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Molecular Endocrinology, doi:10.1210/me.2006-0315
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Molecular Endocrinology 21 (1): 197-214
Copyright © 2007 by The Endocrine Society

Transcriptional Control of Receptor Activator of Nuclear Factor-{kappa}B Ligand by the Protein Kinase A Activator Forskolin and the Transmembrane Glycoprotein 130-Activating Cytokine, Oncostatin M, Is Exerted through Multiple Distal Enhancers

Sungtae Kim, Miwa Yamazaki, Nirupama K. Shevde and J. Wesley Pike

Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Address all correspondence and requests for reprints to: Dr. J. W. Pike, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706. E-mail: pike{at}biochem.wisc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Receptor activator of nuclear factor-{kappa}B ligand (RankL) is a potent osteoclastogenic cytokine the expression of which is regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], protein kinase A (PKA) activators such as PTH and transmembrane glycoprotein 130 (gp130)-activating cytokines such as oncostatin M. We recently identified five highly conserved chromatin domains located significant distances upstream of the RankL transcriptional start site that contribute to the ability of 1,25-(OH)2D3 and its receptor to enhance RankL gene output. We therefore screened these five common regulatory regions for their potential ability to mediate the actions of PKA- and gp130-activators using a directed chromatin immunoprecipitation approach employing antibodies to the PKA target cAMP response element-binding protein (CREB) and the gp130 target signal transducer and activator of transcription 3. CREB was identified at each of the upstream regulatory regions; signal transducer and activator of transcription 3, in contrast, was associated with only a subset. Interestingly, only the most distal of these regions demonstrated CREB- and oncostatin M-regulated transcriptional activity in a heterologous transfection system. Mapping studies pointed to two highly conserved cAMP response elements as well as an adjacent regulatory site that bound Runt transcription factor 2 and was able to influence both basal as well as hormone-inducible RankL activity. Surprisingly, PKA and gp130 activation prompted recruitment of RNA polymerase II to the five distal enhancers as well as to the RankL transcriptional start site. Activation was also accompanied by a significant and location-selective rise in histone 4 acetylation. This study demonstrates that the activation of RankL gene expression by PKA- and gp130-inducers is mediated via common regulatory domains that also served to facilitate the activity of 1,25-(OH)2D3.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RECEPTOR ACTIVATOR OF nuclear factor (NF)-{kappa}B ligand (RankL) is a potent osteoclastogenic cytokine (1, 2). Its expression in stromal cells and osteoblasts is regulated transcriptionally by numerous factors including 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (3), protein kinase A (PKA) activators such as PTH (4, 5) and prostaglandin E2 (PGE2) (6), and transmembrane glycoprotein 130 (gp130)-activating cytokines such as IL-6, IL-11, and oncostatin M (OSM) (7, 8, 9). Each of these regulators plays either a normal physiological role in the maintenance of RankL expression or can participate in pathological overexpression of RankL, which often results in net bone loss and osteoporosis (2, 10). The mechanism of RankL action on osteoclast precursors is now well established, wherein the TNF-like factor activates and/or induces the expression of transcription factors such as c-fos, nuclear factor (NF)-{kappa}B, and NFATc1, all of which orchestrate the complex program of osteoclast differentiation, activation, and survival (11, 12). Due to both its physiological and pathophysiological importance, an understanding of the molecular mechanisms relevant to the induction of RankL gene expression and its biological activity continues to be of topical importance.

The calcemic hormones 1,25-(OH)2D3 and PTH are especially important regulators of bone remodeling, acting, in part, to modulate key aspects of osteoclast formation and bone resorption. This regulation is mediated primarily via stromal cells and cells of the osteoblast lineage (13, 14), a phenomenon that contributed to a hypothesis developed years earlier by Rodan and Martin (15) that osteoblasts and hematopoietic osteoclast precursors were in constant communication and that their respective skeletal activities were tightly coupled. Subsequent studies have demonstrated unequivocally that the osteoblastic factor responsible for osteoclastogenic activity in coculture experiments is RankL, a membrane-bound protein belonging to the TNF family of regulators (16, 17). The ability of both 1,25-(OH)2D3 and PTH to induce RankL confirmed that this regulatory protein was indeed the long-sought-after factor responsible for osteoclast formation. More recently, studies focusing on the process of activation of RankL gene expression by 1,25-(OH)2D3 and PTH have revealed significant details regarding the nature of the activation pathways. Not surprisingly, RankL induction by 1,25-(OH)2D3 is mediated by the vitamin D receptor (VDR). Accordingly, genetic deletion of the VDR in mice results in complete loss of RankL sensitivity to 1,25-(OH)2D3 (18). In the case of PTH, extensive studies by O’Brien and co-workers (19) have revealed a central role for the PKA activation pathway and the direct involvement of cAMP response element (CRE)-binding protein (CREB) on RankL induction. Unfortunately, whereas much has been learned recently regarding the roles of these pathways on RankL activation, the details whereby the downstream activators of these pathways converge on the regulatory regions of the RankL gene are only now emerging.

RankL is also induced by osteoclastogenic cytokines such as IL-6, IL-11, and OSM (7, 8, 9, 19, 20). In opposition to the activities of the calcemic hormones, cytokine activation arises largely as a result of various inflammatory events and often produces pathological bone loss (21). The cytokine signaling pathways have also been characterized, revealing the involvement of not only the gp130 coreceptor but also the downstream Jak/signal transducer and activator of transcription (Stat) signal transduction pathway as well (20). These studies established an integral role for the Stat3 transcription factor in the regulation of RankL gene expression. The control sequences located within the RankL gene that mediate the actions of these cytokines, however, remain uncharacterized.

We recently described a mechanism whereby the vitamin D hormone 1,25-(OH)2D3 induces RankL gene expression in the mouse stromal cell line ST2 (22). Using DNA enriched for VDR/retinoid X receptor (RXR) binding sites via chromatin immunoprecipitation (ChIP), we screened a large region of the RankL gene locus at 50 bp resolution using a ChIP-DNA microarray (chip) approach. This screening revealed five sites of VDR/RXR interaction, all located at significant distances upstream of the RankL transcriptional start site (TSS). Additional analyses revealed that the most distal of these sites, which we termed the RL-DCR (RankL distal control region), contained regulatory elements for the VDR/RXR heterodimer, the glucocorticoid receptor, and other potential transcriptional regulators. Interestingly, Fu et al. (23) also uncovered the RL-DCR while studying the actions of cAMP using an alternative approach that focused upon the use of a bacterial artificial chromosome containing the RankL gene locus. Importantly, these investigators were able to delete the RL-DCR from the mouse genome and to demonstrate that the activity of this region was central to the ability of PTH to regulate RankL expression both in vivo and ex vivo.

