Calmodulin-Dependent Kinase IV Stimulates Vitamin D Receptor-Mediated Transcription

Tara I. Ellison, Diane R. Dowd and Paul N. MacDonald

Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Paul N. MacDonald, Ph.D., Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106. E-mail: pnm2{at}case.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] promotes intestinal absorption of calcium primarily by binding to the vitamin D receptor (VDR) and regulating gene expression. 1,25-(OH)2D3 also exerts rapid actions at the cell membrane that include increasing intracellular calcium levels and activating protein kinase cascades. To explore potential cross talk between calcium signaling elicited by the nongenomic actions of 1,25-(OH)2D3 and the genomic pathway mediated by VDR, we examined the effects of activated Ca2+/calmodulin-dependent kinases (CaMKs) on 1,25-(OH)2D3/VDR-mediated transcription. Expression of a constitutively active form of CaMKIV dramatically stimulated 1,25-(OH)2D3-activated reporter gene expression in COS-7, HeLa, and ROS17/2.8 cell lines. Metabolic labeling studies indicated that CaMKIV increased VDR phosphorylation levels. In addition, CaMKIV increased the independent transcription activity of the VDR coactivator SRC (steroid receptor coactivator) 1, and promoted ligand-dependent interaction between VDR and SRC coactivator proteins in mammalian two-hybrid studies. The functional consequences of this multifaceted mechanism of CaMKIV action were revealed by reporter gene studies, which showed that CaMKIV and select SRC coactivators synergistically enhanced VDR-mediated transcription. These studies support a model in which CaMKIV signaling stimulates VDR-mediated transcription by increasing phosphorylation levels of VDR and enhancing autonomous SRC activity, resulting in higher 1,25-(OH)2D3-dependent interaction between VDR and SRC coactivators.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE VITAMIN D RECEPTOR (VDR) is a member of the nuclear receptor family of transcription factors. 1,25-(OH)2D3, the most biologically active form of vitamin D, is the endogenous ligand for the VDR. Ligand-bound VDR heterodimerizes with the retinoid X receptor (RXR) and binds to DNA response elements (VDREs) in the promoter regions of 1,25-(OH)2D3-regulated target genes to alter transcription (1). Nuclear receptor coactivator proteins, such as steroid receptor coactivators (SRCs) (2), NCoA-62 (3, 4, 5), and mediator complex (6) stimulate transcription by recruiting RNA polymerase II, general transcriptional machinery, chromatin remodeling factors and other associated factors to the promoter template.

The major physiological role of VDR is to promote calcium absorption in the intestine and maintain calcium homeostasis in the body (1). Despite the essential role VDR plays in regulating calcium homeostasis, interplays between intracellular calcium levels and VDR transcriptional activity have not been studied extensively. Intracellular calcium levels regulate a variety of distinct cellular signaling pathways including ligand-gated ion channels, cell cycle progression, apoptosis, and transcriptional processes (7, 8, 9). Calmodulin (CaM) is a calcium sensor and an effector protein that functions in many of these calcium-dependent processes. Calcium binding induces a conformational change in CaM resulting in the activation of a variety of protein kinases, phosphatases, ion channels, receptors, and NO synthases (10). The kinases activated by CaM include myosin light chain kinase and the multifunctional Ca2+/calmodulin-dependent kinases I, II, and IV (CaMKI, II, and IV). CaMKI and II are ubiquitously expressed in mammalian cells, whereas CaMKIV expression is more restricted. The brain, T lymphocytes, testes, ovary, adrenal glands, skin, and bone marrow contain high levels of CaMKIV (11, 12, 13). The intracellular localization of the CaM kinases also differs, with CaMKI and II primarily residing in the cytoplasm and CaMKIV residing primarily in the nucleus (14, 15, 16). CaMKIV consists of a catalytic domain that contains the active site, and an autoinhibitory domain that regulates the activity of the kinase (17). Ca2+/CaM activates CaMKIV by binding to its regulatory region (11), thereby revealing its catalytic domain. Ca2+/CaM kinase kinase phosphorylates CaMKIV, which induces full activity of CaMKIV and allows it to act independently of Ca2+/CaM (17). Phosphorylated CaMKIV activates a variety of proteins, including cAMP response element binding protein (CREB), serum response factor, activating transcription factor 1, and CREB binding protein (10).

