Nongenotropic, Anti-Apoptotic Signaling of 1α,25(OH)2-Vitamin D3 and Analogs through the Ligand Binding Domain of the Vitamin D Receptor in Osteoblasts and Osteocytes

Because sex steroids regulate the life span of bone cells by modulating cytoplasmic kinase activity via a nongenotropic action of their classical receptors, we have explored the possibility that the vitamin D nuclear receptor (VDR) might exhibit similar nongenotropic actions. We report that the conformationally flexible full VDR agonist, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), and the 6-s-cis-locked 1α,25(OH)2-lumisterol3 (JN) analog, also acting through the VDR but with poor transcriptional activity, protected murine osteoblastic or osteocytic cells from apoptosis. This effect was reproduced in HeLa cells transiently transfected with either wild type VDR or a mutant consisting of only the VDR ligand binding domain. The VDR ligand binding domain bound [3H]1α,25(OH)2D3 as effectively as wild type VDR but did not induce vitamin D response element-mediated transcription. The anti-apoptotic effects of 1α,25(OH)2D3 and the 6-s-cis-locked 1α,25(OH)2-lumisterol3 analog in calvaria cells were blocked by three cytoplasmic kinase inhibitors: Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), phosphatidylinositol 3 kinase inhibitor Wortmannin, and the JNK kinase inhibitor SP600125. However, inhibition of p38 with SB203580 or ERK with either U0126 or a transfected dominant negative MEK did not interfere with these anti-apoptotic actions. Further, 1α,25(OH)2D3 induced rapid (5 min) association of VDR with Src kinase in OB-6 cells. Finally, actinomycin D or cycloheximide prevented the anti-apoptotic effect of 1α,25(OH)2D3, indicating that transcriptional events are also required. These findings suggest that nongenotropic modulation of kinase activity is also a general property of the VDR and that ligands that activate nongenotropic signals, but lack transcriptional activity, display different biological profiles from the steroid hormone 1α,25(OH)2D3.

Changes in the birth or death of osteoblasts and/or osteoclasts represent fundamental pathophysiologic changes in most acquired metabolic bone diseases, including the osteoporosis that results from sex steroid deficiency, glucocorticoid excess, or old age (1)(2)(3)(4)(5)(6). Furthermore, pharmacotherapeutics used commonly for the treatment of metabolic bone diseases exert their beneficial effects on bone by regulating the rate of birth of new osteoclasts or osteoblasts or their apoptosis (6 -8).
We have recently shown that estrogens and androgens, acting via their classical nuclear receptors (ER␣, 1 ER␤, or AR), attenuate the apoptosis of several different cell types, including osteoblasts and osteocytes, by rapidly activating the Src/Shc/ ERK and phosphatidylinositol 3-kinase (PI3K) and down-regulating the JNK signaling pathways. This effect requires only the ligand binding, not the DNA binding, domain of the receptor, and, unlike its classical transcriptional action, it is eliminated by nuclear targeting of the receptor (9). Activation of ERKs leads to the rapid translocation of the kinases into the nucleus where they phosphorylate common transcription factors like Elk-1, CCAAT enhancer-binding protein-␤, and cAMPresponse element-binding protein. These transcription factors in turn up-regulate gene expression, as exemplified by the up-regulation of the early growth response-1 protein gene, an ERK/serum response element target gene. Likewise, suppression of the JNK signaling cascade by sex steroids leads to downregulation of c-Jun expression (10). We have earlier used a ligand that potently and selectively activates nongenotropic actions of the classical ER or AR and thereby activates kinases and their downstream transcription factors and target genes with only minimal effects on classical estrogen response element-mediated genotropic transcription. Furthermore, we have demonstrated that, although such classical genotropic actions of sex steroid receptors are essential for their effects on reproductive tissues, they are dispensable for their bone protective effects (2).
