Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M208494200 on October 2, 2002

J. Biol. Chem., Vol. 277, Issue 50, 48868-48875, December 13, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/50/48868    most recent
M208494200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fu, Q.
Right arrow Articles by O'Brien, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fu, Q.
Right arrow Articles by O'Brien, C. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Qiang FuDagger , Robert L. JilkaDagger §, Stavros C. ManolagasDagger §, and Charles A. O'BrienDagger

From the Dagger  Division of Endocrinology & Metabolism, Center for Osteoporosis & Metabolic Bone Diseases and the § Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Received for publication, August 20, 2002, and in revised form, September 18, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Parathyroid hormone (PTH) stimulates osteoclast formation by binding to its receptor on stromal/osteoblastic cells and stimulating the production of receptor activator of NFkappa B 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-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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 NFkappa B-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 (3-5). 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Cells-- Bovine PTH (1-34) (NLE-8-18-TYR-34), dibutyryl-cAMP (db-cAMP), phorbol 12-myristate 13-acetate (PMA), cycloheximide, actinomycin D, and PD98059 were purchased from Sigma. Murine oncostatin M was purchased from R&D Systems (Minneapolis, MN). 1,25-(OH)2 vitamin D3 (1,25(OH)2D3), and H-89 were from BIOMOL (Plymouth Meeting, PA). The UAMS-32 cell line and its derivatives were maintained in alpha -minimal essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT) and 1% each of penicillin, streptomycin, and glutamine.

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 p7×AP1 was purchased from Stratagene (La Jolla, CA), and pCRE-luc was created by inserting the NheI-HindIII fragment containing a 3×CRE-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).

Northern Blot Analysis-- Northern blots were performed and analyzed as previously described (16). The following cDNA probes were utilized: a 2000-bp cDNA encoding murine RANKL (17), a 1.3-kb cDNA coding for rat OPG (9), an 800-bp cDNA encoding v-fos (18), and a 900-bp cDNA coding for murine ribosomal protein S2 (ChoB) (19).

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 × 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 CaCl2, 2 mM MgCl2, 1% Nonidet P-40). Cells were lysed in a Dounce homogenizer, and nuclei were collected by centrifugation at 500 × 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 MgCl2. Run-on transcription was performed by mixing 200 µl of nuclei with 200 µl of 2× transcription buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl, 5 mM dithiothreitol, 1 mM ATP, GTP, and CTP, and 100 µCi of [alpha -32P]UTP, 3000 Ci/mmol) and incubating at 30 °C for 30 min. Radiolabeled RNA from the reaction was extracted with UltraspecTM (Biotecx Laboratories, Houston, TX), precipitated in isopropanol, and resuspended in 100 µl of 10 mM EDTA. Equivalent counts (~500,000 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 IIKS+ (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.

Immunoblotting-- Immunoblots of extracts from UAMS-32P cells were performed as previously described (20). Antibodies against ERK 1 (K-23), phospho-ERK (E-4), CREB-1 (C-21), and phospho-CREB-1 (Ser-133) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were used at a dilution of 1:1000.

Protein Kinase A Assay-- UAMS-32P cells were grown to confluence and incubated with and without 30 µM 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 × 104 cells/well in 24-well plates and transiently transfected with reporter constructs (total of 0.4 µg 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 ± S.D.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 1.   Introduction of the human PTHR1 into UAMS-32 cells increases responsiveness to PTH. A, UAMS-32 or UAMS-32P cells were treated with vehicle, 20 ng/ml OSM, or 10-7 M PTH for 4 or 8 h. Total RNA was analyzed by Northern blot with probes for RANKL, OPG, hPTHR1, and ChoB (ribosomal protein S2). B, UAMS-32 or UAMS-32P cells were co-cultured with non-adherent bone marrow cells in the presence of increasing concentrations (10-12 to 10-7 M) of PTH for 6 days. The cultures were then stained for the presence of tartrate-resistant acid phosphatase (red color) and photographed.

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 µg/ml) inhibited [3H]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 PTH-inhibition of OPG but not PTH stimulation of RANKL.