Two studies suggest a potential role for CaMKIV in nuclear receptor-mediated transcription. Kane and Means (18) demonstrated that CaMKIV augments the constitutive activity of the retinoid-related orphan receptors (RORs) and other orphan nuclear receptors. A second study indicated that thyroid hormone receptor activity is modestly activated by CaMKIV, but this effect requires ROR{alpha} (19). A direct role for CaMKIV in ligand-activated nuclear receptor-mediated transcription and specifically, in 1,25-(OH)2D3/VDR-activated transcription, has not been addressed. The potential for CaM/CaMK involvement in vitamin D signaling is supported by the well-known capacity of 1,25-(OH)2D3 to elicit rapid, membrane-initiated calcium transients in numerous cell types (20, 21, 22). However, there is scant evidence linking the 1,25-(OH)2D3-induced calcium transients to VDR transcriptional activity. Because VDR transactivation is a target for a variety of kinases including casein kinase II (23), protein kinase C (24), and the MAPK pathway (25), and because 1,25-(OH)2D3-induced intracellular calcium levels could activate CaMKIV, we tested the hypothesis that CaMKIV regulates the activity of VDR. Here, we provide new evidence that active CaMKIV stimulates 1,25-(OH)2D3- and VDR-mediated transcription by phosphorylating VDR, increasing the activity of SRC1, and by promoting the interaction between VDR and SRC coactivator proteins. These data suggest a potential intersection between two critical 1,25-(OH)2D3 signaling pathways, namely the well-established genomic mechanism mediated through the VDR and the emerging nongenomic pathways involving membrane-initiated stimulation of second messenger pathways, kinase cascades, and opening of ion channels.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CaMKIV Selectively Enhances 1,25-(OH)2D3/VDR-Mediated Transcription
We initially explored the effect of CaMKIV on VDR-mediated transcription using the (VDRE)4-TATA-Luc reporter gene in HeLa cells. Full-length VDR was expressed in the presence or absence of a constitutively active form of CaMKIV containing amino acids 1–313 of the rat sequence (CaMKIV1–313) (26). In the absence of CaMKIV1–313, 1,25-(OH)2D3 activated reporter gene expression 16-fold. When CaMKIV1–313 was ectopically expressed, 1,25-(OH)2D3-stimulated luciferase activity was induced 55-fold, a 4-fold activation compared with vector control (Fig. 1AGo). This increase in luciferase activity was dependent upon the 1,25-(OH)2D3 ligand, as expression of the reporter gene was unaffected by CaMKIV1–313 alone. Binding of 1,25-(OH)2D3 by the VDR ligand binding domain (LBD) causes a conformation change, creating an interaction surface for RXR and a variety of comodulatory proteins. To determine whether this domain also mediates VDR activation by CaMKIV1–313, the VDR LBD (amino acids 93–427) was expressed as a fusion protein with a heterologous gal4 DNA binding domain (DBD). The gal4-VDR fusion protein activated a gal4-dependent reporter gene [(gal4)5-TATA-Luc] 9-fold in the presence of 1,25-(OH)2D3 (Fig. 1AGo). Coexpression of CaMKIV1–313 enhanced 1,25-(OH)2D3 activation of the reporter construct 63-fold, seven times the activation observed in the absence of CaMKIV1–313. Thus, the LBD of VDR was sufficient to mediate the effects of CaMKIV1–313 on 1,25-(OH)2D3-activated reporter gene expression in this system.



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Fig. 1. CaMKIV Selectively Enhances 1,25-(OH)2D3/VDR-Mediated Transcription

A, Constitutively active CaMKIV activates both the full-length VDR and the VDR LBD fused to a heterologous gal4 DBD. HeLa cells were transfected with 1 µg of (VDRE)4-TATA-Luc, 3 ng of SG5-hVDR, and 1 µg of SG5 or SG5-CaMKIV1–313 per well, or with 1 µg of (gal4)5-TATA-Luc, 30 ng SG5-gal4-VDR, and 1 µg of SG5 or SG5-CaMKIV1–313 per well, as described in Materials and Methods. After a 24-h treatment with ethanol vehicle or 10–8 M 1,25-(OH)2D3, cell extracts were prepared and analyzed for luciferase expression. Data are graphed as the mean of duplicate samples ± SD. B, CaMKIV1–313 produces little effect in the absence of VDR or vitamin D response elements. COS-7 cells were transfected with 3 ng of SG5-hVDR, 1 µg of SG5 or SG5-CaMKIV1–313, and 1 µg of (VDRE)4-TATA-Luc or (gal4)5-TATA-Luc. Cells were treated and extracts prepared as described in part A. C, CaMKIV1–313 activates VDR-mediated transcription in a dose-dependent manner. Fold activation by CaMKIV1–313 was calculated as the luciferase activity obtained in the presence of SG5-CAMKIV1–313 divided by the luciferase activity observed with the SG5 vector control. COS-7 cells were transfected with 1 µg of (VDRE)4-TATA-Luc, 3 ng of SG5-VDR and increasing amounts of SG5-CaMKIV1–313 or SG5 vector control, treated with ethanol or 10–8 M 1,25-(OH)2D3 for 24 h, and analyzed as described in part A. D, CaMKIV1–313 activates the VDRE reporter in a 1,25-(OH)2D3 concentration-dependent manner. COS-7 cells were transfected with 1 µg of (VDRE)4-TATA-Luc, 3 ng of SG5-VDR and 300 ng of SG5-CaMKIV1–313 or vector control, treated with the indicated concentrations of ethanol or 1,25-(OH)2D3 for 24 h, and analyzed as described in part A.

 
CaMKIV1–313 also activated VDR-mediated transcription in COS-7 cells, which express low levels of endogenous VDR (less than 500 transcripts per cell) (Fig. 1BGo). Here, CaMKIV1–313 enhanced 1,25-(OH)2D3-activated transcription 6-fold compared with vector control. CaMKIV1–313 exerted minimal effects on the (VDRE)4-TATA-Luc reporter in the absence of VDR. Residual activation was likely due to low levels of endogenous VDR in this system. In addition, full-length VDR activation due to 1,25-(OH)2D3 or CaMKIV1–313 was not apparent using a reporter lacking vitamin D response elements [(gal4)5-TATA-Luc, Fig. 1BGo]. These data show that CaMKIV1–313 selectively augments 1,25-(OH)2D3-mediated transcription in a VDR- and VDRE-dependent manner.

CaMKIV1–313 activated VDR in a dose-dependent manner (Fig. 1CGo). In the presence of 1,25-(OH)2D3, 100 ng of SG5-CaMKIV1–313 activated the VDRE-reporter 4-fold compared with vector control. A maximal activation of approximately 14-fold was observed at 1000 ng of SG5-CaMKIV1–313 expression plasmid. However, amounts of CaMKIV1–313 greater than this exhibited lower efficacy, probably the result of squelching effects from the large quantity of plasmid. SG5-CaMKIV1–313 had little effect in the absence of 1,25-(OH)2D3 (open bars in Fig. 1CGo). The CaMKIV1–313 activation of VDR also depended upon the concentration of 1,25-(OH)2D3 (Fig. 1DGo). CaMKIV1–313 augmented VDRE-dependent transcription 6-fold at ligand concentrations as low as 10–9 M 1,25-(OH)2D3, and the response dramatically increased to 13-fold and 17-fold at 10–8 M and 10–7 M 1,25-(OH)2D3, respectively. Taken together, these data establish a potent and selective effect of constitutively active CaMKIV on 1,25-(OH)2D3- and VDR-induced promoter activity.

CaMKIV and CaMKII Impact 1,25-(OH)2D3-Activated Transcription from Synthetic and Natural Promoters in a Cell-Selective Manner
CaMKIV belongs to a three-member family of multifunctional Ca2+/CaM-dependent kinases, the best-characterized member being CaMKII. Because CaMKII and CaMKIV exhibit some overlapping substrate specificity, we tested whether CaMKII would similarly affect VDR-mediated transcription. Surprisingly, whereas CaMKIV1–313 expression in COS-7 cells increased 1,25-(OH)2D3-activated transcription 8-fold, expression of constitutively active CaMKII (amino acids 1–290) produced minimal effects on 1,25-(OH)2D3-activated transcription in this VDR/VDRE-driven reporter gene system (Fig. 2AGo). However, when these studies were expanded to ROS 17/2.8 rat osteosarcoma cells, a 1,25-(OH)2D3 target cell line that expresses more than 20,000 copies of VDR (27), both CaMKIV1–313 and CaMKII1–290 strongly induced 1,25-(OH)2D3-activated reporter gene expression mediated by endogenous VDR (Fig. 2BGo). Therefore, both CaMKIV1–313 and CaMKII1–290 augment VDR-mediated transcription, although the magnitude of the CaMKII1–290 effect varies by cell type.



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Fig. 2. CaMKIV and CaMKII Impact 1,25-(OH)2D3-Activated Transcription from Synthetic and Natural Promoters in a Cell-Selective Manner

A, COS-7 cells were transfected with 680 ng of (VDRE)4-TATA-Luc, 200 ng of phRG-TK, 3 ng of SG5-VDR expression plasmid, and 1 µg of either SG5, SG5-CaMKIV1–313, or SG5-CaMKII1–290 as described in Materials and Methods. Luciferase activity was measured 24 h after treatment with 10–8 M 1,25-(OH)2D3 or ethanol vehicle. Data are graphed as the mean of duplicate measurements ± SD. B, ROS17/2.8 osteosarcoma cells were transfected with 680 ng of (VDRE)4-TATA-Luc, 200 ng of phRG-TK, and 1 µg of either SG5, SG5-CaMKIV1–313, or SG5-CaMKII1–290. Cells were treated and extracts prepared as described in part A. C, ROS17/2.8 osteosarcoma cells were transfected with 680 ng of the rat24OHase-Luc reporter, 200 ng phRG-TK, and 1 µg of either SG5, SG5-CaMKIV1–313, or SG5-CaMKII1–290. Cells were treated and extracts prepared as described in part A.

 
The studies described thus far used heterologous reporter constructs containing minimal vitamin D response elements. To extend these studies to a more natural VDR target system, a reporter gene construct containing the native 482-bp promoter of the rat 24-hydroxylase gene was introduced into ROS 17/2.8 rat osteosarcoma cells. Expression of constitutively active CaMKIV in this context activated the 24-hydroxylase promoter 3.5-fold, and CaMKII1–290 activated the promoter 5.5-fold in the presence of 1,25-(OH)2D3 (Fig. 2CGo). Neither kinase altered unliganded transcriptional activity from the 24-hydroxylase promoter. Thus, CaMKIV1–313 and CaMKII1–290 activate both a synthetic and a natural promoter construct in the native cellular context of a VDR target cell.

CaMKIV1–313 Increases Phosphorylation of VDR
To address potential mechanisms underlying the observed increase in VDR/1,25-(OH)2D3-activated transcription, we focused on CaMKIV1–313 and the COS-7 cell system because CaMKIV1–313 displayed the widest cell activity profile and because COS-7 cells express minimal levels of endogenous VDR. One potential mechanism may involve direct or indirect phosphorylation of VDR by CaMKIV1–313. Sequence analysis of VDR revealed a putative CaMKIV consensus phosphorylation site at serine residue 172 (167PSRPNSRHTP176). The consensus CaMKIV site is (Hyd-x-R-x-x-S/T-x-x-x-Hyd) in which Hyd refers to any hydrophobic amino acid, R is arginine, S/T is a serine or threonine at the phosphorylation site, and x refers to any amino acid. VDR contains a proline residue in each of the hydrophobic positions, and an arginine at amino acid 169. To explore the importance of the VDR serine 172, site-directed mutagenesis was used to change this serine to an alanine. As shown in Fig. 3AGo, this mutation had no effect on 1,25-(OH)2D3 responsiveness of VDR because 1,25-(OH)2D3 induced an 8-fold activation of the (VDRE)4-TATA-Luc reporter compared with vehicle treatment in both the wild-type VDR and S172A VDR-transfected cells. The wild-type and S172A VDR also responded similarly to CaMKIV1–313 expression, as both were activated approximately 10- to 11-fold in the presence of 1,25-(OH)2D3. Western blot showed equivalent expression of the wild-type and S172A mutant VDR (data not shown). These data suggest that S172 is not required for 1,25-(OH)2D3 responsiveness and that S172 is not involved in CaMKIV1–313-mediated activation of VDR-dependent gene expression.



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Fig. 3. CaMKIV1–313 Increases Phosphorylation of VDR

A, VDR does not require serine 172 for activation by CaMKIV1–313. COS-7 cells were transfected with 680 ng of (VDRE)4-TATA-Luc reporter, 200 ng of phRG-TK, 3 ng of SG5-VDR (WT or S172A), and 500 ng of SG5-CaMKIV1–313 or SG5 vector control. Luciferase activity was measured 24 h after treatment with 10–8 M 1,25-(OH)2D3 or ethanol vehicle. Data are graphed as the average of duplicate samples ± SD. B, Phosphorylation of VDR was examined using metabolic labeling with 32P-orthophosphate. COS-7 cells were transfected with 500 ng of CMV-Flag-VDR and 1 µg of either SG5-CaMKIV1–313 or SG5. Cells were incubated with [32P]-orthophosphate and 10–8 M 1,25-(OH)2D3 or ethanol vehicle in phosphate-free media for 2 h. Cells were harvested and Flag-VDR immunoprecipitated. The autoradiogram in the upper row shows immunoprecipitated 32P-labeled VDR and the lower row is a Western blot of total VDR protein levels. Normalized signal states the quantitated intensity of 32P-labeled VDR divided by intensity of the VDR Western for each lane, relative to the ratio of the bands in lane 1. The data shown are representative of three independent experiments.

 
To test whether other amino acids are targeted by CaMKIV, 32P-labeling studies were used to examine overall phosphorylation of VDR. COS-7 cells expressing Flag-tagged VDR were labeled with 32P-orthophosphate in the absence and presence of CaMKIV1–313, and cell extracts were subjected to immunoprecipitation with an anti-Flag antibody. Expression of CaMKIV1–313 increased the phosphorylation level of VDR 4-fold (Fig. 3BGo), both in the absence of 1,25-(OH)2D3 treatment (compare lane 3 with 1) and the presence of 1,25-(OH)2D3 treatment (compare lane 4 with 2). However, this increase in VDR phosphorylation was accompanied by a 2-fold increase in VDR expression as assessed by Western immunoblots of crude protein samples. When normalized to VDR protein levels, CaMKIV1–313 expression enhanced both unliganded and liganded VDR phosphorylation, by 2.2- and 1.8-fold, respectively.

CaMKIV1–313 Increases the Intrinsic Transactivation of SRC1
In addition to up-regulating phosphorylation of VDR, it is possible that CaMKIV1–313 phosphorylates other proteins involved in activation of VDR-mediated transcription. SRC1, a coactivator of several nuclear receptors, is phosphorylated at multiple sites by the MAPK family member Erk-2 (28). This phosphorylation augments the intrinsic transactivation activity of SRC1 and its coactivation of the progesterone receptor (29). To determine whether CaMKIV1–313 impacts the intrinsic transactivation potential of SRC1, full-length SRC1 was expressed as a fusion protein with the DBD (amino acids 1–147) of the heterologous gal4 transcription factor in COS-7 cells (Fig. 4Go). The gal4-SRC1 fusion exhibited constitutive activity compared with the gal4 DBD alone, whereas the gal4-SRC1 receptor interaction domain (RID), containing only the LXXLL motifs, lacked this activity (open bars in Fig. 4Go). Coexpression of CaMKIV1–313 increased gal4-SRC1 activity 6.8-fold compared with vector control, whereas CaMKIV1–313 did not affect the activity of the gal4-SRC1 RID that lacks activation domains. In this system, CaMKIV1–313 did not affect the activity of gal4-VP16 (virion protein 16), an unrelated transactivator with an acidic activation domain (data not shown). In addition, CaMKII1–290 failed to augment transactivation of gal4-SRC1 and gal4-SRC1 RID, an expected outcome given the selective effect of CaMKII1–290 on VDR-mediated transcription in ROS17/2.8 cells but not COS-7 cells. Taken together, these data show that CaMKIV1–313 selectively augments the intrinsic transactivation of SRC1 in COS-7 cells.



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Fig. 4. CaMKIV1–313 Increases the Intrinsic Transactivation of SRC1

COS-7 cells were transfected with 680 ng of (gal4)5-TATA-Luc reporter, 200 ng of phRG-TK, 150 ng of SG5-gal4, SG5-gal4-SRC1, or SG5-gal4-SRC1 RID, and 300 ng of SG5-CaMKIV1–313, SG5-CaMKII1–290, or SG5 control. Luciferase activity was measured 42 h after transfection, and data are graphed as the average of triplicate samples ± SD.

 
CaMKIV1–313 Increases VDR Interaction with the SRC Family of Coactivators in a Mammalian Two-Hybrid System
The extent of VDR interaction with its heterodimer partner RXR and with the SRC coactivators determines, in part, the degree of VDR transcriptional activity. Thus, the mammalian two-hybrid assay was used to determine whether phosphorylation events by CaMKIV1–313 enhanced these interactions. In this assay, the VDR LBD was expressed as a VP16 activation domain fusion protein, whereas the RXR{gamma} LBD and the LXXLL-containing RIDs of the coactivators were fused to the gal4 DBD. Protein-protein interactions were monitored using the (gal4)5-TATA-luciferase reporter. As illustrated in Fig. 5Go (left panel), CaMKIV1–313 did not dramatically alter the interaction between VDR and RXR{gamma}. However, expression of CaMKIV1–313 increased the interaction between VDR and the LXXLL-containing domains of SRC1 and SRC2 by 3.5-fold and 3-fold, respectively. This enhanced interaction with SRC1 and SRC2 was dependent upon 1,25-(OH)2D3 because in the absence of ligand, VDR did not interact with SRC1 or SRC2, regardless of the expression of CaMKIV1–313. Little interaction was observed between VDR and the RID of SRC3 (Fig. 5Go, right panel; note the expanded y-axis). CaMKIV1–313 also increased this weak interaction 2- to 3-fold. In contrast, CaMKIV1–313 did not affect the low basal interaction of VDR with the gal4 control protein. These data indicate that CaMKIV1–313 increases interaction of VDR with the SRC family of coactivators, likely yielding enhanced transcriptional activity of VDR.



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Fig. 5. CaMKIV1–313 Increases Interaction of VDR with the RIDs of the SRC Family of Coactivators in a Mammalian Two-Hybrid System

COS-7 cells were transfected with 680 ng of (gal4)5-TATA-Luc reporter, 150 ng of VP16-VDR, and 100 ng of SG5-gal4, SG5-gal4-RXR{gamma} LBD, SG5-gal4-SRC1 RID, SG5-gal4-SRC2 RID, or SG5-gal4-SRC3 RID and 500 ng of SG5-CaMKIV1–313 or SG5. Luciferase activity was measured 24 h after treatment with 10–8 M 1,25-(OH)2D3 or ethanol vehicle. Data are graphed as the average of duplicate samples SD.

 
CaMKIV1–313 Synergizes with SRC1 and SRC2 to Activate VDR-Mediated Transcription
We have shown that CaMKIV1–313 increases VDR phosphorylation and enhances both SRC1 intrinsic transactivation and VDR/coactivator interactions. Figure 6Go illustrates the functional consequences of these interplays between CaMKIV1–313 and the SRC proteins on VDR-activated reporter gene expression. In the absence of coactivator (SG5 control), CaMKIV1–313 augmented the 1,25-(OH)2D3-response of a VDRE-containing reporter 4.5-fold. SRC1 increased the 1,25-(OH)2D3-response 1.5-fold, and coexpression of SRC1 and CaMKIV1–313 produced a 7.3-fold synergistic level of VDR-mediated transcription compared with vector control. Similarly, SRC2 increased the activity of ligand-activated VDR 2.2-fold, and coexpression of SRC2 and CaMKIV1–313 activated liganded VDR 8-fold compared with vector alone, confirming the synergy between CaMKIV1–313 and SRC2. In contrast, CaMKIV1–313 and NCoA-62, an unrelated coactivator that lacks LXXLL motifs, activated VDR-mediated transcription in an additive manner. NCoA-62 increased the 1,25-(OH)2D3-response of VDR 1.2-fold, and together with CaMKIV1–313 the two proteins produced a 3.2-fold increase in activation. Unsurprisingly, given the weak interaction observed in the mammalian two-hybrid assay, SRC3 produced minimal coactivation of VDR-mediated transcription regardless of CaMKIV1–313 expression. Cumulatively, these studies suggest that the increased activity of VDR in the presence of CaMKIV1–313 stems from increased VDR interaction with SRC1 and SRC2, whereas SRC3 and NCoA-62 do not play an important role in the mechanism of CaMKIV1–313 action in COS-7 cells.



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Fig. 6. CaMKIV1–313 Synergizes with SRC1 and SRC2 to Activate VDR-Mediated Transcription

COS-7 cells were transfected with 680 ng (VDRE)4-TATA-Luc reporter, 200 ng of phRG-TK, 3 ng SG5-VDR, and 25 ng of SG5-SRC1, SG5-SRC2, SG5-SRC3, SG5-NCoA62, or SG5 vector in the presence of 300 ng of SG5-CaMKIV1–313 or SG5 vector control. After a 24-h treatment with ethanol or 10–8 M 1,25-(OH)2D3, cell extracts were prepared and analyzed for luciferase expression. Data are graphed as the mean of normalized duplicate samples ± SD.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1,25-(OH)2D3 exerts its biological effects through a multifaceted set of mechanisms. The genomic pathway involves 1,25-(OH)2D3 binding to VDR, VDR heterodimerization with the RXR, and heterodimer binding to specific DNA response elements in the promoters of 1,25-(OH)2D3 target genes, which leads to altered rates of transcription of selected target genes (1). A second established pathway involves membrane-initiated, nongenomic effects of 1,25-(OH)2D3 (reviewed in Refs.30 and 31). Indeed, numerous intracellular signaling pathways are rapidly activated (within seconds to minutes) by 1,25-(OH)2D3, including fluctuations in intracellular calcium levels (21), alterations in calcium channel activity (32), protein kinase C activation and translocation (33, 34), and activation of the MAPK pathway (35). The molecular mechanisms governing the rapid cellular responses to 1,25-(OH)2D3 are unresolved. Some reports suggest a VDR-independent pathway (36) that uses a novel membrane-associated receptor for 1,25-(OH)2D3 (37). In contrast, recent data implicate a requirement for functional VDR in the nongenomic response of cells to 1,25-(OH)2D3 (22, 38). Importantly, the potential for cross talk between the genomic and nongenomic pathways of 1,25-(OH)2D3 action is becoming increasingly evident (25, 39). In this manuscript, we provide new evidence for potential cross talk between the genomic and rapid response pathways by showing that mediators of intracellular calcium signaling potentiate 1,25-(OH)2D3-activated genomic responses mediated by the VDR.

Elevated intracellular calcium activates a number of intracellular signaling pathways and kinase cascades. Thus, to focus on Ca2+/CaMKs as downstream mediators of calcium signaling, constitutively active forms of these kinases were used. We showed that CaMKIV1–313 potently activates 1,25-(OH)2D3/VDR-mediated transcription from synthetic and natural promoter-reporter genes. This effect is selective for VDR-activated reporter gene expression as CaMKIV1–313 did not affect basal expression. Thus, it appears that CaMKIV1–313 acts upon select components of nuclear receptor-mediated transcription rather than components of the general transcription machinery. A likely target is VDR itself, but mutation of a putative CaMKIV consensus motif within the VDR LBD did not alter VDR activation by CaMKIV1–313 (Fig. 3AGo). This suggests that the activation of VDR occurs indirectly, perhaps through phosphorylation by another kinase or by phosphorylation of an intermediate protein. Alternatively, previous studies show that both CaMKIV and CaMKII also phosphorylate nonconsensus sequences (40, 41), so the possibility exists that CaMKIV1–313 targets another site on VDR. Consistent with this possibility, whole cell labeling in COS-7 cells shows that CaMKIV1–313 expression augments VDR steady-state phosphorylation approximately 2-fold, relative to the total amount of VDR protein, and also increases VDR protein levels (Fig. 3BGo). Therefore, whereas precise amino acid target identification on VDR will require more intensive research effort, our studies suggest that phosphorylation of VDR by CaMKIV plays a role in regulating VDR protein levels and transactivation.

CaMKIV signaling increases VDR phosphorylation, and it also modulates VDR activity via the SRC family of proteins. SRC proteins are transcriptional coactivators of VDR and other nuclear receptors (2). They associate with VDR in a 1,25-(OH)2D3-dependent manner (42, 43) and remodel chromatin at 1,25-(OH)2D3-responsive promoters by acetylating histones. Targeting SRC proteins to promoter regions independent of nuclear receptors is sufficient for transactivation, as evidenced by their intrinsic transactivation capacity when fused to heterologous DNA binding domains (for example, gal4 DBD) (44). In this study, we found that CaMKIV1–313 augments the intrinsic transactivation of SRC1. Importantly, this activation occurs independently of VDR and 1,25-(OH)2D3. Therefore, these studies suggest a role for SRC phosphorylation by CaMKIV in augmenting VDR-mediated transcription.

We hypothesized that CaMKIV1–313 promotes the ligand-dependent interaction between VDR and the SRC family of coactivators because the VDR LBD, which also recruits coactivators, was sufficient to mediate the enhanced 1,25-(OH)2D3-activated transcription by CaMKIV1–313 (Fig. 1AGo). Indeed, CaMKIV1–313 increases interaction of VDR with the SRC proteins without affecting interactions between VDR and its heterodimer partner RXR{gamma} (Fig. 5Go). Processes that promote VDR-SRC interaction are expected to enhance the transactivation potential of the VDR, an effect that is illustrated by the synergistic activation of a 1,25-(OH)2D3-responsive reporter gene by CaMKIV1–313 and SRC1 and SRC2 (Fig. 6Go). CaMKIV1–313 minimally affected the coactivator activity of SRC3, perhaps because VDR/SRC3 interaction and SRC3 coactivator activity is weak in this system. Functional synergy was not evident between CaMKIV1–313 and NCoA62/SKIP, an AF2-independent nuclear receptor coactivator that is unrelated to the SRCs and that coactivates VDR through distinct mechanisms (3). Thus, the effects of CaMKIV1–313 on VDR-activated transcription are restricted to select coactivators. Importantly, because CaMKIV1–313 fails to change VDR interaction with its heterodimer partner RXR{gamma} (Fig. 5Go), it is unlikely that the increased interactions observed for VDR with SRC1 and SRC2 are caused by a general effect such as increased expression of VDR.

In addition to the activation of 1,25-(OH)2D3/VDR-mediated transcription by CaMKIV1–313, we also observed a striking increase by constitutively active CaMKII in ROS17/2.8 cells that was not apparent in COS-7 cells (Fig. 2Go). Other studies have shown that CaMKIV and CaMKII mediate calcium-dependent activation of transcription factors such as CREB, although the activation varies greatly depending on cell type and which CRE-containing promoters are studied (45). Several possibilities exist to explain the differential effects of CaMKII in COS-7 and ROS17/2.8 cells. Species-related differences may play a role because COS-7 cell experiments used human VDR and experiments in ROS17/2.8 cells used rat VDR. Despite high sequence homology between human and rat VDRs, it is possible that CaMKII1–290 phosphorylates residues within rat VDR that are not present in the human variant. Alternatively, coactivator expression profiles differ greatly between cell types, and we show that CaM kinases selectively target discrete coactivators in VDR-mediated transcription (Fig. 6Go). Thus, the cohort of coactivator proteins expressed in a particular cell may determine the degree to which that cell responds to CaMKIV or CaMKII in augmenting vitamin D responsiveness. Ultimately, the physiologic effects of CaMKIV and CaMKII depend on which cell types coexpress VDR with one or more of the CaM kinases. CaMKIV has a restricted cell expression pattern, limiting the number of tissues in which it can influence VDR-activated transcription. In contrast, CaMKII and VDR are coexpressed in a variety of cell types, providing CaMKII ample opportunity to regulate VDR activity in a physiologic setting.

A previous study demonstrated that constitutively active orphan nuclear receptors including RORs and COUP-TFI (chicken ovalbumin upstream promoter-transcription factor 1) are activated by CaMKIV1–313 in keratinocyte cell lines (18). The authors suggest that the effect of CaMKIV1–313 is selective for the constitutively active orphan receptors because CaMKIV1–313 does not affect thyroid hormone receptor or estrogen receptor-mediated transcription. Importantly, our studies clearly extend a role for CaMKIV1–313 to liganded nuclear receptors, namely 1,25-(OH)2D3-activated transcriptional processes mediated by the VDR. In addition, our data provide strong evidence that CaMKII1–290 influences the actions of VDR in a cell-selective manner. Activation by CaMKIV1–313 and CaMKII1–290 was striking and selective for VDR-activated processes compared with the general transcriptional processes studied here. Importantly, this is the first indication that CaMKIV signaling results in phosphorylation of a nuclear receptor, although this may occur indirectly. Our studies also implicate novel actions of CaMKIV on the SRC family of proteins, resulting in increased interactions with VDR and augmented intrinsic transactivation. SRC1 contains several putative consensus sites for CaMKIV and CaMKII, possibly providing the opportunity for the CaM kinases to phosphorylate these residues.

In conclusion, we have shown that CaMKIV1–313 augments 1,25-(OH)2D3/VDR-mediated transcription through multiple mechanisms, including promoting phosphorylation of VDR, increasing VDR protein expression, stimulating the intrinsic transactivation of SRC1 and increasing the interaction of VDR with SRC1 and SRC2. These multiple effects culminate in synergistic enhancement of VDR-mediated transcription by CaMKIV and SRC proteins. Further studies will be necessary to demonstrate the roles that calcium and the CaMKs play in the genomic actions of the VDR. However, our studies suggest the potential for convergence of calcium signaling cascades elicited by 1,25-(OH)2D3 with the genomic 1,25-(OH)2D3-activated processes mediated by the VDR because nongenomic pathways dramatically enhance intracellular calcium levels in a variety of 1,25-(OH)2D3 target cells. Cross talk between the two pathways may provide an important means to augment 1,25-(OH)2D3 action in target cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
SG5-gal4-VDR was constructed as described (46). The LBD of RXR{gamma} (amino acids 209–463) was amplified using the PCR primers 5'-GCGCGAATTCGTGCAAGAAGAAAGACAG-3' and 5'-GCGCGGATCCCCCAGGGGTCATCCTGGG-3', and cloned into the SG5 vector (Stratagene, La Jolla, CA) containing amino acids 1–147 of the gal4 protein. VP16-VDR was constructed by digesting the VDR LBD from SG5-gal4-VDR using EcoRI/BamHI restriction digest sites and cloning the fragment into pVP16 (CLONTECH, Palo Alto, CA). SG5-CaMKII1–290 was constructed by inserting the 1.5-kb EcoRI fragment of pCDNA-CaMKII into SG5 (Stratagene).

(gal4)5-TATA-Luc contains five copies of the gal4 response element upstream of the TATA box from the E1b promoter, cloned into pGL3 (Promega, Madison, WI). (VDRE)4-TATA-Luc was created by cloning the VDRE (4)-TATA fragment (NdeI/BamHI) from (VDRE)4-TATA-GH (47) into SmaI and BglII sites of pGL3 basic (Promega).

The rat 1,25-(OH)2D3 24-hydroxylase promoter (–369 to +113 relative to the transcriptional start site) was cloned by PCR using ROS 17/2.8 genomic DNA. The forward and reverse primers used for PCR were 5'-AGCGCTCGAGTGTCCTCAGGGACCTTGC-3' and 5'-CGATAAGCTTTGGCAGCTATGGGGAGAG-3', respectively. The PCR product was cloned into pGL3 basic (Promega), and the final construct was confirmed by sequencing.

The construction of SG5-VDR was described previously (24). CMV-Flag-VDR was constructed by ligating the 1.6-kb EcoRI/Klenow/BamHI-digested fragment of SG5-VDR into Flag-CMV2 (Sigma, St. Louis, MO). This resulted in an in-frame fusion of VDR with an N-terminal FLAG epitope. SG5-gal4-SRC2 (RID) contains amino acids 595–780 encompassing the three nuclear receptor-interacting boxes of SRC2 fused to the gal4 DBD (amino acids 1–147) in the SG5-gal4 vector. SG5-gal4-SRC1 (RID) contains amino acids 595–840 fused to gal4 (amino acids 1–147) in the SG5-gal4 vector (4). Amino acids 588–774 of SRC3 composing the RID were amplified by PCR from SRC3 CMX-SRC3 (48) and cloned in-frame into the SG5-gal4 vector. SG5-gal4-SRC1 was constructed by cloning the SRC1 fragment from pCR-SRC1 (49) in-frame into SG5-gal4. SG5-SRC1 was constructed by inserting the SRC1 fragment from pCR-SRC1 into the SG5 vector (Stratagene), and SG5-SRC2/GRIP (glucocorticoid receptor-interacting protein) was described previously (50). SG5-SRC3 was constructed by inserting the SRC3 cDNA from CMX-SRC3 (48) into SG5 (Stratagene).

SG5-VDR (S172) was made using the GeneEditor Site Directed Mutagenesis System (Promega) and the primer 5'-GTGTGTCTGGCGTTGGGCCTG-3'. Briefly, the primer was phosphorylated and annealed to the denatured SG5-VDR DNA template. The mixture was incubated for 1 h for mutant strand synthesis and ligation, and DNA was transformed into a mismatch repair-deficient Escherichia coli strain for DNA production. DNA was purified using the Wizard Plus SV Miniprep kit (Promega), transformed into DH5{alpha} E. coli, and plated for colony selection. Accuracy of the S172A SG5-VDR mutation and surrounding sequence was confirmed by sequencing.

Cell Culture and Transfections
All cells were grown in 5% CO2 at 37 C. COS-7 cells were cultured in DMEM supplemented with 5% charcoal-stripped calf bovine serum (CBS) for 4 d before transfection. COS-7 cells were seeded for transfection in six-well plates at a density of 7 x 104 cells per well. HeLa cells were maintained in DMEM with 10% fetal bovine serum and seeded for transfection in six-well plates at a density of 7 x 104 cells per well. ROS 17/2.8 cells were maintained in DMEM with 5% charcoal-stripped CBS and seeded for transfection in six-well plates at a density of 8 x 104 cells per well. Cells were transfected by standard calcium phosphate precipitation procedures (27). The phRG-TK vector (Promega) was cotransfected for normalization of transfection efficiency, as indicated. After 18 h, cells were washed twice with PBS and media were replaced, then cells were treated with 1,25-(OH)2D3 or ethanol control, as indicated. After 24 h, cells were washed twice with PBS and lysed in 300 µl Passive Lysis Buffer (Promega) per well. Luciferase activity was determined from 20 µl samples of cell lysate using the Dual Luciferase Assay System (Promega) and a LMax luminometer (Molecular Devices, Sunnyvale, CA).

Whole Cell Labeling with 32P Orthophosphate
COS-7 cells were seeded in 60-mm plates at a density of 1.8 x 105 cells per plate. Cells were grown for 24 h in DMEM (Invitrogen, Carlsbad, CA) supplemented with 5% charcoal-stripped CBS, then transfected with CMV-Flag-VDR in the absence or presence of SG5-CaMKIV1–313 by standard calcium phosphate precipitation techniques. After 18 h, the precipitate was removed by washing twice with PBS, pH 7.2 (PBS), followed by replenishment of the media. After 24 h, the cells were washed in sterile Tris-buffered saline pH 7.5 (TBS) and phosphate-free DMEM was added, supplemented with 2% CBS dialyzed against TBS. [32P]-orthophosphate (0.06 mCi) was added to each plate in the absence or presence of 10 nM 1,25-(OH)2D3. The cells were labeled for 2 h, then washed twice with ice-cold TBS and harvested in 500 µl ice-cold lysis buffer [25 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, with 0.1% Nonidet P-40, 1 mM dithiothreitol, 50 mM NaF, 0.65 mM NaVO3, and a protease inhibitor cocktail (Sigma, St. Louis, MO)]. The cells were lysed for 10 min with rocking at 4 C, scraped, and transferred to microfuge tubes. After a 10-min centrifugation at 4 C, the supernatant was transferred to a new tube, a crude sample was reserved, and 50 µl of 50% M2 affinity gel slurry (Sigma) was added. After a 2-h incubation at 4 C, the M2 beads were collected and washed three times with cold lysis buffer containing 0.1% Nonidet P-40. The washed pellet was solubilized in SDS-PAGE sample buffer and separated on 10% denaturing polyacrylamide gels. The gel was dried and exposed to X-OMAT-ARS film for 8 h. A second gel containing crude samples was transferred to polyvinylidene difluoride membrane and processed by standard Western blotting procedures using the 9A7 rat monoclonal antibody raised against VDR and chemiluminescent detection. No 32P signal was observed from the exposure time used for Western blot. Quantitation of 32P and Western signal was performed from film scans by the GeneGenius imaging system (Syngene, Frederick, MD), using GeneSnap and GeneTools (Syngene) software for analysis.


    ACKNOWLEDGMENTS
 
We thank B. W. O’Malley (Baylor University) for the generous contribution of pCR3.1-SRC, and A. R. Means (Duke University) for SG5-CaMKIV1–313 and pCDNA-CaMKII1–290. We also thank D. C. Schultz for generous use of equipment, and A. L. M. Sutton for thoughtful discussion of the manuscript.


    FOOTNOTES
 
This work was supported by Grant RO1DK50348 and RO1DK53980 from The National Institute of Diabetes and Digestive and Kidney Diseases. T.I.E. is supported by an Institutional National Research Service Award from the National Institutes of General Medical Sciences (T32GM 08803). Portions of this work were presented at the 25th Annual Meeting of the American Society for Bone and Mineral Research, September 2003.

First Published Online May 26, 2005

Abbreviations: CaM, Calmodulin; CaMK, calcium/CaM-dependent kinase; CBS, calf bovine serum; CREB, cAMP response element binding protein; DBD, DNA binding domain; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; LBD, ligand binding domain; RID, receptor interaction domain; ROR, retinoid-related orphan receptor; RXR, retinoid X receptor; SRC, steroid receptor coactivator; VDR, vitamin D receptor; VDREs, vitamin D response elements; VP16, virion protein 16.

Received for publication September 27, 2004. Accepted for publication May 17, 2005.


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