Parathyroid Hormone Stimulates Receptor Activator of NFκB Ligand and Inhibits Osteoprotegerin Expression via Protein Kinase A Activation of cAMP-response Element-binding Protein*

Parathyroid hormone (PTH) stimulates osteoclast formation by binding to its receptor on stromal/osteoblastic cells and stimulating the production of receptor activator of NFκB ligand (RANKL) and inhibiting the expression of osteoprotegerin (OPG). However, the mechanisms through which PTH regulates these genes remain unknown. Here we report that PTH stimulated RANKL gene transcription and increased RANKL mRNA stability in murine stromal/osteoblastic cells stably expressing human PTH/PTH-related protein receptor 1. PTH also potently suppressed OPG mRNA in these cells. Cycloheximide did not block the effects of PTH on RANKL but did inhibit the suppression of OPG mRNA. Activation of protein kinase A (PKA) was necessary and sufficient for the effect of PTH on both genes. Conditional expression of a dominant-negative form of the transcription factor CREB, but not c-fos or Runx2, significantly reduced PTH stimulation of RANKL. CREB activity was also required for full stimulation of RANKL by oncostatin M or 1,25-dihydroxyvitamin D3. Dominant-negative forms of CREB and c-fosreduced the suppression of OPG by PTH. These results demonstrate that PTH directly stimulates RANKL expression via a PKA-CREB pathway and that CREB may be a central regulator of RANKL expression. Furthermore, they suggest that PTH suppression of OPG involves CREB and c-fos.

Parathyroid hormone (PTH) stimulates osteoclast formation by binding to its receptor on stromal/osteoblastic cells and stimulating the production of receptor activator of NFB ligand (RANKL) and inhibiting the expression of osteoprotegerin (OPG). However, the mechanisms through which PTH regulates these genes remain unknown. Here we report that PTH stimulated RANKL gene transcription and increased RANKL mRNA stability in murine stromal/osteoblastic cells stably expressing human PTH/PTH-related protein receptor 1. PTH also potently suppressed OPG mRNA in these cells. Cycloheximide did not block the effects of PTH on RANKL but did inhibit the suppression of OPG mRNA. Activation of protein kinase A (PKA) was necessary and sufficient for the effect of PTH on both genes. Conditional expression of a dominant-negative form of the transcription factor CREB, but not c-fos or Runx2, significantly reduced PTH stimulation of RANKL. CREB activity was also required for full stimulation of RANKL by oncostatin M or 1,25-dihydroxyvitamin D 3 . Dominant-negative forms of CREB and c-fos reduced the suppression of OPG by PTH. These results demonstrate that PTH directly stimulates RANKL expression via a PKA-CREB pathway and that CREB may be a central regulator of RANKL expression. Furthermore, they suggest that PTH suppression of OPG involves CREB and c-fos.
Parathyroid hormone (PTH) 1 maintains calcium homeostasis in part by increasing the production of bone-resorbing osteoclasts to release calcium from the skeleton. It does so indirectly by binding to its G-protein-coupled receptor, PTH/PTH-related protein receptor 1 (PTHR1), on stromal/osteoblastic cells (1). These cells support osteoclast formation via expression of M-CSF and receptor activator of NFB-ligand (RANKL) (2). Mice deficient in either are incapable of forming osteoclasts, and together these proteins are sufficient to stimulate osteoclast formation in vitro in the absence of stromal/osteoblastic cells (3)(4)(5). In vitro and in vivo studies have demonstrated that the magnitude of osteoclast formation is directly proportional to the level of RANKL expression, whereas M-CSF appears to play primarily a permissive role (4,6,7). RANKL binds to its receptor RANK on hematopoietic precursors of osteoclasts and stimulates their differentiation and survival (8). The action of RANKL is blocked by osteoprotegerin (OPG), which functions as a soluble decoy receptor (9). PTH simultaneously stimulates RANKL and inhibits OPG expression in primary cultures of stromal/osteoblastic cells and in bone tissue from parathyroidectomized rats infused with this hormone (10,11). Based on this, it has been proposed that PTH influences osteoclast formation primarily by regulation of the RANKL/OPG ratio (2). However, the signaling pathways and molecular mechanisms utilized by PTH to regulate these genes remain largely unknown.