In the present work, we describe the results of additional studies that focus on the activities of PKA activators and OSM at the RankL gene locus. Using forskolin- or OSM-activated ST2 cells, we screened, using a directed ChIP approach, the five previously identified upstream enhancer regions of the RankL gene for the presence of CREB and Stat3 binding sites. To our surprise, we discovered that CREB was associated with not only the far upstream RL-DCR, as reported by Fu et al. (23), but also with the four downstream enhancers of the RankL gene as well. Stat3, in contrast, was localized to a subset of these distal enhancers. Additional studies characterize the properties of these enhancers with respect to forskolin and OSM induction. We conclude that these five mouse RankL regulatory regions contain cis clusters, mediate the actions of 1,25-(OH)2D3, PTH, and OSM, and may facilitate the activity of additional modulatory factors as well.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PKA Activators and OSM Induce RankL in ST2 Cells
ST2 cells represent a useful model with which to explore RankL activation by 1,25-(OH)2D3, PKA activators such as forskolin, cAMP, PTH, and the PGE2, and gp130 activators such as IL-6, IL-11, and OSM (1, 24). Indeed, previous studies using ST2 cells have firmly established that these regulators induce not only RankL, but facilitate the ability of these stromal osteoblasts to direct the formation of new osteoclasts in coculture assays containing osteoclast precursors (1). To both establish this model and to confirm the relevance of these pathways, we treated ST2 cells with an optimal dose of 1,25-(OH)2D3, the PKA activator forskolin (a PTH surrogate), PGE2, and the cytokine OSM and assessed the consequence of these treatments on RankL mRNA content using RT-PCR. As can be seen in Fig. 1Go, each of these regulators was capable of inducing RankL mRNA in a time-dependent fashion. Such bona fide 1,25-(OH)2D3 targets as Cyp24a1 and osteopontin (OPN) and the OSM target OPN were also induced as expected (25). Based upon these results and bolstered by our previous studies (22), we embarked upon a series of experiments to define how the PKA activators such as forskolin and cAMP and the osteoclastogenic cytokine OSM also induce this important gene.


Figure 1
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  		Fig. 1. Induction of mRankL mRNA by 1,25-(OH)2D3, Forskolin (FSK), PGE2, and OSM in ST2 Cells

ST2 cells were treated for periods up to 24 h with either 1,25-(OH)2D3 (10–7 M), forskolin (10–6 M), PGE2 (10–7 M), or OSM (20 ng/ml). Total RNA was isolated and subjected to RT-PCR analysis using primers specific to mouse Cyp24a1 (30 cycles), OPN (Opn) (15 cycles), RankL (30 cycles), or ß-actin (20 cycles) as documented in Materials and Methods. The results are typical of multiple similar experiments.

 
The PKA Activators Forskolin and Dibutyryl (db)-cAMP Promote CREB Binding to the Five RankL Distal Enhancers
Our most recent studies in ST2 cells revealed five sites of 1,25-(OH)2D3 activity within the RankL gene locus (22). These regions were highly conserved across multiple species, including human. Enhancers are modular, however, and often mediate or integrate the actions of more than one transactivating factor. Thus, we explored the possibility that these enhancers might interact with CREB, a primary transcription factor target of PKA and a regulatory component known from earlier studies to mediate the activity of PTH on RankL expression as well (19). ST2 cells were treated with vehicle, forskolin, or db-cAMP and subjected 6 h later to ChIP analysis using antibodies specific to mouse CREB. The precipitated DNA was then analyzed by PCR using primer sets capable of assessing the presence of unique regions of the RankL gene extending from the TSS to more than 150 kb upstream as indicated in Fig. 2Go. As can be seen, five of the primer sets detect DNA derived from the five previously identified 1,25-(OH)2D3-regulated regions, and a sixth set was capable of detecting the highly conserved region mouse (m)RLD5b located immediately upstream and contiguous with mRLD5. The additional primers monitored intervening regions or regions extending upstream of mRLD5b. The results documented in Fig. 2Go reveal that CREB does indeed localize to each of the five regulatory regions (mRLD1–mRLD5) as well as mRLD5b in response to the PKA activators. Although the binding of CREB to these sites varies to some degree, and some constitutive interaction is also apparent, the binding of CREB appears to be strongest at mRLD5b and at mRLD2. These results suggest that multiple enhancers located upstream of the RankL TSS mediate PKA activation via CREB. They do not, however, define whether the cross-linking interaction of CREB at these sites is via a direct, DNA binding interaction or arises as a result in an indirect tethering mechanism.


Figure 2
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  		Fig. 2. PKA Activation in ST2 Cells Induces CREB Binding to the Upstream Enhancers of the Mouse RankL Gene

ST2 cells were treated with either vehicle, forskolin (10–6 M), or db-cAMP (1.5 x 10–3 M) for 6 h and then subjected to ChIP analysis using antibodies to CREB or control IgG. The immunoprecipitated DNA was isolated and then amplified using the primer sets the positions of which are illustrated in the top panel and the sequences of which are documented in Table 1Go. Amplification used 31 cycles for mRLD1-mRLD5, mRLD5b, mRLIS1-mRLIS8, and mRLIS9 and 34 cycles for the TSS. PCR analyses were all performed within the linear range of amplification. All results are typical of several separate experiments. FSK, Forskolin.

 
The mRLD5b Region of the Mouse RankL Gene Confers Forskolin Sensitivity to a Heterologous Promoter
The ability of PKA activators to induce CREB binding to the mRLD1–mRLD5 and mRLD5b regions suggests the possibility that these regions might be capable of transferring forskolin sensitivity to a heterologous promoter such as that for thymidine kinase (TK). We therefore cloned each of these DNA segments into a common TK-luciferase reporter plasmid, as documented in Fig. 3AGo, transfected these constructs into ST2 cells, and assessed their capacity to mediate transcriptional response in the presence of an optimal concentration of forskolin. Each construct was also evaluated for response to the cytokine OSM as well, with 1,25-(OH)2D3 serving as a positive control. To our surprise and as documented in Fig. 3BGo, among the mRLD1–mRLD5 fragments, only mRLD4 was capable of mediating a modest response to forskolin. The newly cloned DNA fragment mRLD5b, however, was fully activated and strongly induced by PKA activation. This DNA fragment also exhibited a striking increase in basal activity relative to the other segments examined. Interestingly, both the mRLD4 and mRLD5 regions appeared to mediate response to OSM, although the inducible activity was not particularly striking. 1,25-(OH)2D3 induction was generally limited to the mRLD5 region, a region shown previously to contain a complex and highly active vitamin D response element (VDRE) (22). To test whether both mRLD5 and mRLD5b together would manifest the appropriate 1,25-(OH)2D3, forskolin, and OSM response, we cloned this contiguous DNA fragment into the TK reporter vector, introduced this fragment (termed mRLD5a/b) into ST2 cells, and exposed the cells for 24 h to each of the three stimulators. As can be seen in Fig. 3Go, B and C, the mRLD5a/b fragment manifested a similarly high basal activity and was indeed responsive to each of the inducers tested. Perhaps most importantly, the effects of both 1,25-(OH)2D3 and forskolin were additive (Fig. 3CGo). Collectively, these studies indicate that the mRLD5 region mediates both 1,25-(OH)2D3 and OSM response, the mRLD5b region likely contains DNA elements that mediate forskolin induction, and the extended fragment mediates the activities of all three agents. The simultaneous presence of significant CREB binding at the mRLD1–mRLD5 regions as identified via ChIP, however, suggests that each of these regions participates in the induction of RankL gene expression in response to PKA activation.