View larger version (74K):
[in this window]
[in a new window]
  		Fig. 2.   PTH directly stimulates RANKL mRNA expression and indirectly inhibits OPG mRNA expression. A, UAMS-32P cells were treated with 10-7 M PTH, 1 µg/ml cycloheximide (CHX) or PTH and CHX (the CHX was added 1 h before the PTH). Total RNA was extracted from the cells at the indicated time points. Total RNA was analyzed by Northern blot using probes for RANKL, OPG, and ChoB. B, primary murine bone marrow cells were cultured for 8 days in the presence of 50 µg/ml ascorbic acid and treated as in A.

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 ~4-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).


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 3.   PTH stimulates RANKL gene transcription and mRNA half-life. A, UAMS-32P cells were treated with vehicle or 10-7 M PTH for 4 h, followed by isolation of nuclei and nuclear run-on assay. RNA was subsequently extracted and hybridized to membranes containing RANKL and ChoB cDNAs, or pBluescript IIKS+. B, RANKL mRNA half-life was determined by treating UAMS-32P cells with 10-7 M PTH or vehicle for 24 h, after which actinomycin D was added to a final concentration of 5 µg/ml. At the indicated time points after actinomycin D addition, total RNA was extracted and analyzed by Northern blot with probes for RANKL and ChoB. C, the amount of RANKL mRNA, relative to ChoB, remaining at a given time point after actinomycin D addition was determined by quantitating the band intensity (ImageQuaNT, Applied Biosystems). Closed and open circles indicate vehicle- and PTH-treated cells, respectively.

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 10-8 and 10-7 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).


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 4.   Activation of PKA stimulates RANKL and inhibits OPG mRNA expression. A, UAMS-32P cells were treated for 8 h with vehicle, PTH (10-10 to 10-7 M), db-cAMP (5 × 10-4 to 3 × 10-3 M), or PMA (2.5 × 10-8 to 2 × 10-7 M). Total RNA was extracted and analyzed by Northern blot with probes for RANKL and OPG. B, primary murine bone marrow cells were cultured for 8 days in the presence of 50 µg/ml ascorbic acid and treated with vehicle, 10-7 M PTH, 3 × 10-3 M db-cAMP, or 2 × 10-7 M PMA for 8 h. Total RNA was analyzed as in A. C, UAMS-32P cells were treated with vehicle or 10-7 M PTH in the absence of inhibitors or in the presence of 30 µM H-89 or 10 µM PD98059. The inhibitors were added 30 min prior to addition of PTH. Total RNA was isolated 8 h after PTH addition and analyzed as in A. D, UAMS-32P cells were incubated with and without 30 µM H-89 for 30 min after which vehicle or 10-7 M PTH was added for an additional 2 min. PKA activity is presented as the mean ± S.D. of quadruplicate cultures. Asterisks indicate p < 0.001 versus vehicle-treated cells in the absence of H-89 using one-way analysis of variance. E, UAMS-32P cells, cultured in medium containing 10% FBS, were incubated with vehicle or 10-7 M PTH for 30 min in the presence or absence of 10 µM PD98059. PD98059 was added 30 min prior to PTH treatment. Total cellular protein was analyzed by immunoblot with antibodies for ERK after which the membrane was stripped and incubated with anti-phospho-ERK antibodies.

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 signal-regulated 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).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   CREB is required for PTH regulation of RANKL and OPG. A, UAMS-32P cells were incubated in medium containing 0.5% FBS overnight and treated with PTH for the indicated times. Protein extracts were analyzed by immunoblot with antibodies recognizing CREB or CREB phosphorylated at Ser-133 (P-CREB). The relative intensities of the phospho-CREB bands are indicated in the graph below the immunoblots. B, UAMS-32P cells conditionally expressing A-CREB were cultured in the presence or absence of 100 ng/ml doxycycline (DOX) for 48 h. Cells were then transiently transfected with a luciferase reporter construct containing three cAMP-response elements (3X-CRE-LUC) and treated with vehicle or 10-7 M PTH for 16 h. C, UAMS-32P cells conditionally expressing A-CREB were cultured as in B and subsequently treated with 1.5 mM db-cAMP (db) or 10-7 M PTH for 16 h. Total RNA was analyzed by Northern blot.

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 ± 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 ± 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)2D3 (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.