Binding of PTH to PTHR1 on stromal/osteoblastic cells activates both the protein kinase A (PKA) and protein kinase C (PKC) pathways, and both pathways have been implicated in PTH-induced osteoclast formation in vitro (12). Activation of these pathways by PTH leads to activation of multiple transcription factors, including CREB, AP-1, and Runx2 (12). Specifically, PKA stimulates the ability of DNA-bound CREB to activate transcription by phosphorylation of serine 133 (Ser-133) resulting in CBP/p300 co-activator recruitment (13). PTH stimulation of AP-1 is indirect requiring CREB-mediated transcription of AP-1 family members such as c-fos (14). Finally, it has been suggested that PTH may regulate the transcriptional activity of the RUNT domain transcription factor Runx2 via PKA phosphorylation (15). Whether PTH regulates RANKL or OPG expression via any of these factors is unknown.
The goal of the studies described here was to identify molecular mechanisms involved in PTH regulation of RANKL and OPG expression. To accomplish this, we generated a cell line in which PTH stimulates a robust induction of RANKL and inhibition of OPG. These cells were used to demonstrate that PTH directly stimulates RANKL transcription as well as mRNA stability and indirectly inhibits OPG expression. In addition we found that activation of PKA, but not PKC, is sufficient and required for PTH regulation of both genes. Finally, activation of the transcription factor CREB was shown to be required for full stimulation of RANKL by PTH, and both CREB and AP-1 are involved in PTH-suppression of OPG.
DNA Constructs-The human PTHR1 cDNA was inserted into the filled BglII site of the retroviral expression construct pLEN (16) to generate pLEN-PTHR1. A-CREB, A-fos, and dnRunx2 cDNAs were inserted into the filled BamHI site of the retroviral vector pREV-TRE (Clontech, Palo Alto, CA) to generate pRT-ACREB, pRT-AFOS, and pRT-dnRunx2, respectively. The reporter construct p7ϫAP1 was purchased from Stratagene (La Jolla, CA), and pCRE-luc was created by inserting the NheI-HindIII fragment containing a 3ϫCRE-promoter from pCRE-SEAP (Clontech) into the same sites in pGL3-basic (Promega, Madison, WI).
Osteoclast Formation Assay-Osteoclast formation in co-cultures of non-adherent bone marrow cells and UAMS-32 or UAMS-32P cells was performed as previously described (16).
Nuclear Run-on Assay-UAMS-32P cells, treated with vehicle or PTH for 4 h, were collected in phosphate-buffered saline and centrifuged at 500 ϫ g for 5 min at 4°C. Cell pellets were washed twice with phosphate-buffered saline and placed in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.4, 3 mM CaCl 2 , 2 mM MgCl 2 , 1% Nonidet P-40). Cells were lysed in a Dounce homogenizer, and nuclei were collected by centrifugation at 500 ϫ g for 5 min at 4°C. Pelleted nuclei were resuspended in an equal volume of a buffer containing 40% glycerol, 50 mM Tris-HCl, pH 8.0, and 5 mM MgCl 2 . Run-on transcription was performed by mixing 200 l of nuclei with 200 l of 2ϫ transcription buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 , 300 mM KCl, 5 mM dithiothreitol, 1 mM ATP, GTP, and CTP, and 100 Ci of [␣-32 P]UTP, 3000 Ci/mmol) and incubating at 30°C for 30 min. Radiolabeled RNA from the reaction was extracted with Ultraspec TM (Biotecx Laboratories, Houston, TX), precipitated in isopropanol, and resuspended in 100 l of 10 mM EDTA. Equivalent counts (ϳ500,000 cpm) were hybridized for 2 days to membranes onto which linearized and denatured plasmids containing RANKL or ChoB cDNAs had been previously immobilized. Membranes also contained plasmid Bluescript IIKSϩ (Stratagene) as a negative control.