Figure 3
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  		Fig. 3. The Highly Conserved RL-DCR Region (mRLD5 and mRLD5b) of the mRankL Gene Mediates Transcriptional Induction by Forskolin (FSK) and OSM

A, Location and gene coordinates (distance from the RankL TSS) of the cloned mRLD1-mRLD5, mRLD5b, and mRLD5a/b (RL-DCR) regions of the mRankL gene (February 2006 University of Santa Cruz Genome Assembly). Depiction and designation of individual RankL enhancer constructs prepared in either the pTK or the pmRL (100) minimal RankL promoter vectors. B, Basal and stimulus-induced activity of mRLD1–mRLD5, mRLD5b, and mRLD5a/b in the context of the TK promoter in ST2 cells. ST2 cells were transfected with pCH110-ßgal (50 ng), pcDNA-hVDR (50 ng), and either the pTK control vector (250 ng) or pTK-mRLD1, pTK-mRLD2, pTK-mRLD3, pTK-mRLD4, pTK-mRLD5, pTK-mRLD5b, or pTK-mRLD5a/b. Cells were treated with either vehicle, 1,25-(OH)2D3 (10–7 M), forskolin (10–6 M), or OSM (20 ng/ml) and evaluated 24 h later for both luciferase and ß-gal activity as described in Materials and Methods. Each point represents the normalized RLU average ± SEM for a triplicate set of transfections. *, P < 0.05 compared with control. These data are representative of three or more similar experiments. C, The mRLD5a/b (RL-DCR) region of the RankL gene mediates the activity of 1,25-(OH)2D3, forskolin, and OSM. ST2 cells were transfected as in panel B above with pCH110-ßgal (50 ng), pcDNA-hVDR (50 ng), and either the pTK control vector (250 ng) or pTK-mRLD5a/b. Cells were treated with either vehicle, 1,25-(OH)2D3 (10–7 M), forskolin (10–6 M), OSM (20 ng/ml) or combinations of forskolin (10–6 M) and increasing concentrations of 1,25-(OH)2D3 (10–10 to 10–7 M) and evaluated 24 h later as described in panel B. Each point represents the normalized RLU average ± SEM for a triplicate set of transfections. *, P < 0.05 compared with control; **, P < 0.05 compared with control and *. These data are representative of several similar experiments.

 
mRLD5b Confers Forskolin Sensitivity to a Homologous RankL Minimal Promoter
The failure to secure forskolin-inducible activity from the mRLD1–5 constructs in the context of the TK promoter prompted us to examine the activity of these DNA fragments in the context of an homologous mRL minimal promoter comprised of the RankL promoter sequence –100/+56. We therefore inserted each of the fragments identified in Fig. 3AGo into a pGL3 vector containing the mRL promoter, transfected these constructs into ST2 cells, and assessed individual response to forskolin and OSM. The results, as seen in supplemental Fig. S1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org, indicate that, in this context, a similar forskolin response profile across each of the independent DNA fragments is also observed. Surprisingly, the weak OSM activity seen in the TK context was apparent only in the mRLD5 construct. We conclude from these studies that even in the context of a homologous promoter, the localized contexts of each of the mRL DNA fragments are largely retained. An alternative possibility, however, is that the RL DNA fragments that do not exhibit response to forskolin (or OSM) are activated via an indirect tethering mechanism.

Mapping the Regulatory Sites in mRLD5b that Mediate Forskolin Induction
To localize the region within mRLD5b responsible for both increased basal activity as well as inducibility by forskolin, we prepared a series of DNA fragments wherein a set of 5'-deletion fragments of D5b were fused upstream of mRLD5, as illustrated in the left portion of Fig. 4Go. Fusion of full-length mRLD5b with mRLD5 comprised mRLD5a/b, the RL-DCR enhancer region. The plasmid constructs were introduced into ST2 cells via transfection and evaluated 24 h later for both basal activity and transcriptional response to forskolin as well as OSM. As can be seen in the right portion of Fig. 4Go, whereas modest OSM activity was evident in mRLD5 and generally (although not always) retained as the fragments extended increasingly upstream, only mRLD5-E4 manifested a striking response to forskolin reminiscent of the activity seen with the mRLD5a/b construct alone. The expected response to 1,25-(OH)2D3 was also observed. These results suggest that the DNA element responsible for forskolin-inducible activity in this regulatory region of the RankL gene is fully contained in mRLD5-E4 and is therefore located within the most distal 76 bp of the mRLD5-E4 fragment. mRLD5a/b, however, demonstrated a significant increase in basal activity relative to the shorter constructs. This observation suggests that an element(s) responsible for significant basal activity is likely present in the final 108 bp of mRLD5a/b and can be fully dissociated from the forskolin-inducible activity.


Figure 4
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  		Fig. 4. Enhancer Analysis of the mRLD5a/b Region Reveals the Approximate Location of Transcriptional Response to Forskolin (FSK)

Left panel, Schematic of the mRankL enhancer fragment series used to map basal and forskolin-inducible response within the mRLD5b region. Numbering at the bottom represents distance from the RankL TSS (Feb 2006 Assembly). The RankL VDRE is identified in mRLD5. Right panel, Transcriptional activities of the mRLD5 enhancer fragments in response to vehicle, 1,25-(OH)2D3, forskolin, or OSM. ST2 cells were transfected with pCH110-ßgal (50 ng), pcDNA-hVDR (50 ng), and either the pTK control vector (250 ng) or the pTK-mRLD5 extension series as identified in the left panel. Cells were treated with either vehicle, 1,25-(OH)2D3 (10–7 M), forskolin (10–6 M), or OSM (20 ng/ml) and evaluated after 24 h for both luciferase and ß-gal activity as described in Materials and Methods. *, P < 0.05 compared with control. These results were confirmed in at least two separate experiments.

 
Identification of Forskolin-Inducible CREs Located in mRLD5b
Evaluation of the most distal 180 bp of mRLD5b using the CONSITE (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite) algorithm revealed two potential CREs, as indicated in Fig. 5AGo. These CREs are located within 12 bp of each other at –76567 to –76560 (CRE1) and –76587 to –76580 (CRE2) and are highly evolutionarily conserved across a number of genomes including that of the human (26). To test whether these two elements were capable of mediating forskolin response in the context of mRLD5b, we first introduced 3-bp mutations into each of the potential CREs in both of the mRLD5b and mRLD5a/b segments as indicated in Fig. 5AGo. The mutant mRLD5b and mRLD5a/b constructs, as well as mRLD5b and mRLD5a/b illustrated in the left portion of Fig. 5BGo, were then introduced into ST2 cells, and their activities in response to forskolin were evaluated 24 h later. As can be seen in the right portion of Fig. 5BGo, mutations in either CRE1 or CRE2 reduced the basal activity of mRLD5b and individually reduced mRLD5b response to forskolin. Interestingly, although mutations in CRE1 or in both CRE1 and CRE2 in the context of mRLD5a/b also affected basal activity, neither of the mutations resulted in complete loss of forskolin response. The expected loss of response to 1,25-(OH)2D3 upon mutation of the VDRE in mRLD5a/b was also observed. Finally, we tested the capacity of the small 180-bp fragment of mRDL5b, termed "mRLD5b-CRE1/2," to mediate forskolin inducibility directly. Extending from –76,705 to –76,525, this fragment was cloned into the TK promoter reporter gene and transfected into ST2 cells, and its activity was evaluated in the presence of either vehicle or forskolin. As can be seen in Fig. 5CGo, mRLD5b-CRE1/2 displayed both high basal activity, similar to that of mRLD5b and mRLD5a/b, and full activation in the presence of the PKA inducer forskolin. Scrambled mutagenesis of either CRE1 or CRE2 in this context resulted in complete loss of forskolin activation, and no response was observed in the presence of 1,25-(OH)2D3 or OSM (data not shown). These results suggest that CRE1 and CRE2 located within the mRLD5b region are capable of mediating forskolin response and likely play a significant role in the induction of RankL gene expression by this activator. Interestingly, the similarity in basal activity observed with the RLD5b-CRE1/2, mRLD5b, and mRLD5a/b constructs confirms the potential for a basal element(s) located within the most distal 108 bp of mRLD5b.