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 6.   CREB and PKA are required for RANKL regulation by OSM and 1,25(OH)2D3. A, UAMS-32P cells conditionally expressing the A-CREB protein were cultured in the presence or absence of 100 ng/ml DOX for 48 h and treated with vehicle, 10-7 M PTH, 20 ng/ml OSM, or 10-8 M 1,25(OH)2D3 for 16 h. Total RNA was analyzed by Northern blot with the indicated probes. B, UAMS-32P-A-CREB cells were cultured as in A and treated with vehicle, PTH, or OSM for 30 min. Total RNA was analyzed by Northern blot with a probe that recognizes both a housekeeping gene (fau) and c-fos. C, UAMS-32P cells were transfected with the 3×-CRE-LUC plasmid and treated with the indicated agents (filled circles, PTH; open circles, OSM; triangles, 1,25(OH)2D3) for 4, 8, and 16 h prior to relative light units determination. The values shown represent the mean -fold increase in relative light units relative to vehicle-treated cells ± S.D. D, UAMS-32P cells were cultured in the absence or presence of 30 µM H-89 for 30 min and treated with vehicle, PTH, OSM, or 1,25(OH)2D3 for 16 h, and total RNA was analyzed as in A. E, murine primary bone marrow cells were cultured for 8 days in the presence of 50 µg/ml ascorbic acid. The cells were then incubated in the absence or presence of 30 µM H-89 for 30 min and then treated with vehicle, db-cAMP, PTH, OSM, or 1,25(OH)2D3 for 16 h, and RNA was analyzed as above.

To distinguish between a passive role for CREB in the actions of OSM or 1,25(OH)2D3 and regulation of CREB activity by these agents, we determined whether OSM or 1,25(OH)2D3 could stimulate a CREB-responsive reporter construct. While PTH stimulated the activity of this construct, OSM or 1,25(OH)2D3 had no effect (Fig. 6C). This result suggested that, although CREB may be required for stimulation of RANKL by OSM and 1,25(OH)2D3, 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)2D3 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 OSM- and 1,25(OH)2D3 stimulation of RANKL. Consistent with the A-CREB results, H-89 reduced stimulation of RANKL by both OSM and 1,25(OH)2D3 (Fig. 6D). Similarly, inhibition of PKA activity reduced stimulation of RANKL by db-cAMP, PTH, OSM, or 1,25(OH)2D3 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)2D3 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.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 7.   PTH inhibits OPG via AP-1. A, UAMS-32P cells conditionally expressing the A-fos protein were cultured in the presence or absence of 100 ng/ml DOX for 48 h. Cells were then transiently transfected with a luciferase reporter construct containing seven AP-1 binding sites upstream from a minimal promoter (7X-AP1-LUC) and treated with vehicle or 10-7 M PTH for 16 h. B, UAMS-32P cells conditionally expressing A-fos were cultured as in A and treated with 10-7 M PTH, 20 ng/ml OSM, or 10-8 M 1,25(OH)2D3 for 16 h. Total RNA was analyzed by Northern blot with the indicated probes.

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)2D3 (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).


View larger version (84K):
[in this window]
[in a new window]
  		Fig. 8.   Runx2 is not required for PTH regulation of RANKL or OPG. UAMS-32P cells conditionally expressing the dnRunx2 protein were cultured in the presence or absence of 100 ng/ml DOX for 48 h. The cells were subsequently treated with 10-7 M PTH, 20 ng/ml OSM, or 10-8 M 1,25(OH)2D3 for 16 h, and total RNA was analyzed by Northern blot with probes for RANKL, OPG, osteocalcin (OCN), and ChoB.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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)2D3-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)2D3. 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)2D3, stimulated CREB transcriptional activity suggests that only basal CREB activity is required for the regulation of RANKL by OSM and 1,25(OH)2D3. The lack of CREB transcriptional modulation by OSM or 1,25(OH)2D3 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 tissue-specific 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 (43-47). 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)2D3 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.

    ACKNOWLEDGEMENTS

We thank Drs. E. Schipani, C. Vinson, G. Karsenty, and A. Grigoriadis for cDNAs used in this study and L. Plotkin and S. Kousteni for helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01-AR45241 and by a University of Arkansas for Medical Sciences institutional grant (to C. A. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of Arkansas for Medical Sciences, 4301 W. Markham St., Mail Slot 587, Little Rock, AR 72205. Tel.: 501-686-5607; Fax: 501-686-8148; E-mail: obriencharlesa@uams.edu.