Retroviral Packaging and Infection-Production of retroviral particles and infection of cells were performed as previously described (16). Infections with the Tet-regulated constructs were carried out in the presence of 100 ng/ml doxycycline. Cells infected with retroviral constructs harboring neomycin, blasticidin, or hygromycin resistance genes were selected in the respective antibiotic beginning 48 h after the last infection.
Protein Kinase A Assay-UAMS-32P cells were grown to confluence and incubated with and without 30 M H-89 for 30 min after which the cells were incubated with vehicle or PTH for an additional 2 min. PKA activity was determined using a commercial kit (SignaTECT PKA Assay System, Promega) and values were normalized to total protein (Bio-Rad, Hercules, CA).
Luciferase Reporter Assays-UAMS-32P cells, or their derivatives, were seeded at 2 ϫ 10 4 cells/well in 24-well plates and transiently transfected with reporter constructs (total of 0.4 g of DNA/well) using LipofectAMINE Plus (Invitrogen). Firefly luciferase values were normalized to Renilla luciferase values resulting from co-transfection of the plasmid pRL-SV40 (Promega). Firefly and Renilla luciferase values were determined using the Dual Luciferase Assay (Promega). All values presented are the mean of triplicate determinations Ϯ S.D.

Introduction of the Human PTHR1 into a Murine Stromal/ Osteoblastic Cell Line Confers Robust Responsiveness to PTH-
Some of the mechanistic studies we sought to perform required the use of a continuous cell line in which PTH strongly stimulates RANKL and inhibits OPG expression. However, in contrast to primary cultures, very few cell lines have been reported to support osteoclast formation or express RANKL in response to PTH (1,21). Consistent with this, UAMS-32 stromal/osteoblastic cells, which we have used previously as a model of osteoclast support cells, weakly supported osteoclast formation in response to PTH (16). We attempted to enhance the PTH responsiveness of UAMS-32 cells by introducing the human PTHR1. The resulting cells, designated UAMS-32P, expressed significantly larger amounts of RANKL in response to PTH than the parental cell line (Fig. 1A). In addition, although PTH only minimally suppressed OPG in the parental cell line, it completely suppressed OPG in UAMS-32P cells (Fig. 1A). PTH stimulated significantly more osteoclast formation in cultures containing UAMS-32P cells than in cultures containing UAMS-32 ( Fig. 1B), confirming that the increased effect of PTH on RANKL and OPG expression in UAMS-32P cells translated into an increased osteoclastogenic response.
PTH Directly Stimulates RANKL mRNA Expression and Indirectly Inhibits OPG mRNA Expression-To determine whether intermediate gene expression is required for the effects of PTH on RANKL and OPG expression, UAMS-32P cells were treated with PTH for 4, 8, or 24 h in the absence or presence of the protein synthesis inhibitor cycloheximide ( Fig.  2A). The concentration of cycloheximide utilized (1 g/ml) inhibited [ 3 H]leucine incorporation into total protein by 91% (data not shown). Cycloheximide alone stimulated RANKL mRNA levels at 4 and 8 h but did not block the additional stimulatory action of PTH. Cycloheximide alone significantly inhibited the expression of OPG at 24 h but partially reduced the inhibition of OPG by PTH at 4 and 8 h. Similar results were obtained in primary bone marrow cells (Fig. 2B). These results indicate that new protein synthesis is required for full PTHinhibition of OPG but not PTH stimulation of RANKL. PTH Stimulates RANKL Transcription and mRNA Stability-We next determined whether PTH stimulates RANKL mRNA levels by altering gene transcription and/or mRNA stability. Nuclear run-on analysis revealed that PTH stimulated RANKL transcription by ϳ4-fold (Fig. 3A). Quantitation of RANKL mRNA at various times following inhibition of transcription by actinomycin D demonstrated that PTH also increased the relative half-life of RANKL mRNA (Fig. 3, B and C). We were not able to determine the effect of PTH on OPG transcription, because the level of OPG transcripts was below the level of detection for our nuclear run-on assay. Similarly, we were not able to determine the effect of PTH on OPG mRNA stability, because the very low mRNA levels in the presence of PTH could not be accurately quantitated (data not shown).
Activation of PKA Stimulates RANKL and Inhibits OPG Expression-To identify signaling pathways involved in PTH regulation of RANKL and OPG, we determined whether activation of PKA or PKC mimicked the effects of PTH on RANKL and OPG in UAMS-32P cells. UAMS-32P cells were treated with increasing concentrations of PTH, dibutyryl-cAMP (db-cAMP) to activate PKA, or phorbol 12-myristate 13-acetate (PMA) to activate PKC. PTH strongly stimulated RANKL and inhibited OPG expression at concentrations of 10 Ϫ8 and 10 Ϫ7 M (Fig. 4A). Similarly, treatment of the cells with db-cAMP stimulated RANKL mRNA expression and inhibited OPG expression in a dose-dependant manner. However, PMA only minimally stimulated RANKL at any concentration and, in contrast to either PTH or db-cAMP, also slightly stimulated OPG expression. A similar pattern of responsiveness was obtained with primary bone marrow cells except that db-cAMP was more effective than PTH at stimulating RANKL (Fig. 4B).
To determine whether PKA mediates the effects of PTH on RANKL and OPG, UAMS-32P cells were preincubated with a chemical inhibitor of PKA, H-89. H-89 significantly reduced PTH stimulation of RANKL in UAMS-32P cells (Fig. 4C). In addition, H-89 reduced PTH suppression of OPG (Fig. 4C).
Direct analysis of PKA activity confirmed that H-89 inhibited both basal and PTH-stimulated PKA activity in these cells (Fig.  4D). These results suggest that PTH regulates RANKL and OPG via the cAMP-PKA pathway. However, it has been reported that H-89 is not completely specific for PKA in that it also potently blocks mitogen and stress-activated protein kinase (MSK1) (22), a kinase activated by extracellular signalregulated kinases (ERKs) (23). Furthermore, in some cell types cAMP has been shown to stimulate ERKs via Epac (exchange protein directly activated by cAMP), independent of PKA (24). To address the possibility that PTH regulates RANKL and OPG via a cAMP-ERK-MSK1 pathway, we determined whether ERK activation is required for PTH stimulation of RANKL or suppression of OPG. Preincubation of UAMS-32P cells with PD98059, a chemical inhibitor of MAPK/ERK kinase (MEK), did not alter PTH stimulation of RANKL or suppression of OPG (Fig. 4C). Immunoblot analysis with a phospho-ERK-specific antibody confirmed that PD98059 effectively blocked ERK activity in these cells (Fig. 4E). From these results we conclude that PTH regulates RANKL and OPG primarily via a cAMP-PKA pathway and not via activation of either PKC or ERKs.
CREB Is Required for Full Stimulation of RANKL and Inhibition of OPG by PTH-We next sought to determine whether CREB is involved in the PTH regulation of RANKL and/or OPG. PTH stimulated phosphorylation of CREB at Ser-133 in UAMS-32P cells as early as 15 min with maximal phosphorylation at 60 min and return to basal levels by 8 h (Fig. 5A). The phospho-specific antibody used for these studies recognizes the phosphorylated form of CREB and its related family members, ATF-1 and CREM (25). However, PTH did not lead to significant phosphorylation of proteins whose molecular sizes correspond to ATF-1 (35 kDa) or CREM (34 -36 kDa) in UAMS-32P cells (data not shown).
To determine whether CREB is required for PTH regulation of RANKL or OPG, we expressed a dominant-negative form of CREB in UAMS-32P cells. This protein, known as A-CREB, blocks endogenous CREB action by forming heterodimers that are unable to bind DNA and effectively blocks CREB-, ATF-1-, and CREM-mediated transcription (26). Conditional expression of A-CREB using the tetracycline regulation system was confirmed by demonstrating that removal of doxycycline from the culture medium blocked PTH induction of a CREB-responsive reporter construct (Fig. 5B).
Induction of A-CREB significantly inhibited stimulation of RANKL by either db-cAMP or PTH (Fig. 5C). A-CREB reduced PTH-induced RANKL expression to an average of 34 Ϯ 9% that of control levels in three experiments. The ability of A-CREB to inhibit the actions of db-cAMP indicates that the effect of A-CREB is not due simply to inhibition of PTH1R expression or activation. A-CREB also partially blocked inhibition of OPG by PTH or db-cAMP (Fig. 5C). A-CREB increased the levels OPG in the presence of PTH by an average of 747 Ϯ 106% in three experiments. Taken together, these results indicate that CREB is required for both full stimulation of RANKL and full inhibition of OPG expression by PTH.
Surprisingly, A-CREB also significantly inhibited stimulation of RANKL by OSM and 1,25(OH) 2 D 3 (Fig. 6A). This result suggested that CREB may play a general role in gp130 or VDR signaling in these cells or that CREB may play a central role specifically in the regulation of RANKL by a variety of agents. To address the first of these possibilities, we determined whether A-CREB altered the OSM regulation of a different target gene, c-fos. A-CREB did not alter OSM stimulation of c-fos, but, consistent with previous studies (27), it did significantly reduce stimulation of c-fos by PTH (Fig. 6B). These results indicate that CREB is not a universal requirement for downstream targets of gp130 signaling.
To distinguish between a passive role for CREB in the actions of OSM or 1,25(OH) 2 D 3 and regulation of CREB activity by these agents, we determined whether OSM or 1,25(OH) 2 D 3 could stimulate a CREB-responsive reporter construct. While PTH stimulated the activity of this construct, OSM or 1,25(OH) 2 D 3 had no effect (Fig. 6C). This result suggested that, although CREB may be required for stimulation of RANKL by OSM and 1,25(OH) 2 D 3 , these agents do not actively regulate CREB transcriptional activity.
We next wanted to determine whether basal CREB activity was required for the actions of OSM and 1,25(OH) 2 D 3 on RANKL expression. Based on the assumption that basal CREB activity may be due to basal PKA activity, we pretreated UAMS-32P cells with H-89 and determined the effect on OSMand 1,25(OH) 2 D 3 stimulation of RANKL. Consistent with the A-CREB results, H-89 reduced stimulation of RANKL by both OSM and 1,25(OH) 2 D 3 (Fig. 6D). Similarly, inhibition of PKA activity reduced stimulation of RANKL by db-cAMP, PTH, OSM, or 1,25(OH) 2 D 3 in primary murine bone marrow cultures (Fig. 6E). These results suggest that a basal level of PKA activity is required for the regulation of RANKL by a variety of stimuli.
PTH Inhibits OPG Expression via Activation of AP-1-PTH has also been shown to activate the transcription factor AP-1 (12). Therefore, we conditionally expressed a dominant-negative form of c-fos in UAMS-32P cells. The A-fos protein is able to heterodimerize with all members of the Jun family and is thus sufficient to block AP-1 activity (28). Induction of the A-fos protein inhibited PTH stimulation of a reporter construct containing multiple AP-1 binding sites (Fig. 7A). Expression of the A-fos protein did not alter the ability of PTH, OSM, or 1,25(OH) 2 D 3 to stimulate RANKL (Fig. 7B). However, induction of A-fos did result in a striking increase in the basal level of OPG mRNA (Fig. 7B). This result indicates that under basal conditions AP-1 activity suppresses OPG expression. Furthermore, the ability of PTH to inhibit OPG expression was partially reduced in the presence of the A-fos protein (Fig. 7B). These results, combined with the finding that PTH strongly stimulates AP-1 activity in UAMS-32P cells (Fig. 7A), suggest that PTH inhibits OPG expression in part via activation of AP-1 and that this may occur indirectly via CREB.
Finally, because some studies indicate that PTH can regulate the transcriptional activity of the Runx2 transcription factor, we conditionally expressed a dominant-negative form of this protein (designated dnRunx2) in UAMS-32P cells. The dnRunx2 protein consists of the DNA binding domain only of Runx2 and thus lacks transcriptional activity (29). Induction of dnRunx2 protein did not alter the regulation of RANKL or OPG by PTH, OSM, or 1,25(OH) 2 D 3 (Fig. 8). Confirmation of dnRunx2 activity was indicated by a 4-fold down-regulation of osteocalcin mRNA expression, which is known to be positively regulated by Runx2 (Fig. 8). DISCUSSION Our results in UAMS-32P and primary bone marrow cells indicate that PTH directly stimulates RANKL transcription via cAMP activation of PKA. Furthermore, PTH suppression of OPG also requires activation of PKA but differs from regulation of RANKL in that intermediate gene expression is required. Although previous work indicated that PTH stimulation of RANKL and inhibition of OPG involves a cAMP-activated pathway (30 -32), it remained unclear whether the downstream mediator of cAMP is the well-characterized PKA pathway or the recently described Epac pathway (24). Our demonstration that inhibition of MEK did not alter PTH regulation of either RANKL or OPG argues against involvement of the Epac pathway.
Consistent with our studies, recent work with a different murine stromal cell line (MS1) also indicates that PKA, but not PKC, is required for simultaneous stimulation of RANKL and suppression of OPG after a 7-day PTH exposure (33). The previous studies with this cell line suggesting that both PKA and PKC are required for PTH-stimulated osteoclast formation utilized 3-week co-cultures of MS1 and spleen cells (1,34). It is possible that pro-osteoclastogenic cytokines that utilize PKC were produced during this comparatively long culture period and, via stimulation of RANKL, contributed along with PTH to the total number of osteoclasts formed.
The transcription factors CREB, c-fos, and Runx2 have been shown to mediate at least some of the gene regulatory effects of PTH (12). Our results indicate that CREB, but not c-fos or Runx2, is involved in the PTH stimulation of RANKL. The lack of c-fos involvement is not unexpected, because new protein synthesis was not required for PTH stimulation of RANKL and previous work has shown that PTH stimulates c-fos indirectly via CREB (27). In addition, the lack of Runx2 involvement is consistent with our previous study indicating that this factor is not required for basal, OSM-, or 1,25(OH) 2 D 3 -stimulated RANKL expression (35). Expression of A-CREB did not completely block PTH stimulation of RANKL. This residual stimulation may be due to an inability of the dominant-negative protein to completely block endogenous CREB activation. Alternatively, the residual stimulation may be due solely to effects of PTH on RANKL mRNA stability, which may not involve CREB.
We were surprised to find that PKA and CREB activity are required for regulation of RANKL by OSM and 1,25(OH) 2 D 3 . These results suggest that the PKA-CREB pathway is a necessary component of transcriptional activation of the RANKL gene regardless of the nature of the stimulus. Although our results could have been due to A-CREB inhibition of genes required for gp130 or VDR action, our finding that OSM stimulated a different target gene, namely c-fos, in the presence of A-CREB argues against this possibility. It is also possible that the protein complex consisting of A-CREB and wild-type CREB sequestered co-factors essential for STAT3 and VDR action such as p300/CBP (36). However, the A-fos protein, which functions via a mechanism similar to A-CREB, had no effect on RANKL expression in response to any of the stimuli used, suggesting that this class of dominant-negative B-ZIP factors does not limit co-factor availability.