Figure 5
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  		Fig. 5. Identifying the mRLD5b-CREs

A, Location and DNA sequence of putative CREs in the mRLD5b region revealed by in silico analysis (CONSITE (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite). The nucleotide numbering represents the boundaries of the mRLD5b-CREs relative to the RankL TSS (Feb 2006 Assembly). Triplet base alterations introduced by site-directed mutagenesis into each of the two putative CREs (CRE1 and CRE2) or the VDRE are indicated above the mouse sequence. B, Mutations in the putative CREs located in mRLD5b compromise basal and forskolin-inducible response. ST2 cells were transfected with pCH110-ßgal (50 ng), pcDNA-hVDR (50 ng), and either pTK control vector (250 ng) or one of the individual wild-type or mutant versions of pTK-mRLD5b or pTK-mRLD5a/b as depicted in the left panel. Cells were treated with either vehicle, 1,25-(OH)2D3 (10–7 M) or forskolin (10–6 M) and evaluated after 24 h for both luciferase and ß-gal activity as seen in the right panel. The X depicts the presence of the mutation. *, P < 0.05 compared with control. These results were repeated at least twice with similar results. C, Basal and forskolin-inducible activity of mRLD5b-CRE1/2. ST2 cells were transfected with pCH110-ßgal (50 ng) and either pTK control vector (250 ng), pTK-mRTLD5b, or mRLD5b-CRE1/2 as indicated to the left. Cells were treated with either vehicle or forskolin (10–6 M) and evaluated 24 h later for both luciferase and ß-gal activity as seen in the right panel. *, P < 0.05 compared with control. FSK, Forskolin.

 
CREB Interacts Directly with the Functional CREs Identified in mRLD5b
We also examined whether CREB could bind to the CREs we had identified functionally in the above studies. To this end, we carried out EMSA using duplex oligonucleotides comprised of either CRE1 or CRE2. As can be seen in supplemental Fig. S2, published as supplemental data on The Endocrine Society’s Journals Online web site, a component in forskolin-stimulated ST2 cell nuclear extracts was capable of interacting directly with each of the CREs. The ability of cold CRE1 and CRE2 to complete in an almost equivalent fashion with labeled CRE1 or CRE2 indicates that the relative affinity of the component for each of the binding sites is similar. Finally, the ability of a CREB antibody, but not an irrelevant antibody, to disrupt this interaction provides direct support for the presence of CREB within the complex. Taken together with the above studies, our results suggest that the two CREs located at –76567 to –76560 (CRE1) and –76587 to –76580 (CRE2) likely represent bona fide regulatory elements capable of both binding CREB and mediating the transactivation potential of this complex in transfected cells.

Properties of CREB Activation and Interaction with RankL CREs
The interaction of CREB with DNA sequence elements at target genes is exceedingly complex (27). Indeed, although stimulus-induced binding of CREB to DNA frequently occurs, recent studies suggest that CREB occupancy at numerous gene targets is more frequently constitutive (28). A key feature of CREB’s ability to transactivate is its capacity to recruit coregulators, particularly, but not exclusively, those such as CREB-binding protein and/or p300 (29). Indeed, the phosphorylation of CREB at serine-133 within the kinase-inducible domain of this protein appears to be a key determinant of CREB-binding protein/p300 recruitment (27). To explore whether CREB is phosphorylated when bound to RankL regulatory regions, we treated ST2 cells with either vehicle or forskolin and conducted a ChIP analysis 6 h later using antibodies to both CREB and phosphoserine 133-specific CREB. As can be seen in Fig. 6Go, treatment with forskolin resulted in the localization of CREB to three relevant regions of the RankL locus (mRLD5 and mRLD5b regions as well as to mRLD4). Interestingly, phosphoserine 133-CREB also localized to these three regions as well (phosphoserine 133-CREB was not detectable at mRLD1–mRLD3; data not shown). These observations suggest the possibility that forskolin-induced binding of CREB to regulatory regions of the RankL gene, particularly those located within the mRLD5a/b region, involves direct phosphorylation of CREB at serine-133. We also examined whether ACREB, a dominant-negative form of wild-type CREB (30), could block forskolin-induced CREB activation of mRLD5b transcription. Accordingly, ACREB (or empty vector) was cotransfected together with pTK-mRLD5b into ST2 cells, and the response to forskolin was evaluated after 24 h. The results, as documented in supplemental Fig. S3A, published as supplemental data on The Endocrine Society’s Journals Online web site, demonstrate that ACREB can fully block mRLD5b-mediated transcriptional activity induced by forskolin. Because this dominant-negative mutant interacts directly with wild-type CREB and functions, in part, to prevent preferential CREB DNA binding, the data support the idea that transactivation by CREB requires direct DNA binding to the CREs located in the mRLD5b region (27). In a final experiment, we asked whether other members of the B-zip family could be detected at the mRLD5b region in response to forskolin, particularly ATF4, which has been suggested to bind to more proximal regions at the RankL promoter (31). ChIP analysis was therefore performed as above using antibodies not only to CREB and phosphoserine 133-CREB, but also to ATF2 and ATF4. As can be seen in supplemental Fig. S3B, neither of these CREB-like factors appears to associate across the mRLD5b region, although some binding was seen with ATF2. Interestingly, these results reveal that 1,25-(OH)2D3 also induces CREB binding to the mRLD5b region of the RankL gene and that forskolin, in turn, enhances VDR binding to the mRLD5 region. The former observation suggests that the vitamin D hormone may be able, in some manner, to impact the PKA pathway and is consistent with the finding by O’Brien and colleagues (19) that the PKA inhibitor H89 blocks not only PKA-activated RankL gene expression, but 1,25-(OH)2D3-mediated induction as well. That forskolin can increase VDR binding on gene subsets is also consistent with our previous observations (32). Collectively, however, these results suggest that CREB is indeed the predominant transcription factor that mediates the actions of forskolin at the RankL gene locus, and that the actions of CREB involve both phosphorylation at serine-133 and specific DNA binding.


Figure 6
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  		Fig. 6. Forskolin (FSK)-Induced CREB Binding to the RankL Regulatory Is Associated with CREB Phosphorylation at Serine-133

ST2 cells were treated with vehicle or forskolin (10–6 M) for 6 h and then subjected to ChIP analysis using antibodies to CREB, phosphoserine 133-CREB, or control IgG. The immunoprecipitated DNA was isolated and then amplified using primer sets, the locations of which relative to the RankL upstream regions are identified in the upper panel of Fig. 2Go. PCR analyses were all performed within the linear range of amplification using 31 cycles. All results are typical of several separate experiments.