Published, JBC Papers in Press, October 2, 2002, DOI 10.1074/jbc.M208494200

    ABBREVIATIONS

The abbreviations used are: PTH, parathyroid hormone; PTHR1, PTH/PTH-related protein receptor 1; RANKL, receptor activator of NFkappa B ligand; OPG, osteoprotegerin; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; db-cAMP, dibutyryl-cAMP; 1, 25(OH)2D3, 1,25-dihydroxyvitamin D3; IL, interleukin; OSM, oncostatin M; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MSK1, mitogen and stress-activated protein kinase 1; MEK, MAP/ERK kinase; VDR, vitamin D receptor; CRE, cAMP-response element; CREB, CRE-binding protein; CREM, CRE modulator; FBS, fetal bovine serum; DOX, doxycycline; STAT, signal transducers and activators of transcription; Epac, exchange protein directly activated by cAMP.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Liu, B. Y., Guo, J., Lanske, B., Divieti, P., Kronenberg, H. M., and Bringhurst, F. R. (1998) Endocrinology 139, 1952-1964[Abstract/Free Full Text]
2. Khosla, S. (2001) Endocrinology 142, 5050-5055[Abstract/Free Full Text]
3. Yoshida, H., Hayashi, S., Kunisada, T., Ogawa, M., Nishikawa, S., Okamura, H., Sudo, and Shultz, L. D. (1990) Nature 345, 442-444[CrossRef][Medline] [Order article via Infotrieve]
4. Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H., Sullivan, J., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian, Y. X., Kaufman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J., Delaney, J., and Boyle, W. J. (1998) Cell 93, 165-176[CrossRef][Medline] [Order article via Infotrieve]
5. Kong, Y. Y., Yoshida, H., Sarosi, I., Tan, H. L., Timms, E., Capparelli, C., Morony, S., Oliveira, d. S. A., Van, G., Itie, A., Khoo, W., Wakeham, A., Dunstan, C. R., Lacey, D. L., Mak, T. W., Boyle, W. J., and Penninger, J. M. (1999) Nature 397, 315-323[CrossRef][Medline] [Order article via Infotrieve]
6. Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M., Mochizuki, S., Tomoyasu, A., Yano, K., Goto, M., Murakami, A., Tsuda, E., Morinaga, T., Higashio, Udagawa, N., Takahashi, N., and Suda, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3597-3602[Abstract/Free Full Text]
7. Fan, X., Fan, D., Gewant, H., Royce, C. L., Nanes, M. S., and Rubin, J. (2001) Am. J. Physiol. 280, E103-E111
8. Hsu, H. L., Lacey, D. L., Dunstan, C. R., Solovyev, I., Colombero, A., Timms, E., Tan, H. L., Elliott, G., Kelley, M. J., Sarosi, I., Wang, L., Xia, X. Z., Elliott, R., Chiu, L., Black, T., Scully, S., Capparelli, C., Morony, S., Shimamoto, G., Bass, M. B., and Boyle, W. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3540-3545[Abstract/Free Full Text]
9. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M. S., Luthy, R., Nguyen, H. Q., Wooden, S., Bennett, L., Boone, T., Shimamoto, G., DeRose, M., Elliott, R., Colombero, A., Tan, H. L., Trail, G., Sullivan, J., Davy, E., Bucay, N, Renshaw-Gegg, L., Hughes, T. M., Hill, D., Pattison, W., Campbell, P., Sander, S., Van, G., Tarpley, J., Derby, P., Lee, R., and Boyle, W. J. (1997) Cell 89, 309-319[CrossRef][Medline] [Order article via Infotrieve]
10. Lee, S. K., and Lorenzo, J. A. (1999) Endocrinology 140, 3552-3561[Abstract/Free Full Text]
11. Ma, Y. L., Cain, R. L., Halladay, D. L., Yang, X., Zeng, Q., Miles, R. R., Chandrasekhar, S., Martin, T. J., and Onyia, J. E. (2001) Endocrinology 142, 4047-4054[Abstract/Free Full Text]
12. Swarthout, J. T., D'Alonzo, R. C., Selvamurugan, N., and Partridge, N. C. (2002) Gene (Amst.) 282, 1-17[Medline] [Order article via Infotrieve]