In our earlier studies, we demonstrated that 1␣,25(OH) 2 D 3 prevented apoptosis of HeLa cells that were transfected either with the VDR or the retinoic acid receptor (RXR), but not in untransfected cells or cells transfected with the ER␣ or the AR (9). 1␣,25(OH) 2 D 3 has also been shown to inhibit ultraviolet B-induced apoptosis and Jun kinase activation in human keratinocytes (21) and also to up-regulate Bcl-2 expression and protect thyrocytes from apoptosis (22). In the present study, we have followed up these preliminary observations and show that 1␣,25(OH) 2 D 3 , as well as other natural metabolites of vitamin D and synthetic analogs of 1␣,25(OH) 2 D 3 incapable of inducing vitamin D-responsive element (VDRE)-mediated transcription, attenuates osteoblast and osteocyte apoptosis. Cell Cultures-Osteoblastic cells were isolated from neonatal murine calvaria and cultured as previously described (3). OB-6 cells, HeLa cells, and osteocytic MLOY-4 cells were cultured as previously described (2)(3)(4)10). Osteoblastic and osteocytic cells were treated at 80% confluence with either vehicle or the appropriate kinase inhibitor for 30 min prior to the addition of 1␣,25(OH) 2 D 3 or the indicated 1␣,25(OH) 2 D 3 metabolites or its synthetic analogs for an additional 1 h in the presence of 10% serum. To induce apoptosis, etoposide was added to the cells (final concentration of 50 and 100 M for calvaria or MLO-Y4 cells and OB-6 cells, respectively), and the cultures were continued for 6 h. CV-1 monkey kidney cells were seeded at 1.5 ϫ 10 6 cells/150-mm culture dishes and cultured in minimal Eagle's medium with Earl's buffered salts and non-essential amino acids (Mediatech Inc., Herndon, VA) with 10% fetal bovine serum (Mediatech Inc., Herndon, VA). Cos-1 monkey kidney cells were seeded at 1 ϫ 10 6 cells/150-mm culture dishes in Dulbecco's modified Eagle's medium (Mediatech Inc.) with 10% fetal bovine serum.
Plasmids-pcDNA plasmid was purchased from Invitrogen. The human VDR and RXR plasmids were obtained from D. McDonnell (Duke University, Durham, NC) and D. Mangelsdorf (University of Texas, Southwestern Medical Center, Dallas, TX), respectively. The nuclear green fluorescent protein (GFP) was obtained by attaching the SV40 large T antigen nuclear localization sequence to the N terminus of the cDNA construct encoding GFP (7). Wild type ER␣ was obtained from B. Katzenellenbogen (University of Illinois, Urbana, IL). The wild type and dominant negative MEK plasmids were donated by Natalie Ahn from the University of Colorado at Boulder (23). The VDRE-secreted alkaline phosphatase (SEAP) reporter was constructed by removing the promoter and untranslated regions from the osteocalcin VDRE-human growth hormone reporter and cloning into the pSEAP2-Basic plasmid (Stratagene, Palo Alto, CA) (24). This reporter contains the osteocalcin gene VDRE (235-219 bp with respect to the transcription start site) and the calcitonin promoter, having both the Octomer element (167-149 bp) and the Sp1 element (81-76 bp). An N-terminal fluorescent tag was positioned in-frame with the wtVDR or VDR ligand binding domain (LBD-VDR) by using PCR-generated restriction enzyme-flanked inserts included into the pECFP-Nuc plasmid (Stratagene). The full-length wtVDR (residues 4 -427) was inserted into pECFP-Nuc, and the nuclear localization signal was removed. The LBD-VDR residues 119 -427 were inserted into the pECFP-NUC plasmid, and the nuclear localization signal was also removed. Expression of the plasmid constructs was confirmed by fluorescent microscopy.