How might CREB be required for the actions of multiple signaling pathways on RANKL expression? Our finding that PTH, but not OSM or 1,25(OH) 2 D 3 , stimulated CREB transcriptional activity suggests that only basal CREB activity is required for the regulation of RANKL by OSM and 1,25(OH) 2 D 3 . The lack of CREB transcriptional modulation by OSM or 1,25(OH) 2 D 3 also suggests that these agents do not alter PKA activity in UAMS-32P cells. Nonetheless, PKA activity was required for gp130 and VDR action on RANKL. Therefore, we hypothesize that basal PKA activity is a prerequisite for the effects of both the gp130 and VDR pathways on RANKL. Basal PKA activity may result from factors present in the serum-containing medium or autocrine production of factors that activate PKA. Basal PKA activity may in turn maintain basal CREB phosphorylation, which by itself is not sufficient for high level RANKL expression but, together with activated STAT3 or VDR, is able to induce RANKL transcription. In the case of PTH, strong activation of PKA may lead to levels of CREB phosphorylation that are sufficient to stimulate RANKL transcription without additional stimulus-dependant factors.
The ability of osteoclastogenic cytokines and hormones to stimulate RANKL expression appears to be restricted to stromal/osteoblastic cells and mammary epithelium (37). However, both CREB and STAT3 are broadly expressed and activated by many agents (25,38), and the VDR can be activated in a variety of different tissues as well (39). These observations raise the question of how activation of broadly expressed factors leads to RANKL expression specifically in stromal/osteoblastic cells. We postulate that cell type-specific factors function together with broadly expressed transcription factors to regulate RANKL expression. The finding that both AP-1 and Runx2 binding sites are required for PTH stimulation of the collagenase 3 promoter illustrates one mechanism whereby a tissuespecific transcription factor (Runx2) acts in concert with a broadly expressed factor (AP-1) to control osteoblast-specific expression of a gene (40). In addition, although CREB is broadly expressed, it appears to be involved only in the differentiation and/or survival of specific cell types, such as neurons, T cells, and adipocytes (41,42).
PTH transiently stimulates gp130-utilizing cytokines such as IL-6, IL-11, and leukemia inhibitory factor in primary osteoblasts and cell lines (43)(44)(45)(46)(47). These cytokines stimulate RANKL expression and osteoclast formation in co-cultures of stromal/osteoblastic and hematopoietic cells (6,48). Furthermore, blockade of IL-6 or gp130 signaling reduced osteoclast formation or bone resorption induced by PTH or 1,25(OH) 2 D 3 in vitro and in vivo (45,49,50). Based on these studies, it has been suggested that PTH might stimulate RANKL and/or inhibit OPG by stimulating expression of IL-6-type cytokines. Our demonstration that intermediate gene expression is not required for PTH stimulation of RANKL argues against this scenario and is consistent with our earlier finding that blockade of gp130 signaling specifically in stromal/osteoblastic cells did not block PTH-induced osteoclastogenesis (16). It is unlikely that gp130 cytokines are involved in PTH suppression of OPG, because OSM did not inhibit OPG in UAMS-32P cells.
Given these results, what role might IL-6 type cytokines play in PTH-induced bone resorption? IL-6 may be required for maximal osteoclast precursor proliferation (51,52). Alternatively, IL-6 may be required for PTH-induced bone resorption via a mechanism that does not involve regulation of osteoclast formation. IL-6 is capable of prolonging the lifespan of cells of the monocyte/macrophage lineage (53), and gp130 cytokines augment RANKL/M-CSF-induced osteoclast formation (54). Therefore, the role of gp130 cytokines in PTH-induced bone resorption may be to prolong the lifespan of differentiated osteoclasts.
In conclusion, this study provides evidence that PTH stimulates osteoclast formation via PKA-mediated induction of RANKL and suppression of OPG in stromal/osteoblastic cells and that stimulation of RANKL involves CREB-mediated transcription. Furthermore, PTH appears to inhibit OPG expression via a PKA-CREB-AP-1 pathway. Because PKA activation of CREB occurs in a variety of cell types, our work suggests that other factors limit the expression of RANKL to cells of the osteoblastic lineage. Identification of the cis-acting elements in the RANKL gene responsible for cell type-specific RANKL expression, as well as regulation by osteoclastogenic agents, should shed light on the mechanisms whereby CREB, together with STAT3 and the VDR, regulate RANKL expression.