13. Shaywitz, A. J., and Greenberg, M. E. (1999) Annu. Rev. Biochem. 68, 821-861[CrossRef][Medline] [Order article via Infotrieve]
14. Pearman, A. T., Chou, W. Y., Bergman, K. D., Pulumati, M. R., and Partridge, N. C. (1996) J. Biol. Chem. 271, 25715-25721[Abstract/Free Full Text]
15. Selvamurugan, N., Pulumati, M. R., Tyson, D. R., and Partridge, N. C. (2000) J. Biol. Chem. 275, 5037-5042[Abstract/Free Full Text]
16. O'Brien, C. A., Gubrij, I., Lin, S. C., Saylors, R. L., and Manolagas, S. C. (1999) J. Biol. Chem. 274, 19301-19308[Abstract/Free Full Text]
17. Anderson, D. M., Maraskovsky, E., Billingsley, W. L., Dougall, W. C., Tometsko, M. E., Roux, E. R., Teepe, M. C., DuBose, R. F., Cosman, D., and Galibert, L. (1997) Nature 390, 175-179[CrossRef][Medline] [Order article via Infotrieve]
18. Grigoriadis, A. E., Schellander, K., Wang, Z. Q., and Wagner, E. F. (1993) J. Cell Biol. 122, 685-701[Abstract/Free Full Text]
19. Harpold, M. M., Evans, R. M., Salditt-Georgieff, M., and Darnell, J. E. (1979) Cell 17, 1025-1035[CrossRef][Medline] [Order article via Infotrieve]
20. Lin, S. C., Yamate, T., Taguchi, Y., Borba, V. Z., Girasole, G., O'Brien, C. A., Bellido, T., Abe, E., and Manolagas, S. C. (1997) J. Clin. Invest. 100, 1980-1990[Medline] [Order article via Infotrieve]
21. Yamashita, T., Asano, K., Takahashi, N., Akatsu, T., Udagawa, N., Sasaki, T., Martin, T. J., and Suda, T. (1990) J. Cell. Physiol. 145, 587-595[CrossRef][Medline] [Order article via Infotrieve]
22. Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Biochem. J. 351, 95-105[CrossRef][Medline] [Order article via Infotrieve]
23. Deak, M., Clifton, A. D., Lucocq, L. M., and Alessi, D. R. (1998) EMBO J. 17, 4426-4441[CrossRef][Medline] [Order article via Infotrieve]
24. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J. L. (1998) Nature 396, 474-477[CrossRef][Medline] [Order article via Infotrieve]
25. Mayr, B., and Montminy, M. (2001) Nat. Rev. Mol. Cell Biol. 2, 599-609[CrossRef][Medline] [Order article via Infotrieve]
26. Ahn, S., Olive, M., Aggarwal, S., Krylov, D., Ginty, D. D., and Vinson, C. (1998) Mol. Cell. Biol. 18, 967-977[Abstract/Free Full Text]
27. Tyson, D. R., Swarthout, J. T., and Partridge, N. C. (1999) Endocrinology 140, 1255-1261[Abstract/Free Full Text]
28. Olive, M., Krylov, D., Echlin, D. R., Gardner, K., Taparowsky, E., and Vinson, C. (1997) J. Biol. Chem. 272, 18586-18594[Abstract/Free Full Text]
29. Ducy, P., Starbuck, M., Priemel, M., Shen, J., Pinero, G., Geoffroy, V., Amling, M., and Karsenty, G. (1999) Genes Dev. 13, 1025-1036[Abstract/Free Full Text]
30. Takami, M., Takahashi, N., Udagawa, N., Miyaura, C., Suda, K., Woo, J. T., Martin, T. J., Nagai, K., and Suda, T. (2000) Endocrinology 141, 4711-4719[Abstract/Free Full Text]
31. Kanzawa, M., Sugimoto, T., Kanatani, M., and Chihara, K. (2000) Eur. J. Endocrinol. 142, 661-664[Abstract]
32. Halladay, D. L., Miles, R. R., Thirunavukkarasu, K., Chandrasekhar, S., Martin, T. J., and Onyia, J. E. (2001) J. Cell. Biochem. 84, 1-11[CrossRef][Medline] [Order article via Infotrieve]
33. Kondo, H., Guo, J., and Bringhurst, F. R. (2002) J. Bone Miner. Res. 17, 1667-1679[CrossRef][Medline] [Order article via Infotrieve]
34. Liu, B. Y., Wu, P. W., Bringhurst, F. R., and Wang, J. T. (2002) Endocrinology 143, 627-635[Abstract/Free Full Text]
35. O'Brien, C. A., Kern, B., Gubrij, I., Karsenty, G., and Manolagas, S. C. (2002) Bone 30, 453-462[Medline] [Order article via Infotrieve]