 
The Basal Activity of mRLD5b Requires an Osteoblast-Specific Element (OSE2)
The in silico analysis conducted earlier using the CONSITE (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite) algorithm also revealed the presence of a potential, highly conserved Runt transcription factor 2 (Runx2) binding site or OSE2 (33) 12 bp immediately upstream of CRE2 at –76606 to –76614 as indicated in Fig. 7AGo. Note that this site is absent in mRLD5-E4 but present in mRLD5a/b, wherein a striking increase in basal activity is apparent (see Fig. 4Go). We therefore explored the impact of this OSE2 on the relative transcriptional activity of mRLD5b and mRLD5a/b by introducing mutations into the OSE, as indicated in Fig. 7AGo, and evaluating the transcriptional consequence of this mutagenesis in response to forskolin after transfection into ST2 cells. Response to 1,25-(OH)2D3 was used as a control. As can be seen in Fig. 7BGo, mutations in the OSE2 resulted in a dramatic reduction in basal activity and, surprisingly, a blunting of the response to forskolin. These effects were largely recapitulated when the activity of the mRLD5a/b fragment was evaluated; here, the overall responses to both forskolin and 1,25-(OH)2D3 were reduced, although the most dramatic effect was clearly upon the underlying basal activity. We also explored the potential presence of Runx2 at the OSE2 using ChIP analysis. This analysis revealed that Runx2 was indeed present at the mRLD5b region (Fig. 7CGo), particularly following treatment with forskolin. Runx2 binding was also apparent at mRLD2 as well. The level of Runx2 cross-linking at these sites was not particularly striking, however. We conclude from these studies that the Runx2 transcription factor may play an important role in RankL basal expression as well as activation by forskolin. The role of Runx2 in RankL remains controversial (34). Because basal RankL expression does not appear to be affected in the Runx2 null mouse (34, 35), however, it seems likely that transcriptional redundancy involving other members of this transcription factor family may occur to account for the presence of Runx2 observed in this experiment.


Figure 7
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  		Fig. 7. Runx2 Contributes to the Basal and Forskolin (FSK)-Inducible Activity of mRLD5b

A, DNA sequence of putative OSE2 revealed by in silico analysis (CONSITE (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite) in the mRLD5b region. The nucleotide numbering represents the boundaries of the mRLD5b-OSE2 relative to the RankL TSS (Feb 2006 Assembly). Triplet base alterations introduced by site-directed mutagenesis into the OSE2 are indicated above the sequence. The proximal CREs are indicated as well. B, Effect of mutations in the OSE2 on mRLD5b or mRLD5a/b transcriptional activity. ST2 cells were transfected with pCH110-ßgal (50 ng) and either pTK control vector (250 ng), pTK-mRLD5b, pTK-mRLD5b-OSEm, pTK-mRLD5a/b, or pTK-mRLD5a/b-OSEm and then treated with either vehicle, 1,25-(OH)2D3 (10–7 M) or forskolin (10–6 M) as seen in the left panel. Luciferase and ß-gal activity were measured in extracts after 24 h. *, P < 0.05 compared with control. C, Runx2 is associated with the mRLD5b enhancer in vivo. ST2 cells were treated with either vehicle, db-cAMP (1.5 x 10–3 M) or forskolin (10]–6 M) for 6 h and then subjected to ChIP analysis using antibodies to either Runx2 or control IgG. The immunoprecipitated DNA was isolated and then amplified using primer sets, the positions of which are illustrated in the top panel of Fig. 2Go. PCR analyses were all performed within the linear range of amplification using 31 cycles. These results are typical of several separate experiments.

 
OSM Promotes Stat3 Binding to a Subset of the RankL Distal Enhancers
Our earlier studies of the transcriptional activities of mRLD1–mRLD5 and mRLD5b (see Fig. 3Go) suggested that the inducible activity of OSM might be mediated through elements located in the mRLD4 and mRLD5 regions of the RankL gene, although this activity was modest at best. Stat3 is the primary target of gp130 activation and is known to direct the induction of RankL by OSM as well as by IL-6 and IL-11 (20). We therefore explored whether treatment with OSM could promote Stat3 binding to mRLD4 and mRLD5 and surrounding regions of the RankL gene locus. ST2 cells were treated with either vehicle or OSM and subjected 6 h later to ChIP analysis using antibodies specific to Stat3. The precipitated DNA was then analyzed by PCR using the primer sets described in Fig. 2AGo. As observed in Fig. 8Go, time-dependent Stat3 was indeed detected at the mRLD4 and mRLD5 regions of the RankL gene as well as at mRLD5b, although the activity appeared most prevalent at mRDL5. Time-dependent Stat3 binding, however, was not detected at mRLD1–mRLD3, at sites located between these functional regions, or at the RankL TSS, although some residual binding, perhaps representative of background, was observed. These results provide further evidence that the distal enhancers we originally identified by ChIP-DNA microarray analysis using antibodies to the VDR are indeed modular in nature and capable of mediating differential response to several independent regulators.


Figure 8
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  		Fig. 8. OSM Induces Stat3 Binding to a Subset of the RankL Enhancers

ST2 cells were treated for periods up to 6 h with either vehicle or OSM (20 ng/ml) and then subjected to ChIP analysis using antibodies to Stat3 or control IgG. The immunoprecipitated DNA was isolated and then amplified using primer sets, the locations of which relative to the RankL upstream regions are indicated in the upper panel of Fig. 2Go. PCR analyses were all performed within the linear range of amplification using 31 cycles. All results are typical of several separate experiments.

 
Forskolin and OSM Induce the Recruitment of RNA Polymerase II (RNA pol II) to the Distal RankL Enhancers
Previous studies of the five distal RankL enhancers revealed that each was capable of recruiting RNA pol II in the presence of 1,25-(OH)2D3 (22). We therefore explored the possibility that forskolin and perhaps OSM might also increase the abundance of RNA pol II at each of these regulatory regions as well. ST2 cells were treated with forskolin or OSM for increasing periods of time, and the cells were then harvested and subjected to ChIP analysis using an antibody that recognizes both phosphorylated and hypophosphorylated forms of RNA pol II. As can be seen in Fig. 9Go, RNA pol II was indeed recruited in response to forskolin not only to the RankL TSS, but to each of the five enhancer regions as well. Interestingly, similar studies conducted using the inducer OSM revealed that RNA pol II was also recruited, although this recruitment was limited to mRLD5b, mRLD5, mRLD4, and, to a modest extent, to the TSS. Lower levels of RNA pol II were also evident at some sites in the intervening regions. Whether these levels represent ChIP background or arise as a result of the biological presence of RNA pol II within these regions remains to be determined. Despite this, our data suggest that activation of the upstream enhancer regions of the RankL gene by 1,25-(OH)2D3, PKA stimulators, and gp130 activators leads to VDR-, CREB-, and Stat3-mediated recruitment of RNA pol II in a fashion similar to that seen for the RankL gene at the TSS.


Figure 9
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  		Fig. 9. Forskolin (FSK) and OSM Promote the Recruitment of RNA pol II to Regulatory Regions of the RankL Upstream Regions

ST2 cells were treated for 6 h with either vehicle, forskolin (10–6 M), or OSM (20 ng/ml) and then subjected to ChIP analysis using antibodies to RNA pol II or control IgG. The immunoprecipitated DNA was isolated and then amplified using primer sets, the locations of which relative to the RankL upstream regions are indicated in the top panel of Fig. 2Go. PCR analyses were all performed within the linear range of amplification using 30 cycles. The results are typical of several separate experiments.

