes in VDR mirror the exact scenario that is reflective of in vivo renal VDR regulation that occur during hypo-and hypercalcemia conditions (47,51,52). It will be interesting to examine the dual impact of Ca versus PTH signaling in HK-2G cells with regard to molecular components of renal vitamin D metabolism.
Importantly, the concentration of [Ca 2ϩ ] e used to study signaling and VDR induction in HK-2G cells was within the physiological range of 3 mM indicating that activation of CaSR was likely. Furthermore, use of calcium channel blockers did not preclude the [Ca 2ϩ ] e -mediated regulation of VDR (data not shown), strengthening the hypothesis for CaSR involvement. CaSR activation in other cell models has been demonstrated to result in ERK phosphorylation that is tied to effects of [Ca 2ϩ ] e on targets in the central nervous system and cell survival and proliferation (53)(54)(55)(56). Thus, it was reasonable to examine this signaling pathway in the context of VDR regulation. The results in Figs. 3 and 5 demonstrate that [Ca 2ϩ ] e -mediated ERK phosphorylation does occur in the HK-2G cells, but blocking ERK phosphorylation failed to confirm it plays a direct role in VDR up-regulation, as was the case with p38 phosphorylation. Nevertheless, ERK phosphorylation seems to have some involvement with regulating HK-2G cell VDR levels since levels of phospho-ERK are dramatically increased when CaSR is knocked-down by siRNA treatment (Fig. 8). We also acquired data (not shown) indicating an up-regulation of VDR promoter in the presence of the PD98059 inhibitor of phospho-ERK, but these results cannot be explained in the framework of the present study.
The most significant effect of [Ca 2ϩ ] e in the HK-2G model was rapid and highly significant phosphoactivation of p38 that was critical to the induction of VDR (see Figs. 3C and 4D). Numerous reports also demonstrate the ability of CaSR activation to lead to an increase of phospho-p38 (56 -58) that results in changes in PTHrP release during humoral hypercalcemia, for example. As well, the repression of VDR promoter activity by treatment with SB203580 emphasizes an important role of phospho-p38 to maintain constitutive VDR expression in the HK-2G cell type. We further dissected the p38 MAPK signaling in regulating high [Ca 2ϩ ] e -mediated VDR up-regulation in HK-2G cells. Using another specific pharmacological inhibitor that affects both p38␣, p38␤ isoforms, SB202190; and using an siRNA specifically targeting p38␣, we demonstrated that p38␣, not the p38␤ MAPK, is involved in high [Ca 2ϩ ] e -mediated VDR regulation in HK-2G cells.
SB inhibitors are pyridinyl immidazole compounds have been widely used in investigation of the biological function of p38 MAPK (30). These compounds are also well known for their ability to inhibit TGF␤ receptor activation (59). TGF␤ is an important regulator of 1,25(OH) 2 D 3 -mediated VDR trans- Cell extracts were immunoprecipitated with a pan anti-p38 antibody and immunoblotted with specific antibodies for either p38␣ or p38␤. C, high [Ca 2ϩ ] e specifically activates p38␣ MAPK isoform in HK-2G cells. High [Ca 2ϩ ] e induced activation of p38␣ was detected by immunoprecipitating phosphorylated p38 using phospho-specific anti-p38 antibody and immunoblotting with antibodies specific for p38␣ (blot 1) or p38␤ (blot 2). D, induced activation of p38␣ MAPK by high [Ca 2ϩ ] e was further confirmed by immunoprecipitating total phosphorylated p38 using an immobilized anti-phospho-p38 MAPK (Thr 180 /Tyr 182 ) mAb followed by immunoblotting with antibodies specific for p38␣ and p38␤ MAPK isoforms. E, specific knock-down of p38␣ MAPK with a targeted siRNA blocks high [Ca 2ϩ ] e -mediated up-regulation of VDR in HK-2G cells. HK-2G cells were transfected with 100 nM SMARTpool Plus si-p38␣ MAPK or scrambled SMARTpool siRNA control for 48 h. Cells were serum-starved overnight and stimulated with or without [Ca 2ϩ ] e treatment for 12 h. Cell extracts were used for Western blot analysis using VDR, anti-p38␣ and anti-tubulin antibodies. The expression of p38␣ is selectively abrogated in si-p38␣ treated cells (middle panel) which in turn abolished high [Ca 2ϩ ] e -mediated VDR increase (top panel). activation on target genes (60). SB203580 belongs to a class of pyridinyl imidazoles that inhibits the stress-activated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38 beta 2 but not other mitogen-activated protein kinase family members. As with inhibitors of other protein kinases, SB203580 binds in the ATP-binding pocket of SAPK2a/p38␣. The type I TGF␤ receptor, which has serine at the position equivalent to Thr 106 of SAPK2a/p38␣ and SAPK2b/p38␤, is inhibited by SB203580. In the present study, 1,25(OH) 2 D 3 effects were not studied and treatment of HK-2G cells with the SB inhibitors was only used to demonstrate the involvement of p38 in [Ca 2ϩ ] e -mediated increase in VDR. Control of this effect by [Ca 2ϩ ] e was confirmed to involve transcriptional regulation of VDR itself. We showed that activation of the upstream intermediate of p38, MKK3/6, and p38-mediated downstream transcriptional control by ATF2 were both involved. Thus, we can rule out the interference of TGF␤ in [Ca 2ϩ ] e -mediated VDR gene expression.
During this study, we did not have access to pharmaceutical CaSR inhibitors or the CaSR mimetic drugs; we therefore used siRNA to knock down CaSR expression to examine its intermediary effect on VDR induction. A SMARTpool PLUS siRNA specifically and effectively targeted the CaSR in HK-2G cells and dramatically inhibited CaSR protein expression. This molecular tool, which includes a combination of 3 siRNA constructs, was successfully utilized to demonstrate the involvement of CaSR in both the Ga 3ϩ -and [Ca 2ϩ ] e -mediated inductions of VDR in proximal cells, respectively (see Fig. 7C). Further study will be needed to determine which actual si-construct(s) was responsible for the interference signal against CaSR. A recent report published by Rodriguez et al., (61) demonstrated a positive inductive action of the calcimimetic R-568 on VDR levels in the parathyroid gland (PTG). Given that Garfia et al., (62) have shown a positive correlation of PTG VDR with exposure to [Ca 2ϩ ] e in the PTG and an inverse regulation of VDR with blood PTH in PTG, it is now plausible to predict that the proximal convoluted tubule and the PTG share common effecters for coupling of G-protein, respectively, for Ca and PTH signaling in these cell types. These findings provide strong rationale for the use of potent calcimimetic drugs that specifically target the CaSR for treatment of chronic kidney disease and severe secondary hyperparathyroidism. Caution should be applied if it can be determined that CaSR activation leads to weakened 1,25(OH) 2 D 3 responsiveness in distal convoluted tubules, bone cells and extrarenal tissues where [Ca 2ϩ ] e signaling may decrease cellular VDR concentrations (see Fig. 1). Osteoblasts were previously reported to express functional CaSR, and also to sense fluctuations in local [Ca 2ϩ ] e resulting in induced osteoblast activity (63).
Additionally, the present study indicates that the CaSR utilizes G q/11 for coupling to its major signaling pathways. The data in Fig. 7 demonstrated that knocking down endogenous CaSR, resulted in suppression of VDR in response to high [Ca 2ϩ ] e , while activating CaSR with gadolinium, a known polycationic agonist of CaSR, increased VDR protein levels. This confirms a direct role of CaSR to mediate [Ca 2ϩ ] e induced up-regulation of VDR in proximal kidney cells. Thus in a high [Ca 2ϩ ] e condition (Fig. 9), CaSR couples to the G q/11 system, leading to PLC activation and phosphorylation of p38␣ MAPK, which in turn may activate ATF2 to promote the gene expression of VDR. This last step is currently under investigation (Fig. 9).
In conclusion, high extracellular Ca 2ϩ plays a critical role in up-regulating renal proximal cell VDR through a mechanism that involves activation of p38␣ MAPK. The data herein demonstrate that CaSR signaling is key to the mechanism of VDR increase in HK-2G cells. Our data suggest that serum [Ca 2ϩ ] e concentrations may be counter-regulatory to actions of PTH in renal proximal cells.  FIGURE 9. Proposed model for mechanisms underlying CaSR-induced activation of VDR in HK-2G cells. Activation of the seven transmembrane spanning CaSR by [Ca 2ϩ ] e treatment or with Gd 3ϩ as CaSR-specific agonist, results in a PTX-insensitive, presumably G q/11 -mediated activation of PLC and resultant stimulation of the p-38 MAPK cascade. Activation of ERK1/2 is not linked to VDR induction while phosphorylation of p38, specifically p38␣, causes activation of ATF2, and results in VDR up-regulation.