Transient Transfections-HeLa cells were transiently transfected at 80% confluency with 0.1 g of nuclear GFP and 0.1 g of either ER␣, wtVDR, VDR 119-C, RXR, or pCDNA using Lipofectamine Plus (Invitrogen). Cells were allowed to recover overnight and then treated with vehicle, 1␣,25(OH) 2 D 3 , or the indicated 1␣,25(OH) 2 D 3 analog 1 h prior to exposure to stimulation of apoptosis by etoposide. Subsequently, etoposide was added to a final concentration of 100 M, and the cultures were continued (in the presence of the D-compounds or vehicle) for an additional 6 h. Transient transfection of CV-1 cells was performed at 60% confluency and involved 10 min of pretreatment with 0.2 mg/ml DEAE-dextran (Sigma) in phosphate-buffered saline. Pretreated cells were washed in phosphate-buffered saline and incubated for 30 min with phosphate-buffered saline containing 0.1 g/well pcDNA3, wt hVDR, or the appropriate mutants of hVDR and 0.5 g/well of the VDRE-Luc reporter. Transfected cells were incubated in 80 M chloroquine in minimal Eagle's medium with Earl's buffered salts and nonessential amino acids (Mediatech Inc., Herndon, VA) with 4.5% charcoal-stripped fetal bovine serum for 4 h followed by the same culture medium without chloroquine for 24 h. Twenty-eight hours after transfection, the cell medium was replaced with the same medium containing vehicle or 1␣,25(OH) 2 D 3 . Twenty-two hours later, cell medium was harvested to measure secreted alkaline phosphatase using the Phospha-Light TM kit (Tropix, Bedford, MA). The data are presented as dose response curves of the reporter with raw luminometer units plotted versus the log dose of ligand. All experiments were carried out on quadruplicate samples with data expressed as the mean Ϯ S.E.
Quantification of Apoptotic Cells in Vitro-HeLa cell apoptosis was quantified by direct visualization of changes in nuclear morphology as previously described (6,9,10,25). Visualization of pyknotic or fragmented nuclei was facilitated by co-transfections of cells plated on glass coverslips with nuclear GFP. The percentage of apoptosis was determined by determining the nuclear morphology in 200 -500 transfected (fluorescent) cells. Apoptosis of calvaria cells, OB-6 osteoblastic cells, and MLO-Y4 osteocytes was quantified by trypan blue staining.
Equilibrium Saturation Binding Assay-Cos-1 cells were grown to 80% confluency and were then transfected with 3 g of pcDNA3, wt hVDR, or the appropriate mutants of hVDR as described for CV-1 cells. Sixty-six hours following transfection, cells were washed twice in phosphate-buffered saline and harvested by scraping in TED (10 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.4) hypotonic buffer with Complete EDTA-free protease inhibitor mixture (Roche Diagnostics). Cos-1 cell lysate (0.2 ml) was incubated with increasing concentrations of [ 3 H]1␣,25(OH) 2 D 3 106 Ci/mmol in the presence or absence of excess nonradioactive 1␣,25(OH) 2 D 3 for 4 h at 0°C. Samples were performed in triplicate. After equilibrium was established, 200 l of 50% hydroxyapatite solution in TED was added and washed three times in 1 ml of TED plus 0.5% Triton-X100. Bound [ 3 H]1␣,25(OH) 2 D 3 was eluted with 1 ml of ethanol, and the tritium content was determined in 7 ml of Liquiscint TM scintillation mixture using a Beckman LS6500. The dissociation constant K D for binding of 1␣,25(OH) 2 D 3 to a given VDR construct was determined by non-linear regression of the curve generated by plotting specific binding versus concentration of [ 3 H]1␣,25(OH) 2 D 3 using a one-site binding equation (26).
Immunoprecipitation of the Src/VDR Complex and Western Blot Analysis-OB-6 osteoblastic cells were treated with 1␣,25(OH) 2 D 3 for the indicated periods of time, and cells were lyzed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 5 g/ml leupeptin, 0.14 units/ml aprotinin, 10 mM NaF, 1 mM NA orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. Cell lysates were collected and were either used to determine total protein concentration with the Bio-Rad 500 -0001 protein assay kit or were incubated with a rabbit polyclonal anti-VDR antibody N-20 (SC-1009; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. An equal amount of goat anti-rabbit IgG antibody (SC-2004; Santa Cruz Biotechnology) was added to the lysate for 30 min, followed by the addition of 50% suspension of protein G-Sepharose for 30 min. Samples were centrifuged, and the pellets were size fractionated with SDS-PAGE on a 10% gel. Immunoblotting was performed using a mouse monoclonal anti-v-Src antibody (1:2000, catalogue number OP07; Oncogene Research Products, San Diego, CA). The primary antibody was detected with a horseradish peroxidase-conjugated secondary antibody (1:3000; Santa Cruz Biotechnology) and enhanced chemiluminescence using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The phosphorylation status of ERK1/2 in OB-6 cells was analyzed by immunoblotting. The antibodies used were a mouse monoclonal antibody recognizing tyrosine-phosphorylated ERK1/2 and a rabbit polyclonal antibody recognizing total ERK1/2 (Santa Cruz Biotechnology).