36. Chan, H. M., and La Thangue, N. B. (2001) J. Cell Sci. 114, 2363-2373[Abstract/Free Full Text]
37. Fata, J. E., Kong, Y. Y., Li, J., Sasaki, T., Irie-Sasaki, J., Moorehead, R. A., Elliott, R., Scully, S., Voura, E. B., Lacey, D. L., Boyle, W. J., Khokha, R., and Penninger, J. M. (2000) Cell 103, 41-50[CrossRef][Medline] [Order article via Infotrieve]
38. Ihle, J. N. (1996) Cell 84, 331-334[CrossRef][Medline] [Order article via Infotrieve]
39. Issa, L. L., Leong, G. M., and Eisman, J. A. (1998) Inflamm. Res. 47, 451-475[CrossRef][Medline] [Order article via Infotrieve]
40. Selvamurugan, N., Chou, W. Y., Pearman, A. T., Pulumati, M. R., and Partridge, N. C. (1998) J. Biol. Chem. 273, 10647-10657[Abstract/Free Full Text]
41. Rudolph, D., Tafuri, A., Gass, P., Hammerling, G. J., Arnold, B., and Schutz, G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4481-4486[Abstract/Free Full Text]
42. Reusch, J. E., Colton, L. A., and Klemm, D. J. (2000) Mol. Cell. Biol. 20, 1008-1020[Abstract/Free Full Text]
43. Feyen, J. H., Elford, P., Di, P. F. E., and Trechsel, U. (1989) J. Bone Miner. Res. 4, 633-638[Medline] [Order article via Infotrieve]
44. Lowik, C. W., van der Pluijm, G., Bloys, H., Hoekman, K., Bijvoet, O. L., Aarden, L. A., and Papapoulos, S. E. (1989) Biochem. Biophys. Res. Commun. 162, 1546-1552[CrossRef][Medline] [Order article via Infotrieve]
45. Girasole, G., Passeri, G., Jilka, R. L., and Manolagas, S. C. (1994) J. Clin. Invest. 93, 1516-1524[Medline] [Order article via Infotrieve]
46. Onyia, J. E., Bidwell, J., Herring, J., Hulman, J., and Hock, J. M. (1995) Bone 17, 479-484[Medline] [Order article via Infotrieve]
47. Pollock, J. H., Blaha, M. J., Lavish, S. A., Stevenson, S., and Greenfield, E. M. (1996) J. Bone Miner. Res. 11, 754-759[Medline] [Order article via Infotrieve]
48. Tamura, T., Udagawa, N., Takahashi, N., Miyaura, C., Tanaka, S., Yamada, Y., Koishihara, Y., Ohsugi, Y., Kumaki, K., Taga, T., Kishimoto, T., and Suda, T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11924-11928[Abstract/Free Full Text]
49. Romas, E., Udagawa, N., Zhou, H., Tamura, T., Saito, M., Taga, T., Hilton, D. J., Suda, T., Ng, K. W., and Martin, T. J. (1996) J. Exp. Med. 183, 2581-2591[Abstract/Free Full Text]
50. Grey, A., Mitnick, M. A., Masiukiewicz, U., Sun, B. H., Rudikoff, S., Jilka, R. L., Manolagas, S. C., and Insogna, K. (1999) Endocrinology 140, 4683-4690[Abstract/Free Full Text]
51. Roodman, G. D. (2001) J. Clin. Oncol. 19, 3562-3571[Abstract/Free Full Text]
52. Jilka, R. L. (1998) Bone 23, 75-81[Medline] [Order article via Infotrieve]
53. Kerst, J. M., Slaper-Cortenbach, I. C., van der Schoot, C. E., Hooibrink, B., dem Borne, A. E., and van Oers, R. H. (1993) Exp. Hematol. 21, 1550-1557[Medline] [Order article via Infotrieve]
54. Cappellen, D., Luong-Nguyen, N. H., Bongiovanni, S., Grenet, O., Wanke, C., and Susa, M. (2002) J. Biol. Chem. 277, 21971-21982[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C. Galli, Q. Fu, W. Wang, B. R. Olsen, S. C. Manolagas, R. L. Jilka, and C. A. O'Brien
Commitment to the Osteoblast Lineage Is Not Required for RANKL Gene Expression
J. Biol. Chem., May 8, 2009; 284(19): 12654 - 12662.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
G. Rashid, E. Plotkin, O. Klein, J. Green, J. Bernheim, and S. Benchetrit
Parathyroid hormone decreases endothelial osteoprotegerin secretion: role of protein kinase A and C
Am J Physiol Renal Physiol, January 1, 2009; 296(1): F60 - F66.
[Abstract] [Full Text] [PDF]