 
Forskolin and OSM Induce Broad Changes in H4 Acetylation within the RankL Locus
The localization of transcription factors to enhancers in response to inducers is believed to facilitate the recruitment of coregulators, many of which are capable of covalently modifying histone and thus influencing chromatin structure and transcriptional output (36, 37, 38). To examine whether the activation of RankL gene expression by forskolin and OSM was associated with changes in histone acetylation at this locus, we treated ST2 cells with either forskolin or OSM for increasing time periods, and then subjected the harvested cells to ChIP analysis using antibodies to tetra-acetylated histone 4 (H4). As seen in Fig. 10Go, A and B, and quantitated in supplemental Fig. S4, published as supplemental data on The Endocrine Society’s Journals Online web site, forskolin induced a highly reproducible, time-dependent increase in acetylated H4 levels within the RankL locus. These changes, however, were not generally observed at the enhancers, but rather at sites that we amplified that were located either upstream of the most distal enhancer (mRLD5b), between the five regulatory regions or at the RankL TSS. Indeed, residual H4 acetylation at each of the enhancer regions was observed to be quite high relative to the sites that we evaluated outside these regions. Interestingly, forskolin-induced changes in regional H4 acetylation were detected more than 200 kb upstream of RankL’s TSS, suggesting the possibility that the expression of the RankL gene’s nearest upstream neighbor Akap11 (protein kinase A anchoring protein 11) might also be affected. The results in Fig. 10CGo suggest that this is indeed the case; forskolin induces a time-dependent increase in Akap11 mRNA expression. Interestingly, the actions of OSM on histone acetylation were much more modest and focused almost exclusively at the region upstream of mRLD5b and the intervening region between mRLD5 and mRLD4 (data not shown). We interpret these findings to suggest that forskolin- or OSM-induced binding of CREB or Stat3 to RankL gene enhancers initiates a secondary process that culminates in the acetylation of H4 at sites relatively distant from the sites of transactivator interaction.


Figure 10
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  		Fig. 10. Forskolin (FSK) Induces Histone Acetylation in the Upstream Regions of the mRankL Gene

A and B, Forskolin induces H4 acetylation. ST2 cells were treated with forskolin (10–6 M) for periods up to 6 h and then subjected to ChIP analysis using antibodies to tetraacetylated H4 or IgG. The immunoprecipitated DNA was isolated and then amplified by PCR using primer sets, the locations of which relative to the RankL upstream regions are depicted in panel B. PCR analyses were all performed within the linear range of amplification using 30 cycles. All results are typical of three similar experiments. C, Forskolin induces Akap11 (AK129178). ST2 cells were treated with forskolin for periods up to 24 h. Total RNA was isolated and subjected to RT-PCR analysis using primers specific to mouse Akap11 (30 cycles) or ß-actin (20 cycles) as documented in Materials and Methods. The results are typical of two similar experiments.

 
Forskolin-Induced CREB Binding and Transcriptional Activation via the mRLD5b Is Conserved in the Human Gene
The RLD5a/b region is highly conserved across several genomes including that of the human (26). Indeed, previous studies indicated that the human (h)RLD5 region located at –96 kb contains a VDRE that is structurally and functionally equivalent to that found in the mRLD5 region (22). We therefore asked whether the human equivalent to mRLD5b was also regulated by forskolin. A 542-bp hRLD5b fragment from –98,716 to –99,258 was cloned into the TK vector and transfected into ST2 cells, and its enhancer activity was assessed in response to forskolin as well as 1,25-(OH)2D3 and OSM. As can be seen in Fig. 11Go, this fragment displayed a response that was identical to that of the mRLD5b region; although unresponsive to either OSM or 1,25-(OH)2D3, hRLD5b conferred a significant forskolin sensitivity to the TK promoter. Importantly, when a human version of mRLD5a/b was cloned and evaluated, forskolin activation was not only retained, but both 1,25-(OH)2D3 and OSM activities were also recovered (data not shown). These results suggest that like mRLD5b, hRLD5b mediates, at least in part, the ability of PKA activators to induce RankL gene expression.


Figure 11
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  		Fig. 11. The Highly Evolutionarily Conserved hRLD5b Region Mediates the Transcriptional Activity of Forskolin (CREB) in the Human RANKL Gene

A, Position, size, and coordinates of hRLD5b relative to hRLD5 and the hRL TSS. B, Forskolin-inducible activity of hRLD5b. ST2 cells were transfected with pCH110-ßgal (50 ng), pcDNA-hVDR (50 ng), and either the pTK control vector (250 ng) or pTK-hRLD5b. Cells were treated with either vehicle, 1,25-(OH)2D3 (10–7 M), forskolin (10–6 M), or OSM (20 ng/ml) and evaluated after 24 h for both luciferase and ß-gal activity. Each point represents the normalized RLU average ± SEM for a triplicate set of transfections. *, P < 0.05 compared with control. FSK, Forskolin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The importance of RankL in the formation, activation, and survival of bone-resorbing osteoclasts provides a compelling case for elucidating the mechanisms that underlie transcriptional control of this gene. As an extension of our earlier studies in which we examined the activity of 1,25-(OH)2D3 on RankL gene expression, our current investigation sought to delineate the mechanisms whereby activators of the PKA and gp130 pathways induce this gene as well. We focused on five regions of the RankL gene located upstream of the gene’s TSS that had been identified previously as responsible for vitamin D activation (22). Accordingly, we found that these regions also mediated the activity of the PKA- and gp130-signaling pathways. The most distal of these regions, located at –76 kb and termed the RL-DCR, comprised a distal component that bound CREB and mediated PKA-inducible activity and a more proximal segment that bound the Stat3 transcription factor and mediated OSM activity. Two CREs as well as an OSE2 that contributed basal activity to the DNA fragment were located and characterized in this distal region. ChIP analysis further demonstrated that CREB binding to the RankL CREs was accompanied by phosphorylation of CREB at serine-133. Stat3 binding in response to OSM activation was also observed, although this binding was limited to the mRLD4 and mRLD5 regions. Finally, activation via these enhancer regions was accompanied by localized recruitment of RNA pol II as well as broad acetylation across the RankL gene upstream region. These studies solidify the functional relevance of the five distal enhancers and confirm and extend recent studies by Fu et al. (23). Thus, they provide an important advance in our understanding of the mechanisms that underlie induction of RankL gene expression by key regulatory factors active both in vivo and in vitro.

Gene-regulatory regions are often modular in nature, containing clusters of cis elements that coordinately regulate the expression of a specific gene in response to various local or environmental inputs (39). In fact, it was this characteristic of regulatory regions that prompted us to examine the five RankL enhancers for potential activation by inducers of PKA and gp130. In the case of mRLD5a/b (RL-DCR), it is clear that this region comprises a distinct chromatin domain that is integral to the regulatory control of RankL gene expression. Moreover, RL-DCR is indeed modular, containing functional binding sites for multiple regulators including VDR, RXR, glucocorticoid receptor, and CCAAT-enhancer binding protein ß (22) as well as CREB, Stat3, and Runx2 family members (Ref. 23 and references therein). The activity that arises from the binding of these factors to this region can be both additive [forskolin and 1,25-(OH)2D3, herein] or synergistic [1,25-(OH)2D3 and glucocorticoids (22)]. The RL-DCR enhancer also manifests considerable transcriptional activity in the absence of inducer. This finding suggests the presence of regulatory elements within the RL-DCR that are capable of contributing to the basal level of expression of RankL in the osteoblast. One such element appears to be the OSE2. Finally, the RL-DCR is highly evolutionarily conserved across multiple species (26). Accordingly, a human RankL gene DNA fragment homologous to that of the mRL-DCR also displays both 1,25-(OH)2D3-and forskolin-inducible activities. Interestingly, unlike the mouse RankL gene, the two DNA segments hRLD5 and hRLD5b that make up the hRL-DCR are separated by several kilobases of unconserved DNA. Collectively, however, our results support the idea that the RL-DCR is a critical regulatory component of both mouse and human RankL genes.

Fu et al. (23) also demonstrated the presence of two functional CREs and an OSE2 in the upstream mRLD5b region. Following a bacterial artificial chromosome clone/deletion approach, these authors mapped a region similar to mRLD5b that was responsive to cAMP, and then pinpointed both a proximal CRE (CRE1) and a distal CRE (CRE2) as well as the OSE2. Perhaps most important, the biological relevance of this region was more fully defined through a genetic deletion approach wherein a region equivalent to mRLD5a/b was deleted in the mouse using homologous recombination. Interestingly, an initial examination of the phenotype of this mouse strain did not reveal the striking osteopetrotic phenotype seen in the RankL null mouse (40). Moreover, basal levels of RankL in bone cells remained unchanged. The animals were unable, however, to up-regulate RankL expression when placed on a short-term low-calcium diet that raised serum PTH and is known to increase 1,25-(OH)2D3 levels. A detailed analysis of these animals is likely to follow and should provide a more complete view of both the phenotype and the RankL responsiveness of these animals to 1,25-(OH)2D3 and PTH.

Interestingly, many of the features characteristic of the RL-DCR can also be seen in the additional four RankL regulatory regions. All are highly conserved across multiple species, and all are capable of mediating the activity of several different inducers. Whether these enhancers function in a purely redundant manner or retain activities that are unique remain to be determined. The selective interaction of the glucocorticoid receptor with a subset of these regions seen previously (22) and the selective activity of Stat3 at mRLD4 and mRLD5 observed in the current studies may, however, serve as examples of the latter. If so, it is possible that the function of these multiple regions is to integrate diverse regulatory inputs at the RankL gene TSS. CREB, for example, can be activated by not only stimulators of the PKA pathway, but also by inducers of calmodulin kinase pathways as well (26, 41). It is therefore possible that each of the enhancer regions might be activated differentially, depending upon the nature of the CREB stimulus. Perhaps most interesting is the capacity of forskolin, OSM, and 1,25-(OH)2D3 and their attendant transcription factors to promote common localized recruitment of RNA pol II to both the upstream enhancers and to the RankL TSS. Indeed, recent studies suggest that the recruitment of RNA pol II to these types of enhancers as well as to bona fide promoter sites is highly prevalent across mammalian genomes (42, 43, 44). Such observations are prompting further studies aimed at determining the role of RNA pol II at these sites and delineating the differences between enhancers and authentic promoters (45). The recruitment of RNA pol II to these upstream enhancers suggests the possibility that this recruitment may result in the generation of novel RNA transcripts that could play a role in RankL expression. A second consequence of RankL enhancer activation by not only 1,25-(OH)2D3 but forskolin and OSM as well is the induction of H4 acetylation across the RankL upstream regions. Interestingly, this change does not occur in a dramatic way within the enhancer regions themselves, but rather at sites located between the enhancers and also at sites upstream of the RL-DCR. It seems therefore unlikely that these changes result from the capacity of transactivators such as VDR/RXR or CREB to recruit coactivators with histone acetyltransferase activity, but rather that these changes may be secondary to the recruitment of RNA pol II and basal transcriptional machinery.

The capacity of both forskolin and 1,25-(OH)2D3 to induce changes in H4 acetylation significant distances upstream of the RankL TSS may provide clues as to the actual regulatory boundaries of this important gene. Indeed, whereas changes in H4 acetylation downstream of the final RankL exon are limited, changes in H4 acetylation induced by forskolin [and 1,25-(OH)2D3] extend upstream of the RankL TSS over 200 kb, thereby approaching the Akap11 gene locus, RankL’s nearest annotated upstream neighbor (26). Additional work will be required to define the exact boundaries of the RankL gene regulatory region and the mechanisms that define these boundaries (46, 47). Interestingly, we found that forskolin was also capable of inducing Akap11. Whether the mechanism of this induction involves the ability of forskolin [or 1,25-(OH)2D3] to induce broad changes upstream of the RankL gene locus (a bystander effect) (48) or is due to a direct action by CREB and/or the VDR/RXR heterodimer remains to be determined. It is striking, nevertheless, that regulatory factors such as forskolin and 1,25-(OH)2D3 are able to promote such extended changes in chromatin structure upstream of the RankL gene.

Finally, the molecular activity of OSM is different from that for forskolin or 1,25-(OH)2D3 in that only mRLD5 and mRLD4 appear to be involved. Interestingly, RankL mRNA is not as rapidly induced by OSM as it is by forskolin. Perhaps this is due to the ability of OSM to activate only a subset of the RankL regulatory domains. Fu et al. (23) proposed that Stat3 activates RankL gene expression via a region located upstream of the RL-DCR (mRLD5a/b). The ability of other regulatory factors to promote changes in H4 acetylation in this far upstream region, as discussed above, strongly supports the potential for this region to contain additional regulatory capability. Because we have not explored this region of the RankL locus in detail, however, we are unable to confirm currently the relevance of this region to OSM-mediated RankL induction.

In conclusion, we have shown that activators of the PKA and gp130 pathways modulate the expression of RankL via highly conserved regions located significantly upstream of the RankL TSS. Two CREs located less than 1 kb upstream of the distal RankL VDRE and that mediate forskolin response were identified. Like 1,25-(OH)2D3, both forskolin and OSM induced the recruitment of RNA pol II to two or more of these sites and induced significant changes in histone acetylation. These consequences of hormone action are manifestations of specific enhancer function and seem likely to be responsible for the increase in RankL gene expression seen in the presence of these agents. Future studies are now focused upon the precise mechanisms whereby these long-range enhancers regulate RankL output.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
General biochemicals were purchased from Fisher Scientific (Pittsburgh, PA) and Sigma Chemical Co. (St. Louis, MO). Forskolin (F3917), db-cAMP (D0627), and rabbit IgG (I5006) were purchased from Sigma Chemical Co (St. Louis, MO). Oncostatin M was obtained from R & D Systems, Inc. (Minneapolis, MN), and prostaglandin E2 was purchased from BIOMOL Research Laboratories, Inc. (Plymouth, PA). 1,25-(OH)2D3 was obtained from Tetrionics (Madison, WI). [32P-{gamma}]-dATP was obtained from NEN Life Science Products, Inc. (Boston, MA). Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, IA). Anti-VDR (Sc-1008), anti-Stat3 (Sc-482), anti-ATF2 (Sc-6233), and anti-ATF4 (Sc-200) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). anti-CREB (06-863), anti-phosphoserine 133-CREB (06-519), and antitetraacetyl H4 antibody (06-866) was obtained from Upstate Biotechnology, Inc. (Charlottesville, VA) and anti-RNA polymerase II antibody (8WG16) was obtained from Berkeley Antibody Co. (Richmond, CA). MEM-{alpha} was obtained from Invitrogen (Carlsbad, CA). Lipofectamine Plus was obtained from Invitrogen.

Cell Culture
Mouse ST2 osteoblastic cells were cultured in MEM-{alpha} supplemented with 10% fetal bovine serum obtained from Hyclone Laboratories, Inc. (Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin. All ligands were added in ethanol (0.1% maximum final concentration) or dimethylsulfoxide.

RNA Isolation and Analysis
Total RNA was isolated from cells using Tri-reagent obtained from Molecular Research Center, Inc. (Cincinnati, OH). The isolated RNA was reverse transcribed using the SuperScript III RNase H Reverse transcriptase kit from Invitrogen and then subjected to PCR analysis using standard PCR methods. Primers used include those for mß-actin (forward, 5'-TGTTTGAGACCTTCAACACCC; reverse, 5'-CGTTGCCAATAGTGATGACCT); mCyp24a1 (forward, 5'-GTGCGGATTTCCTTTGTGATA; reverse, 5'-GGTAGCGTGTATTCACCCAGA); mOpn (forward, 5'-CTAACTACGACCATGAGATTGGCAG; reverse, 5'-CTTTAGTTGACCTCAGAAGATGAA); mRankL (forward, 5'-GAATCCTGAGACTCCATGAAAACGC; reverse, 5'-CCATGAGCCTTCCATCATAGCTGG); and Akap11 (AK129178) (forward, 5'-CCTCATGTTGTGGTGACCCCCAAC; reverse, 5'-GAAACAGTCCAGACACATTTTACT).

ChIP Assay
ChIP assays were performed as previously described (22, 25). Primer sets used for amplifying mouse Cyp24a1 and RankL gene regions of interest are listed in Table 1Go.


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  		Table 1. Primers Used for ChIP Analysis

 
DNA Bandshift Analysis
Nuclear extracts of ST2 cells pretreated for 3 h with forskolin were prepared according to the method of Dignam et al. (49). Duplex oligonucleotide probes comprised of the nucleotide sequence 5'-AAGTTTGATGTCACTGTA-3' (mRL CRE1) and 5'-CCAGTTGGCGTCAGGGAA-3' (mRL CRE2) were end labeled using [{gamma}32P]-dATP and then incubated at room temperature with the indicated concentrations of ST2 cell nuclear extract in 10 mM HEPES, pH 7.4; 1 mM EDTA; 5 mM MgCl2; 10% glycerol; 0.5 mM dithiothreitol; 0.7 mM phenylmethylsulfonylfluoride; and 100 mM KCl for 30 min. The presence of CREB in the protein DNA complex was determined by using either an irrelevant antibody or an antibody to CREB (Upstate; 06-519). The CREB antibody selectively blocks CREB CRE binding. Competition studies were performed using nuclear extracts incubated with labeled CRE1 or CRE2 and increasing concentrations of unlabeled probe as indicated. Complexes were resolved on 6% nondenaturing polyacrylamide gels, dried, and then visualized using autoradiography.

Plasmids
The pCH110-ßgalactosidase reporter plasmid and the pcDNA-hVDR vector were previously described (25). The pCMV500 and pCMV-ACREB vectors were described earlier (30). The parent vectors pTK-luc and pGL3-luc were used in subsequent cloning efforts. mRLD1 (–16.4/–15.2), mRLD2 (–23.1/–21.5), mRLD3 (–60.4/–59.3), mRLD4 (–69.0/–68.1), mRLD5 (–76045/–74973), mRLD5b (–76705/–76045), and mRLD5a/b (–76705/–74973) were amplified from mouse genomic DNA using primers that contained HindIII, HindIII/SalI, or HindIII/BamHI restriction ends and then cloned into the corresponding sites within the pTK-luc vector. 5'-Extended fragments mRLD5-E1 (–76205/–74973), mRLD5-E2 (–76365/–74973), mRLD5-E3 (–76521/–74973) and mRLD5-E4 (–76597/–74973) as well as the CRE2/Runx2RE fragment mRLD5b-CRE1/2 (–76705/–76521) were similarly amplified and cloned into the pTK-luc vector using HindIII/SalI restriction sites. The hRLD5b region of the human RANKL gene (–99258/–98716) was amplified from genomic human DNA and cloned into the HindIII/BamHI sites of the pTK-luc vector to produce pTK-hRLD5b. Point mutations in the CRE1, CRE2, VDRE, and OSE2 sites were created using the Quikchange Mutagenesis Kit from Stratagene (San Diego, CA). The pmRL (100) vector was prepared by introducing an amplified segment of the mouse RankL gene promoter (–101/+54 relative to the RankL TSS) into the pGL3-luc expression vector at the XhoI/HindIII sites. Each of the mRLD regions (mRLD1 (–16.4/–15.2), mRLD2 (–23.1/ –21.5), mRLD3 (–60.4/–59.3), mRLD4 (–69.0/–68.1), mRLD5 (–76045/–74973), mRLD5b (–76705/–76045), and mRLD5a/b (–76705/–74973) were then amplified and cloned upstream of pmRL (100) using the MluI/XhoI restriction sites to prepare the appropriate RankL enhancer constructs. All newly created plasmids were sequenced to verify successful cloning.

Transfection Assays
ST2 cells were seeded into 24-well plates at appropriate densities and cultured in MEM-{alpha} medium containing 10% FBS. Cells were transfected 24 h later with Lipofectamine PLUS in serum and antibiotic-free medium. Individual wells were transfected with 50 ng of pCH110-ßgal normalizing plasmid and 250 ng of RankL enhancer-luciferase reporter vector. In some cases, 50 ng of pcDNA-hVDR, pCMV-ACREB, and/or pCMV500 control vectors were introduced together with the above reporter plasmids. Cells were treated with the appropriate inducers at the indicated concentrations and harvested 24 h later, and the lysates were assayed for luciferase and ß-galactosidase activities as previously described (22, 25). Luciferase activity was normalized to ß-galactosidase activity in all cases.

Statistical Analysis
All values are expressed as mean ± SEM. All statistical calculations were performed using the GraphPad PRISM version 4 statistical software package (GraphPad Software, Inc., San Diego, CA). We evaluated differences between groups through one-way ANOVA or Student’s two-tailed t test. Significance was assessed at P < 0.05.


   ACKNOWLEDGMENTS
 
We thank members of the Pike laboratory for helpful discussions and other contributions to this work. We are grateful to Dr. Charles A. O’Brien for informative discussions regarding PTH action at the RankL gene locus. The gifts of pCMV500 and pCMV-ACREB kindly provided by Dr. C. Vinson are gratefully acknowledged. We thank Ms. Laura Vanderploeg for preparing the artwork and illustrations for this manuscript.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK-74993 (to J.W.P.).

The authors state that they have nothing to declare.

First Published Online October 19, 2006

Abbreviations: {alpha}, Anti-Ig; ACREB, antagonist of CREB; Akap11, protein kinase A anchoring protein 11; ATF, activating transcription factor-4; ChIP, chromatin immunoprecipitation; CRE, cAMP response element; CREB, CRE-binding protein; Cyp24a1, 25-hydroxyvitamin D3-24 hydroxylase gene; db, dibutyryl; gp130, transmembrane glycoprotein 130; H4, histone 4; NF-{kappa}B, nuclear factor of B cells; 1,25-(OH)2D3, 1,25-dihydroxyvitmain D3; Opn, osteopontin; OSE2, osteoblast-specific cis-acting element 2; OSM, oncostatin M; PGE2, prostaglandin E2; PKA, protein kinase A; RankL; receptor activator of NF-{kappa}B ligand; Runx2, Runt transcription factor 2; RNA pol II, RNA polymerase II; RL-DCR, RankL distal control region; RXR, retinoid X receptor; Stat3, signal transducer and activator of transcription 3; TK, thymidine kinase; TSS, transcriptional start site; VDR, vitamin D receptor; VDRE, vitamin D response element.

Received for publication August 1, 2006. Accepted for publication October 13, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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