Statistical Analysis-The data were analyzed by analysis of variance, and the Student-Newman-Kleuss method was used to estimate the level of significance of differences between means.

Attenuation of HeLa and Calvaria Cell Apoptosis by 1␣,25(OH) 2 D 3 and Other
Vitamin D Metabolites-To confirm our earlier observations indicating that 1␣,25(OH) 2 D 3 attenuates apoptosis by a VDR-or RXR-dependent mechanism, we performed a dose response study of the effects of 1␣,25(OH) 2 D 3 in HeLa cells transiently transfected with VDR or RXR. Pretreatment of the cells for 1 h with the hormone dose dependently attenuated HeLa cell apoptosis induced by 6 h of treatment with etoposide (Fig. 1A). As in our earlier studies, 1␣,25(OH) 2 D 3 had no effect on the apoptosis of HeLa cells transfected with an empty vector. In full support of the findings for the HeLa cells, 1␣,25(OH) 2 D 3 as well as 24,25(OH) 2 D 3 or 25(OH)D 3 , but not vehicle, protected primary cultures of osteoblastic cells derived from murine calvaria from apoptosis induced by etoposide. As in the case of the HeLa cells, this effect was dose dependent at concentrations ranging from 10 Ϫ12 to 10 Ϫ7 M (Fig. 1B). The anti-apoptotic effect of 1␣,25(OH) 2 D 3 (at 10 Ϫ8 M) was reproduced in both the OB-6 osteoblastic murine cell line and murine MLO-Y4 osteocytic cells, and it was comparable with the anti-apoptotic effect of estradiol in these cell types (Fig. 1C).
Synthetic Vitamin D Analogs Exhibit Potent Anti-apoptotic Effects That Are Independent of Their Relative Binding Affinity for the VDR-Numerous synthetic 1␣,25(OH) 2 D 3 analogs have been previously classified as genotropic or nongenotropic based on their ability to induce the transcription of VDRE-containing genes like osteocalcin via VDR-VDRE interactions or to induce rapid calcium influxes in the intestine and numerous isolated cell types, via rapid phosphorylation of ERKs (13). Using six such synthetic analogs ( Fig. 2 and Table I), we proceeded to examine whether the anti-apoptotic effects of 1␣,25(OH) 2 D 3 result from genotropic or nongenotropic actions of the hormone. As shown in Fig. 3A, the 6-s-cis-locked analog JN prevented etoposide-induced apoptosis in a dose-dependent manner very similar to the dose response curve for 1␣,25(OH) 2 D 3 (Fig. 1A). Another 6-s-cis-locked analog, JM, also prevented etoposideinduced apoptosis of calvaria cells as effectively as JN and 1␣,25(OH) 2 D 3 (Fig. 3B). Both analogs are potent agonists for the rapid membrane-mediated effects of 1␣,25(OH) 2 D 3 but associate poorly with the classical VDR under equilibrium binding conditions (13,17,27). Additionally, the analog HL, an epimer of 1␣,25(OH) 2 D 3 (28), which is an antagonist of many membrane-initiated rapid effects of 1␣,25(OH) 2 D 3 , but not genomic effects (11), was able to prevent apoptosis when used either alone or in combination with JM or JN. On the other hand, the analog MK, which is an antagonist of the nuclear actions of the VDR (29), was ineffective by itself but was able to block the protective effects of analogs JM and JN (Fig. 3C) and  1␣,25(OH) 2 D 3 (Fig. 3D) in calvarial cells. The 6-s-trans-locked analogs JB and JD, which are weak agonists of both genotropic and nongenotropic actions, also exhibited a modest anti-apoptotic effect in calvaria cells (Fig. 3D) (27). Similar to the cis-analogs, HL prevented apoptosis when used alone or in combination with JB, JD, or 1␣,25(OH) 2 D 3 (Fig. 3D). The antagonist MK was able to block the anti-apoptotic effect of 1␣,25(OH) 2 D 3 , but not that of JB or JD (Fig. 3D).
The Ligand Binding Domain of the VDR Is Sufficient for the Mediation of the Anti-apoptotic Effect-Based on our earlier findings that the DNA binding domain of the ER is dispensable for the anti-apoptotic effects of estradiol, we next investigated whether the DNA binding domain of the VDR is also dispensable for the anti-apoptotic effects of 1␣,25(OH) 2 D 3. To this end we constructed three plasmids carrying the wild type human VDR, wt hVDR, fused to cyan fluorescent protein (ECFP-  wtVDR) or just the ligand binding domain of the VDR fused to ECFP (ECFP-VDR119-C). Using a specific steroid competition assay, we demonstrated that 1␣,25(OH) 2 D 3 binds to the ECFP-wtVDR and the ECFP-VDR119-C constructs similar to the wtVDR (Fig. 4A). Further, these three constructs were assayed for responsiveness to the VDRE by transfecting CV1 monkey cells with an osteocalcin reporter construct. As seen in Fig. 4B, unlike the wtVDR or the ECFP-wtVDR, the construct comprising only the ligand binding domain of the VDR (VDR199-C) did not induce transcription from the osteocalcin promoter. As shown in Fig. 4C, 1␣,25(OH) 2 D 3 was equally effective in preventing etoposide-induced apoptosis in HeLa cells transiently transfected with either the wtVDR or the VDR119-C.
ERK Phosphorylation Is Not Required for the Anti-apoptotic Effects of Vitamin D 3 -It has been previously shown that 1␣,25(OH) 2 D 3 rapidly induces the phosphorylation of ERKs in murine osteoblastic calvaria cells and other cell types (13,30,31). We confirmed this observation here using the OB-6 murine bone marrow-derived osteoblastic cell line (Fig. 5A) and also established that this activity could be blocked by PD98059, the inhibitor of MEK that is the kinase responsible for ERK phosphorylation. We next investigated whether ERK phosphorylation is required for the anti-apoptotic actions of 1␣,25(OH) 2 D 3 . In sharp contrast to the effect of E 2 , the protective effect of 1␣,25(OH) 2 D 3 in OB-6 or calvaria cells was not blocked by PD98059 or another inhibitor of ERK phosphorylation, U0126, respectively (Fig. 5B). Confirming these results, transient transfection of a dominant negative MEK plasmid in VDR-containing HeLa cells did not affect the anti-apoptotic action of 1␣,25(OH) 2 D 3 (Fig. 5C), indicating that ERK phosphorylation is not involved in 1␣,25(OH) 2 D 3 -induced anti-apoptosis.

Activation of Cytoplasmic Kinases and Transcriptiondependent Events Mediate the Anti-Apoptotic Actions of 1␣,25(OH) 2 D 3 -
We have previously shown that nongenotropic control of osteoblastic/osteocytic cell survival by sex steroids involves modulation of the PI3K, ERK, and JNK signaling cascades as well as transcription-dependent events downstream of cytoplasmic kinases (9,10). Therefore, we investigated whether the same signaling pathways as well as transcription and new protein synthesis also mediate the antiapoptotic effects of 1␣,25(OH) 2 D 3 . As shown in Fig. 5D, the protective effect of 1␣,25(OH) 2 D 3 in calvarial osteoblastic cells was abrogated by Wortmannin and SP600125, the specific inhibitors for PI3K and JNK, respectively. However, the p38 inhibitor SB203580 had no effect on 1␣,25(OH) 2 D 3 -induced protection from apoptosis. Wortmannin, SP600125, or SB203580 did not affect anti-apoptosis when added in control cultures of calvaria cells. Moreover, the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide, at doses which we have previously shown to inhibit [ 3 H]uridine or [ 3 H[leucine incorporation, respectively, without affecting cell viability (10), abrogated the protective effect of 1␣,25(OH) 2 D 3 on etoposide-induced apoptosis of murine calvaria-derived osteoblasts, indicating that transcription is required for anti-apoptosis (Fig. 5D, right panel).
A VDR/Src Physical Association Is Required for the Protective Effects of 1␣,25(OH) 2 D 3 -Based on our own previous observations that ER and AR mediate the anti-apoptotic actions of sex steroids via the SH2 and SH3 domains of Src, respectively, as well as published evidence that Src physically interacts with the ER and AR (32) or the VDR (33), we investigated the involvement of this kinase in the protective effects of 1␣,25(OH) 2 D 3 . As shown for E 2 , treatment of calvaria-derived osteoblasts with the Src inhibitor PP1 prevented the 1␣,25(OH) 2 D 3 -induced protection from apoptosis (Fig. 6A). Furthermore, incubation of OB-6 cells with 10 Ϫ8 M 1␣,25(OH) 2 D 3 for 5 min-1 h followed by immunoprecipitation with an anti-VDR antibody and immunoblotting with an anti-Src antibody demonstrated the rapid association of Src with the VDR (Fig. 6B). The kinase and receptor proteins interacted within 5 min, and this interaction resolved after 30 min in the presence of 1␣,25(OH) 2 D 3 . Finally, consistent with the mechanism via which 1␣,25(OH) 2 D 3 exerts its anti-apoptotic actions, the anti-apoptotic effect of the nongenotropic analog JN in calvarial osteoblastic cells required Src, PI3K, and JNK, but not ERK signaling, as it was abrogated by PP1, Wortmannin, and SP600125, but not UO126 (Fig. 6C). DISCUSSION VDR, a member of the nuclear receptor superfamily, mediates a plethora of diverse biologic effects in response to its natural ligand, 1␣,25(OH) 2 D 3 , in over 30 tissues, including All experiments were carried out on quadruplicate samples with data expressed as the mean Ϯ S.E. C, HeLa cells were transiently transfected with either wtVDR or VDR(119-C) and nuclear GFP and treated with 10 Ϫ8 M 1␣,25(OH) 2 D 3 , followed by 6 h of treatment with etoposide (100 mM). Apoptosis was assessed as described in Fig. 1A. bone, intestine, kidney, and the immune system. Binding of 1␣,25(OH) 2 D 3 to the VDR results in conformational changes and the formation of a heterodimer with the RXR to form an active transactivator complex that mediates the classical responses of nuclear receptor action (34). Nonetheless, as in the case of all other ligands of the nuclear receptor superfamily, many effects of 1␣,25(OH) 2 D 3 cannot be explained by the classical genotropic mechanisms. Instead, they have been attributed to rapid nongenotropic actions (11). Heretofore, it was unknown whether such nongenotropic actions are mediated via the classical nuclear VDR or a membrane-associated form of VDR or even a completely distinct membrane protein. Likewise, it remains unknown whether some or all of the nongenotropic actions of 1␣,25(OH) 2 D 3 utilize second messenger signaling pathways or whether they require gene transcription or new protein synthesis.
In addition to the naturally occurring metabolites, many synthetic vitamin D analogs have been created. Analogs are excellent model ligands for investigating the molecular mechanisms of 1␣,25(OH) 2 D 3 (11,34) because they can be used to differentiate the genotropic (VDRE-dependent) effects from nongenotropic (VDRE-independent) effects (27). These analogs vary widely in the effects produced. Some analogs are able to potently activate gene transcription and cell proliferation and differentiation and yet have minimum calcium mobilization effects, whereas others produce the opposite effects (34).
The studies reported here indicate that 1␣,25(OH) 2 D 3 and synthetic analogs (like the 6-s-cis-locked analogs JN or JM that exhibit low binding affinity for the VDR in standard steroid competition assays and lack transcriptional activity but can rapidly induce transcaltachia (27), opening of osteoblast chloride channels (18), calcium mobilization, and ERK phosphorylation) (13) are able to protect murine osteoblasts, OB-6 osteoblastic cells, or MLO-Y4 osteocytic cells from etoposide-induced apoptosis. The LBD of the VDR, which is able to bind 1␣,25(OH) 2 D 3 as effectively as the wtVDR but unlike the fulllength receptor is incapable of inducing VDRE-mediated transcription, transduced these anti-apoptotic effects as effectively as the wtVDR. The transduction of anti-apoptotic signaling by the LBD of the VDR in response to its classical ligand as well as synthetic analogs with low VDR affinity, is a feature identical to that which we had shown previously for the ER (9, 10). Therefore, signaling anti-apoptosis by the LBD is evidently not unique to the ER but rather a common property of nuclear receptors.