Home page
 JDRHome page
F. Clauss, M.-C. Maniere, F. Obry, E. Waltmann, S. Hadj-Rabia, C. Bodemer, Y. Alembik, H. Lesot, and M. Schmittbuhl
Dento-Craniofacial Phenotypes and underlying Molecular Mechanisms in Hypohidrotic Ectodermal Dysplasia (HED): a Review
Journal of Dental Research, December 1, 2008; 87(12): 1089 - 1099.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
A. E. Kearns, S. Khosla, and P. J. Kostenuik
Receptor Activator of Nuclear Factor {kappa}B Ligand and Osteoprotegerin Regulation of Bone Remodeling in Health and Disease
Endocr. Rev., April 1, 2008; 29(2): 155 - 192.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. J. Raggatt, L. Qin, J. Tamasi, S. C. Jefcoat Jr., E. Shimizu, N. Selvamurugan, F. Y. Liew, L. Bevelock, J. H. M. Feyen, and N. C. Partridge
Interleukin-18 Is Regulated by Parathyroid Hormone and Is Required for Its Bone Anabolic Actions
J. Biol. Chem., March 14, 2008; 283(11): 6790 - 6798.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
I. A. Nakchbandi, R. Lang, B. Kinder, and K. L. Insogna
The Role of the Receptor Activator of Nuclear Factor-{kappa}B Ligand/Osteoprotegerin Cytokine System in Primary Hyperparathyroidism
J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 967 - 973.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Galli, L. A. Zella, J. A. Fretz, Q. Fu, J. W. Pike, R. S. Weinstein, S. C. Manolagas, and C. A. O'Brien
Targeted Deletion of a Distant Transcriptional Enhancer of the Receptor Activator of Nuclear Factor-{kappa}B Ligand Gene Reduces Bone Remodeling and Increases Bone Mass
Endocrinology, January 1, 2008; 149(1): 146 - 153.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Ono, K. Nakashima, E. Schipani, T. Hayata, Y. Ezura, K. Soma, H. M. Kronenberg, and M. Noda
Constitutively Active Parathyroid Hormone Receptor Signaling in Cells in Osteoblastic Lineage Suppresses Mechanical Unloading-induced Bone Resorption
J. Biol. Chem., August 31, 2007; 282(35): 25509 - 25516.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Fionda, F. Nappi, M. Piccoli, L. Frati, A. Santoni, and M. Cippitelli
15-Deoxy-{Delta}12,14-Prostaglandin J2 Negatively Regulates rankl Gene Expression in Activated T Lymphocytes: Role of NF-{kappa}B and Early Growth Response Transcription Factors
J. Immunol., April 1, 2007; 178(7): 4039 - 4050.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Kousteni, M. Almeida, L. Han, T. Bellido, R. L. Jilka, and S. C. Manolagas
Induction of Osteoblast Differentiation by Selective Activation of Kinase-Mediated Actions of the Estrogen Receptor
Mol. Cell. Biol., February 15, 2007; 27(4): 1516 - 1530.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
S. Kim, M. Yamazaki, N. K. Shevde, and J. W. Pike
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
Mol. Endocrinol., January 1, 2007; 21(1): 197 - 214.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
Q. Fu, S. C. Manolagas, and C. A. O'Brien
Parathyroid Hormone Controls Receptor Activator of NF-{kappa}B Ligand Gene Expression via a Distant Transcriptional Enhancer.
Mol. Cell. Biol., September 1, 2006; 26(17): 6453 - 6468.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Kim, M. Yamazaki, L. A. Zella, N. K. Shevde, and J. W. Pike
Activation of Receptor Activator of NF-{kappa}B Ligand Gene Expression by 1,25-Dihydroxyvitamin D3 Is Mediated through Multiple Long-Range Enhancers.
Mol. Cell. Biol., September 1, 2006; 26(17): 6469 - 6486.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
I. Villa, E. Mrak, A. Rubinacci, F. Ravasi, and F. Guidobono
CGRP inhibits osteoprotegerin production in human osteoblast-like cells via cAMP/PKA-dependent pathway
Am J Physiol Cell Physiol, September 1, 2006; 291(3): C529 - C537.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. J. Bergh, Y. Shao, E. Puente, R. L. Duncan, and M. C. Farach-Carson
Osteoblast Ca2+ permeability and voltage-sensitive Ca2+ channel expression is temporally regulated by 1,25-dihydroxyvitamin D3
Am J Physiol Cell Physiol, March 1, 2006; 290(3): C822 - C831.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
C. A. O'Brien, R. L. Jilka, Q. Fu, S. Stewart, R. S. Weinstein, and S. C. Manolagas
IL-6 is not required for parathyroid hormone stimulation of RANKL expression, osteoclast formation, and bone loss in mice
Am J Physiol Endocrinol Metab, November 1, 2005; 289(5): E784 - E793.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. V. Komarova
Mathematical Model of Paracrine Interactions between Osteoclasts and Osteoblasts Predicts Anabolic Action of Parathyroid Hormone on Bone
Endocrinology, August 1, 2005; 146(8): 3589 - 3595.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X.-c. Bai, D. Lu, A.-l. Liu, Z.-m. Zhang, X.-m. Li, Z.-p. Zou, W.-s. Zeng, B.-l. Cheng, and S.-q. Luo
Reactive Oxygen Species Stimulates Receptor Activator of NF-{kappa}B Ligand Expression in Osteoblast
J. Biol. Chem., April 29, 2005; 280(17): 17497 - 17506.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. A. Ali, R. S. Weinstein, S. A. Stewart, A. M. Parfitt, S. C. Manolagas, and R. L. Jilka
Rosiglitazone Causes Bone Loss in Mice by Suppressing Osteoblast Differentiation and Bone Formation
Endocrinology, March 1, 2005; 146(3): 1226 - 1235.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst
Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands
Endocr. Rev., February 1, 2005; 26(1): 78 - 113.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
I. C. Ozkurt, F. Q. Pirih, and S. Tetradis
Parathyroid Hormone Induces E4bp4 Messenger Ribonucleic Acid Expression Primarily through Cyclic Adenosine 3',5'-Monophosphate Signaling in Osteoblasts
Endocrinology, August 1, 2004; 145(8): 3696 - 3703.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
E. C. Buxton, W. Yao, and N. E. Lane
Changes in Serum Receptor Activator of Nuclear Factor-{kappa}B Ligand, Osteoprotegerin, and Interleukin-6 Levels in Patients with Glucocorticoid-Induced Osteoporosis Treated with Human Parathyroid Hormone (1-34)
J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3332 - 3336.
[Abstract] [Full Text] [PDF]