In full support of the contention that transduction of kinasemediated signals may be a general property of nuclear receptors, as in the case of the ER (and for that matter, AR), we determined here that in osteoblastic cells the VDR physically associates and activates the Src kinase (32) and that the antiapoptotic actions of 1␣,25(OH) 2 D 3 also required activation of PI3K and JNK kinases, but not p38. However, unlike the anti-apoptotic signaling cascade triggered by the ER or the AR, the anti-apoptotic effects of the 1␣,25(OH) 2 D 3 /VDR complex did not require activation of ERKs. However, in a manner identical to the ER, the anti-apoptotic signal of the VDR required kinase-mediated transcription, as evidenced by the ability of cycloheximide and actinomycin D to abrogate the effects of 1␣,25(OH) 2 D 3 . Collectively, these observations indicate that identical to sex steroids, which regulate the life span of bone cells by modulating the activity of cytoplasmic kinases via nongenotropic actions of their classical receptors, nongenotropic modulation of kinase activity and downstream transcriptional events are independent of the classical direct transcriptional activity of the VDR and are the mediators of the antiapoptotic effects 1␣,25(OH) 2 D 3 in osteoblasts and osteocytes.
Several cytoplasmic kinases, including Src, are clustered in caveolae, specialized membrane invaginations that are enriched in the scaffolding protein caveolin-1. The ER has recently been shown to be co-localized in caveolae with both caveolin and endothelial nitric-oxide synthase, an enzyme activated by estrogen through a nongenotropic mechanism of action (35,36). More recently, an intestinal plasma membrane-binding protein for 1␣,25(OH) 2 D 3 was reported (37); it was subsequently identified as the classical VDR associated with the plasma membrane caveolae fraction (12). Likewise, 1␣,25(OH) 2 D 3 was shown to bind to VDR located at the caveolae-enriched membrane fractions and induce rapid responses (opening of chloride channels) in osteoblasts in vivo (18). Importantly, binding of 1␣,25(OH) 2 D 3 to VDR and these nongenotropic responses were abrogated in VDR knock-out mice (12), indicating that, at least in bone, the nongenotropic actions of 1␣,25(OH) 2 D 3 are mediated by either the classical VDR or a slightly altered VDR isoform that is present at the caveolaeenriched membrane cell fractions. Alternatively, the genomic actions of the VDR may in some fashion be permissive for 1␣,25(OH) 2 D 3 -mediated rapid responses in VDR WT osteoblasts that are missing in VDR knock-outs. However, the results presented in this report show that the expression of the LDB of the VDR is sufficient to allow the nongenomic actions of 1␣,25(OH) 2 D 3 and would appear to be much more definitive than the previously mentioned VDR knock-out experiment (17). Taken together, these observations clearly demonstrate that the ability to trigger kinase signaling cascades through extranuclear functions within preassembled scaffolds is a property shared by both the classical ER and the classical VDR.
ER, AR, and VDR are evidently only a few of the nuclear hormone receptors that upon ligand binding are able to transmit nongenotropic signals via a membrane-associated signalosome that entails these receptors in physical association with kinases and that in turn can elicit a cascade of events that lead to the activation or suppression of kinase phosphorylation and kinase-regulated transcription. Indeed, it is now becoming widely accepted that all steroid hormones are capable of generating rapid nongenotropic responses in a variety of cell models (38 -42).
We have previously shown that nongenotropic (kinase-mediated) actions of the ER or AR can be dissociated from classical cis or trans transcriptional activity of the receptors with synthetic ligands that we have termed ANGELS (Activators of NonGenotropic Estrogen-like Signaling). Such ligands reverse bone loss in sex steroid-deficient mice without affecting the weight of reproductive organs (2,9,10), most likely by stimulating osteoblastogenesis through the potentiation of both Wnt and BMP signaling (43,44). In analogy to the evidence for a distinct biologic profile between classical estrogen and AN-GELS, several studies have shown that 6-s-cis-locked analogs of 1␣,25(OH) 2 D 3 activate membrane-initiated rapid pathways and, as we show here, protect osteoblastic/osteocytic cells from apoptosis and at the same time display a weak genotropic activity. Effective equilibrium binding of these analogs to the classical VDR is only 0.1-1% relative to 1␣,25(OH) 2 D 3 . In addition, it is apparent that the apoptosis inhibition assay cannot be classified to be a "pure" nongenotropic assay. This is not so surprising, given the 6-h duration employed for the assay, the intrinsically complex (multistep) process, and the fact that both actinomycin D and cycloheximide can block the actions of both 1␣,25(OH) 2 D 3 and estradiol (Fig. 5D). This conclusion is supported by our previous findings that transcriptional events downstream of kinase activation are also required for the antiapoptotic actions of sex steroids (10). It is further emphasized by the ability of the genomic antagonist, analog MK, to block the anti-apoptotic actions of 1␣,25(OH) 2 D 3 , JM, or JN, suggesting that the first steps in the overall process are dependent upon true rapid or nongenotropic activation of three cytoplasmic kinases (Src, PI3K, and JNK). The results observed with the 6-s-trans-locked JB and JD are complex in that they displayed partial, but not full, anti-apoptic activity in comparison with 1␣,25(OH) 2 D 3 , which was not inhibited by the nuclear FIG. 6. 1␣,25(OH) 2 D 3 triggers physical association of VDR/Src in osteoblastic cells. A, calvaria-derived osteoblastic cells were incubated with vehicle or 10 Ϫ7 M PP1 for 30 min prior to the addition of 1␣,25(OH) 2 D 3 (10 Ϫ8 M). After 30 min, etoposide was added. Apoptosis was assayed 6 h later as described in Fig. 1B. B, OB-6 cells were treated with 10 Ϫ8 M 1␣,25(OH) 2 D 3 for different lengths of time. Cells were washed with TBSV (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM Na 3 VO 4 ) twice, and lysates were collected. An anti-VDR antibody (VDR N-20; Santa Cruz Biotechnology) and an anti-IgG control antibody were added to 66 g of protein/sample. Samples were electrophoresed on a 10% gel for 2 h and then transferred to a membrane. The membrane was incubated first with the VDR antibody and then stripped and incubated with an anti-v-Src antibody. C, calvaria cells were incubated with vehicle, 10 Ϫ6 M U0126, 10 Ϫ7 M PP1, 30 nm Wortmannin, or 10 M SP600125 for 30 min prior to the addition of 10 Ϫ8 M JN analog. After 1 h, etoposide was added. Apoptosis was assayed 6 h later as in Fig. 1B. antagonist MK. Relevant to this, analog HL, which is known to inhibit the nongenomic actions of 1␣,25(OH) 2 D 3 , can also induce anti-apoptosis. Collectively, these observations may suggest an alternative path of nongenomic action of both of the relatively unstudied analogs of 1␣,25(OH) 2 D 3 , JB and JD, as well as HL.
A new VDR conformational ensemble model that identifies a second, or alternate, ligand binding site on the VDR has been proposed to postulate how the VDR could be responsible for both genotropic and nongenotropic responses (38,45). In that model, two ligand pockets within the ER or VDR along with the position of the last helix, helix 12, in either receptor control the onset of either type of response. Additional work needs to be done to clarify the mechanistic issues of ligand-specific responses. Still, there are striking mechanistic and biological similarities between membrane-initiated, nongenotropic actions of the ER and VDR (and even other nuclear receptors in various cell models) in the protection of osteoblasts/osteocytes from apoptosis. In addition, the existence of ER and VDR ligands that can dissociate the nongenotropic from the transcriptional activity of the receptors provides proof of principle for the development of function-specific, as opposed to tissueselective, pharmacotherapeutics. The discovery that, although inactivation of the glucocorticoid receptor caused lethality in mutant mice, elimination of the transcriptional activity of this receptor did not (literally a difference between life and death) (46) strongly underscores the potential of this approach.