Home page
 CROBMHome page
U. H. Lerner
NEW MOLECULES IN THE TUMOR NECROSIS FACTOR LIGAND AND RECEPTOR SUPERFAMILIES WITH IMPORTANCE FOR PHYSIOLOGICAL AND PATHOLOGICAL BONE RESORPTION
Critical Reviews in Oral Biology & Medicine, March 1, 2004; 15(2): 64 - 81.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. Suda, N. Udagawa, N. Sato, M. Takami, K. Itoh, J.-T. Woo, N. Takahashi, and K. Nagai
Suppression of Osteoprotegerin Expression by Prostaglandin E2 Is Crucially Involved in Lipopolysaccharide-Induced Osteoclast Formation
J. Immunol., February 15, 2004; 172(4): 2504 - 2510.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
L. Buttitta, R. Mo, C.-C. Hui, and C.-M. Fan
Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction
Development, December 22, 2003; 130(25): 6233 - 6243.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Bellido, A. A. Ali, L. I. Plotkin, Q. Fu, I. Gubrij, P. K. Roberson, R. S. Weinstein, C. A. O'Brien, S. C. Manolagas, and R. L. Jilka
Proteasomal Degradation of Runx2 Shortens Parathyroid Hormone-induced Anti-apoptotic Signaling in Osteoblasts: A PUTATIVE EXPLANATION FOR WHY INTERMITTENT ADMINISTRATION IS NEEDED FOR BONE ANABOLISM
J. Biol. Chem., December 12, 2003; 278(50): 50259 - 50272.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/50/48868    most recent
M208494200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fu, Q.
Right arrow Articles by O'Brien, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fu, Q.
Right arrow Articles by O'Brien, C. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement