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PTHrP Modulates Chondrocyte Differentiation through AP-1 and CREB Signaling

2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês

10.1074/jbc.m006564200

1083-351X

Andreia Ionescu, Edward M. Schwarz, Charles Vinson, J. Edward Puzas, Randy N. Rosier, Paul R. Reynolds, Regis J. O’Keefe,

Hedgehog Signaling Pathway Studies

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor β. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation. During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor β. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation. During limb development, a cartilaginous template is formed from mesenchymal condensations that perform the skeletal elements (1Wright E. Hargrave M.R. Christiansen J. Cooper L. Kun J. Evans T. Gangadharan U. Greenfield A. Koopman P. Nat. Genet. 1995; 9: 15-20Crossref PubMed Scopus (560) Google Scholar). Subsequent longitudinal bone growth is dependent upon the process of endochondral ossification whereby chondrocytes sequentially proliferate and differentiate. Chondrocyte differentiation is marked by profound physical and biochemical changes, including a 5–10-fold increase in volume and expression of alkaline phosphatase and type X collagen (2Buckwalter J.A. Mower D. Ungar R. Schaeffer J. Ginsberg B. J. Bone Joint Surg. Am. Vol. 1986; 68: 55-243Crossref PubMed Scopus (161) Google Scholar,3Linsenmayer T.F. Chen Q.A. Gibney E. Gordon M.K. Marchant J.K. Mayne R. Schmid T.M. Development. 1991; 111: 191-196Crossref PubMed Google Scholar). The process of chondrocyte differentiation culminates in calcification of the matrix and cellular apoptosis (4Gibson G.J. Kohler W.J. Schaffler M.B. Dev. Dyn. 1995; 203: 468-476Crossref PubMed Scopus (123) Google Scholar, 5Hatori M. Klatte K.J. Teixeira C.C. Shapiro I.M. J. Bone Miner. Res. 1995; 10: 1960-1968Crossref PubMed Scopus (177) Google Scholar, 6Lewinson D. Silbermann M. Anat. Rec. 1992; 233: 504-514Crossref PubMed Scopus (119) Google Scholar). The calcified matrix subsequently serves as the template for primary bone formation. Endochondral ossification is regulated by local growth factors including PTHrP1, which has been defined as a critical regulator of the rate of endochondral ossification. Mice null for either PTHrP (7Karaplis A.C. Luz A. Glowacki J. Bronson R.T. Tybulewicz V.L. Kronenberg H.M. Mulligan R.C. Genes Dev. 1994; 8: 277-289Crossref PubMed Scopus (1009) Google Scholar) or its receptor (8Lanske B. Karaplis A.C. Lee K. Luz A. Vortkamp A. Pirro A. Karperien M. Defize L.H.K. Ho C. Mulligan R.C. Abou-Samra A.B. Juppner H. Segre G.V. Kronenberg H.M. Science. 1996; 273: 663-666Crossref PubMed Scopus (1140) Google Scholar) display accelerated chondrocyte differentiation and therefore abnormal endochondral bone formation. In contrast, animals that overexpress PTHrP exhibit delay in chondrocyte terminal differentiation (9Weir E.C. Philbrick W.M. Amling M. Neff L.A. Baron R. Broadus A.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10240-10245Crossref PubMed Scopus (401) Google Scholar). Humans with an activating mutation in the PTH/PTHrP receptor have Jansen's metaphyseal chondrodysplasia, characterized by disorganization of the growth plate and delayed chondrocyte terminal differentiation (10Schipani E. Lanske B. Hunzelman J. Luz A. Kovacs C.S. Lee K. Pirro A. Kronenberg H.M. Juppner H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13689-13694Crossref PubMed Scopus (338) Google Scholar). Thus, PTHrP signaling is critical for normal growth plate morphology and function. PTHrP receptor activation stimulates both protein kinase A (PKA) and protein kinase C (PKC) signaling. Activation of these signaling pathways mediates important effects and in chondrocytes PKA signaling is associated with proliferation, stimulation of proteoglycan synthesis, and inhibition of alkaline phosphatase activity (11Zuscik M.J. Puzas J.E. Rosier R.N. Gunter K.K. Gunter T.E. Arch. Biochem. Biophys. 1994; 315: 352-361Crossref PubMed Scopus (31) Google Scholar). PTHrP also stimulates phospholipase C activity with metabolism of membrane phospholipids and production of diacyl glycerol and inositol phosphate (12Zuscik M.J. Gunter T.E. Rosier R.N. Gunter K.K. Puzas J.E. Cell Calcium. 1994; 16: 112-122Crossref PubMed Scopus (12) Google Scholar, 13Segre G.V. Abou-Samra A.B. Juppner H. Schipani E. Force T. Urena P. Freeman M. Kong X.F. Kolakowski Jr., L.F. Hock J. J. Endocrinol. Invest. 1992; 15: 11-17PubMed Google Scholar). Inositol phosphate stimulates the release of intracellular calcium stores while the regeneration of diacylglycerol leads to activation of protein kinase C. While much is known about the upstream signaling events immediately following receptor ligation, there is little information on the downstream transcription factors involved in mediating PTHrP effects in chondrocytes and their relationship with PKA and PKC signaling. One of the potential downstream signaling targets of PKA is the cyclic AMP response element-binding protein (CREB) (14Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (860) Google Scholar). This transcription factor is a CREB/ATF family member that is constitutively present in the nucleus. CREB binds to a DNA consensus cAMP response element (CRE) primarily as a homodimer, via a leucine zipper domain. CREB is activated by PKA-mediated phosphorylation within its P-box domain (15Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2067) Google Scholar), which permits its interaction with p300/CBP and other coactivators, leading to gene transcription. Thymocytes and T cells from transgenic mice expressing a dominant-negative form of CREB specifically in these cells display a profound proliferative defect and G1cell-cycle arrest in response to a number of different activation signals (16Barton K. Muthusamy N. Chanyangam M. Fischer C. Clendenin C. Leiden J.M. Nature. 1996; 379: 81-85Crossref PubMed Scopus (219) Google Scholar). ATF-2 deficiency, another member of the CREB/ATF family, has been shown to induce chondrodysplasia and neurological abnormalities in mutant mice (17Reimold A.M. Grusby M.J. Kosaras B. Fries J.W. Mori R. Maniwa S. Clauss I.M. Collins T. Sidman R.L. Glimcher M.J. Glimcher L.H. Nature. 1996; 379: 262-265Crossref PubMed Scopus (243) Google Scholar). Although the role of CREB in cartilage development or bone formation has not been investigated, the finding that CREB KO mouse has a dwarf phenotype (18Rudolph D. Tafuri A. Gass P. Hammerling G.J. Arnold B. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4481-4486Crossref PubMed Scopus (268) Google Scholar) combined with the established important role of CREB in PKA mediated events in other cells, suggests that this transcription factor may be involved in regulating some of the critical events in chondrocyte differentiation. PTHrP also activates the transcription factor AP-1, a complex formed through interactions between Fos and Jun family members (19Porte D. Tuckermann J. Becker M. Baumann B. Teurich S. Higgins T. Owen M.J. Schorpp-Kistner M. Angel P. Oncogene. 1999; 18: 667-678Crossref PubMed Scopus (138) Google Scholar, 20Selvamurugan N. Chou W.Y. Pearman A.T. Pulumati M.R. Partridge N.C. J. Biol. Chem. 1998; 273: 10647-10657Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). These interactions are also mediated by leucine zipper domains to form Fos/Jun heterodimers or Jun/Jun homodimers (21Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3285) Google Scholar). The protein complex binds to the phorbol 12-myristate 13-acetate response element (TRE), a specific cis-acting DNA consensus sequence in the promoter region of target genes. AP-1-regulated genes appear to have an important role in skeletal physiology. Transgenic mice overexpressing c-Fos develop bone and cartilaginous tumors (22Ruther U. Garber C. Komitowski D. Muller R. Wagner E.F. Nature. 1987; 325: 412-416Crossref PubMed Scopus (269) Google Scholar), while Fos-Jun double transgenic mice develop osteosarcomas at a higher frequency (23Wang Z.Q. Liang J. Schellander K. Wagner E.F. Grigoriadis A.E. Cancer Res. 1995; 55: 6244-6251PubMed Google Scholar). PTHrP stimulates c-Fos mRNA and protein synthesis in osteoblasts (24Clohisy J.C. Scott D.K. Brakenhoff K.D. Quinn C.O. Partridge N.C. Mol. Endocrinol. 1992; 6: 1834-1842Crossref PubMed Scopus (77) Google Scholar), and in chondrocytes PTHrP was found to increase c-Jun and JunD mRNA and protein levels (25Kameda T. Watanabe H. Iba H. Cell Growth Differ. 1997; 8: 495-503PubMed Google Scholar). AP-1 activation has been shown to be mediated by several different signaling pathways including PKA and PKC (26Anouar Y. Lee H.W. Eiden L.E. Mol. Pharmacol. 1999; 56: 162-169Crossref PubMed Scopus (26) Google Scholar,27White B.R. Duval D.L. Mulvaney J.M. Roberson M.S. Clay C.M. Mol. Endocrinol. 1999; 13: 566-577Crossref PubMed Google Scholar). In this work we have characterized CREB and c-Fos as two important regulators of cartilage development and have investigated their activation by PKA and PKC signaling. We have shown that their activation occurs rapidly in response to PTHrP stimulation and that interference with their function results in alteration of cartilage phenotype produced by PTHrP. This represents the first report documenting a critical role for CREB in cartilage development. Embryonic cephalic sternal chondrocytes (day 13) were prepared and cultured as described (28Leboy P.S. Vaias L. Uschmann B. Golub E. Adams S.L. Pacifici M. J. Biol. Chem. 1989; 264: 17281-17286Abstract Full Text PDF PubMed Google Scholar). After isolation and primary culture for 5–7 days, floating cells were plated in secondary cultures at 2.5 × 105cells/cm2 in Dulbecco's modified Eagle's medium containing 10% NuSerum IV (Collaborative Biomedical, Bedford MA), 4 units/ml hyaluronidase (Sigma), and 2 mml-glutamate (Sigma). After 6 days, upper sternal chondrocytes (USC) were harvested and plated in 6-well plates for the transient transfection assay and alkaline phosphatase or 60-mm dishes for Northern analysis. PTHrP (10−7m) was added to the cultures in some experiments. Cell were washed with cold phosphate-buffered saline and lysed on ice in Golden lysis buffer (29Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (324) Google Scholar) supplemented with protease inhibitor mixture tablets (Roche Molecular Biochemicals), 1 mm sodium orthovanadate, 1 mmEGTA, 1 mm NaF, 1 μm microcysteine. Insoluble material was removed by centrifugation at 12,000 × g. The protein concentration of the soluble material was estimated using Coomassie Plus Protein Assay kit (Pierce, Rockford, IL). 25 μg of extracts was assayed by SDS-polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane (Schleicher and Schuell), the blots were probed with the following antibodies: anti-CREB or anti-phospho-CREB antibody (Upstate Biotechnology, Lake Placid, NY) at a dilution 1:500, anti-c-Fos, anti-ATF-2, anti-CREM, and anti-ATF-1 (Santa Cruz) at a dilution 1:1000. In one case, the membrane was striped and reprobed with anti-β-actin (Sigma) in a dilution 1:8000 to serve as a loading control. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibodies (Bio-Rad) were used as secondary antibody. The immune complexes were detected using ECL (Amersham Pharmacia Biotech). Nuclear protein extracts were prepared as previously described (30Schwarz E.M. Salgame P. Bloom B.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7734-7738Crossref PubMed Scopus (40) Google Scholar) were analyzed by electrophoretic mobility shift assay. 5 μg of this nuclear extracts from upper sternal chondrocytes treated with PTHrP for 30 min, 1 h, 2 h, or 4 h were incubated with 1 μg of poly(dI-dC) (Amersham Pharmacia Biotech) and 2 ng of 32P-end-labeled CRE or TRE consensus oligos (Santa Cruz) and run on a 5% polyacrylamide gel. Supershifts were performed with antibodies specific for CREB/ATF and Fos/Jun family members. The incubation with antibodies was 20 min prior to the addition of oligonucleotides. For this set of experiments we have used Stratagene's Path detect in vivosignal transduction pathway reporting systems, which are a series of inducible reporter vectors that contain the firefly luciferase reporter gene driven by a basic promoter (TATA box) plus a defined inducible enhancer element: four repeats of CRE (CRE-Luc) or seven repeats of the TRE (TRE-Luc). The transfection efficiency was controlled for by co-transfecting pRL vector from Promega and determining theRenilla uniformis luciferase activity. The upper sternal chondrocytes were transiently transfected in serum-free media (supplemented with insulin and triiodothyronine) (31Volk S.W. Luvalle P. Leask T. Leboy P.S. J. Bone Miner. Res. 1998; 13: 1521-1529Crossref PubMed Scopus (81) Google Scholar) with these plasmids using the transfection reagent Fugene-6 (Roche Molecular Biochemicals). Twelve hours after transfection, a PTHrP (10−7m) treatment was given to the cells for 24 h. Two plasmids expressing the PKA catalytic subunit and 360–672 amino acids from MEKK were used as positive controls for the CRE/TRE-Luc reporting systems, respectively. The following inhibitors purchased from Calbiochem were used: H-89, 10 μm for PKA inhibition, GO6976, 12 μm for PKC inhibition, 1 μm KN-93 for CaM kinase II, and 50 μmPD98059 for MEKK. We have also used a dominant negative CREB and c-Fos in the pRC-CMV vector, as previously described (32Ahn S. Olive M. Aggarwal S. Krylov D. Ginty D.D. Vinson C. Mol. Cell. Biol. 1998; 18: 967-977Crossref PubMed Scopus (450) Google Scholar). In the co-transfection experiments 1 μg of reporter and 1 μg of dominant negative (or empty vector for control) were co-transfected in a ratio 1:3 with the transfection reagent. Other reporters used were c-Fos promoter and several cyclin D1 promoter constructs from Dr. R. Pestell (Albert Einstein College of Medicine). Constructs employed were the following: 1745-bp full-length cyclin D1 promoter, 66 bp of the promoter encompassing the CRE site situated at 52 bp and 1745 mut and 66 mut that have the CRE site mutated as previously described (33Brown J.R. Nigh E. Lee R.J. Ye H. Thompson M.A. Saudou F. Pestell R.G. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 5609-5619Crossref PubMed Scopus (211) Google Scholar). Luciferase activity in cell lysate was measured using Luciferase assay system (Promega) and an Optocomp luminometer (MGM Instruments). Chick embryonic fibroblasts grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 0.2% fetal chick serum and penicillin/streptomycin were transfected with the replication competent avian sarcoma virus RCASBP(A) alone and RCASBP(A) containing dominant negative c-Fos and CREB inserts. Cells were passaged three times to allow the spreading of the virus. At confluence, growth media was changed to low serum (Dulbecco's modified Eagle's medium with 10% NuSerum) for collection of virus. Viral supernatants were collected at 24-h intervals for 3 days. At the time of secondary plating, chondrocytes were incubated with fresh viral supernatant for 48 h followed by addition of PTHrP in some of the samples. Total RNA was extracted from cultures using the RNAeasy kit (Qiagen, Valencia, CA). 5 μg of the total RNA was run on a 1.2% agarose gel containing 17.5% formaldehyde and transferred to a GeneScreen Plus membrane (PerkinElmer Life Sciences). The RNA was UV cross-linked to the membrane. Northern analysis was performed using denaturing formaldehyde/agarose gels as described (34Ausubel F. Brent R. Kingston R.E. Moore D.D. Sieman J.G. Smith J.A. Short Protocols in Molecular Biology. J. Wiley, and Sons, New York1997Google Scholar). A synthetic type X oligonucleotide was end labeled as previously described (34Ausubel F. Brent R. Kingston R.E. Moore D.D. Sieman J.G. Smith J.A. Short Protocols in Molecular Biology. J. Wiley, and Sons, New York1997Google Scholar). Prehybridization was performed in QuickHyb solution (Stratagene, La Jolla, CA) for 20 min at 68 °C. Hybridization was done at 73 °C for 1 h. The blot was washed twice for 15 min with 2 × SSC and 0.1% SDS, followed by a 30-min wash with 0.1 × SSC and 0.1% SDS. The blot was exposed to X-Omat AR film (Kodak, Rochester, NY) for autoradiography. Alkaline phosphatase activity was measured as previously described (35O'Keefe R.J. Crabb I.D. Puzas J.E. Rosier R.N. J. Bone Joint Surg. Am. Vol. 1989; 71: 607-620Crossref PubMed Scopus (38) Google Scholar). Culture medium was aspirated from chondrocytes cultured in 6-well plates, which then were rinsed with 150 mm NaCl. One ml of reaction buffer containing 0.25m 2-methyl-2-aminopropanol, 1 mmMgCl2, and 2.5 mg/ml p-nitrophenyl phosphate (Sigma) at pH 10.3 was added to the wells at 37 °C. The reaction was stopped after 30 min by the addition of 0.5 ml of 0.3 mNa3PO4 (pH 12.3). The alkaline phosphatase activity was determined spectrophotometrically at 410 nm by comparison with standard solutions of p-nitrophenol and an appropriate blank. Flow cytometry was performed using a fluorescence-activated cell sorter Calibur cytometer and the Cell Quest (Becton-Dickinson, Franklin Lakes, NJ) plotting program. 2–3 × 106 cells were resuspended in 70% EtOH at 4 °C for at least 12 h. The cells were then resuspended in 1 × phosphate-buffered saline with 1 mg/ml RNase and incubated for 30 min at room temperature. Finally cells were stained with propidium iodide (10 μg/ml in phosphate-buffered saline) and run through the flow cytometer. Chondrocytes plated at 200,000 cells/well in a 6-well plate were treated with PTHrP for 24 h and incubated with 2 μCi/ml [3H]thymidine (PerkinElmer Life Sciences) in the last 4 h. Cells were washed with cold 5% trichloroacetic acid two times and then the cells were left in 0.5 ml of 1 n NaOH, 0.1% Triton X-100 overnight at 4 °C. 100-μl aliquots were added to 4 ml of Ecoscint fluid and assayed for 3H incorporation. Statistical comparisons were made between the groups using a way analysis of variance (ANOVA). Significance was considered present when the p value was less than 0.05 and is denoted in each of the figures. The effects of PTHrP stimulation on CREB/ATF activation were investigated in sternal chondrocytes at various times following treatment over a 2-h time course experiment. The level of CREB protein remained constant as determined by Western blotting (Fig.1 A). However, phosphorylated CREB (pCREB) levels were markedly increased following this treatment. The increase in pCREB was detectable within 5 min, maximal by 30 min, and returned to baseline levels 2 h after PTHrP stimulation (Fig.1 A). Following PTHrP stimulation, there was also an increase in c-Fos protein levels that was observed as early as 30 min, was maximal by 1 h, and returned to basal levels by 4 h (Fig.1 B). To determine the effect of PTHrP stimulation on CREB DNA binding activity in chondrocytes, gel mobility shift assays were performed. These experiments revealed minimal differences over a 4-h time course following PTHrP stimulation (Fig.2 A). To characterize this protein-DNA complex, supershift experiments using specific antibodies to members of the CREB/ATF family were performed. In this experiment, only CREB binding to this consensus sequence was detected (Fig.2 B), although ATF-2 and CREM proteins could be detected in these extracts by Western blot (Fig. 2 C). These findings suggest that CREB protein levels and DNA binding activity remain constant following PTHrP treatment. However, PTHrP may modulate gene expression in sternal chondrocytes by inducing CREB phosphorylation and therefore increasing its transcriptional activity.Figure 2PTHrP induces AP-1, but not CREB DNA binding activity. Nuclear extracts (5 μg/lane) obtained from USC stimulated with PTHrP for various time points, were incubated with or without anti-c-Fos antibody, followed by addition of a radioactive labeled consensus AP-1 probe (A). Nuclear extracts (5 μg/lane) from USC were also incubated with radioactively labeled CRE oligonucleotides and then gel shift assay was performed (A). Nuclear extracts (5 μg/lane) from USC were preincubated with antibodies against different CREB/ATF or Fos family members, before addition of radioactive labeled CRE or AP-1 oligonucleotides and then gel shift assay was performed (B). Western blots with antibodies against different CREB/ATF family members were done with whole cell lysates from USC (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess AP-1 activation following PTHrP stimulation, gel mobility shift assays were performed using a probe containing the TRE consensus sequence. PTHrP stimulated a marked increase in DNA binding (Fig.2 A). The increase was detectable at 30 min, peaked at 1 h, and remained elevated 4 h following PTHrP treatment. Addition of an antibody directed against c-Fos protein inhibited DNA binding (Fig. 2 A), showing the presence of c-Fos protein in the gel mobility shift complex, consistent with the increase in c-Fos protein expression observed on Western blot. In contrast, incubation with antibodies directed against Fra1, ATF-2, and CREM did not affect DNA binding (Fig. 2 B). To determine whether this apparent stimulation of CREB and AP-1 signaling is associated with an increase in gene transcription, sternal chondrocytes were transfected with CRE and TRE luciferase reporter constructs and treated with PTHrP (Fig.3). PTHrP (10−7m) treatment resulted in transcriptional activation of both the TRE (23-fold) and CRE (103-fold) constructs. As a positive control we co-transfected the reporter constructs with either activated MAPK kinase kinase (MEKK) or PKA constructs. In these experiments the MEKK construct was a more potent stimulator of the TRE reporter, while the PKA construct caused about a 40-fold stimulation of both the CRE and the TRE reporters (Fig. 3 A). These latter findings indicate the presence of cross-talk between these signaling pathways in chondrocytes following PTHrP stimulation. Furthermore, PKA signaling in these cells results in activation of both CRE and TRE mediated transcription. To determine the relative effects of the PKA, PKC, mitogen-activated protein kinase, and calmodulin kinase signaling pathways on CRE activation, the CRE reporter was transfected into sternal chondrocytes and the cultures were treated with PTHrP in the presence and absence of pharmacological inhibitors of these various pathways (Fig.3 B). Inhibition of PKA signaling with H89 resulted in a 95% inhibition of CRE activation, consistent with the role of PKA as a potent inducer of CREB phosphorylation. Inhibitors of PKC (GO6976, 45% inhibition), mitogen-activated protein kinase (PD98059, 27% inhibition), and calmodulin kinase (KN93, 17% inhibition) all had less potent effects. PKA inhibition also had the greatest effect on the TRE reporter (80% inhibition), while inhibition of MAP kinase signaling resulted in a 37.7% decrease in luciferase activity. In contrast, inhibition of PKC and calmodulin kinase did not affect PTHrP-mediated activation of the TRE reporter (Fig. 3 C). To confirm our findings regarding CREB and AP-1 transcriptional activation following PTHrP treatment, constructs expressing dominant negative c-Fos (AFOS) and CREB (ACREB) were each co-transfected into sternal chondrocytes with reporter constructs for the CRE and TRE (Fig.4). The ACREB construct completely inhibited CRE activation by PTHrP, while the AFOS construct did not inhibit PTHrP activation of the CRE (Fig. 4 A). Moreover, the AFOS construct induced an increase in the PTHrP stimulation of CRE reporter. A possible explanation for this finding is that AP-1 inhibition may result in greater availability of the transcriptional coactivator, CBP (CREB-binding protein), for CREB-mediated signaling. In contrast, both ACREB and AFOS constructs inhibited activation of the TRE reporter following stimulation by PTHrP (Fig. 4 B). Co-transfection of both constructs reduced the detectable luciferase activities to below basal levels. These findings further confirm PTHrP activation of PKA and phosphorylation of CREB, leading to CRE dependent transcription. In contrast, activation of AP-1 by CREB is likely indirect and may be mediated by stimulation of c-Fos transcription. To test this, a c-Fos promoter-reporter construct was transfected into sternal chondrocytes. PTHrP treatment resulted in a 91-fold stimulation in luciferase activity (Fig. 4 C). Co-transfection of these cultures with the ACREB construct blocked this PTHrP stimulation of the cFos promoter, while the AFOS construct had no effect, further supporting a model of PTHrP activation of AP-1 through CREB-dependent transcription of c-Fos. PTHrP regulates the rate of chondrocyte terminal differentiation. To see if PTHrP inhibits terminal differentiation through CREB or AP-1 signaling, we investigated the expression of markers of chondrocyte maturation, type X collagen (colX) mRNA expression, and alkaline phosphatase activity, in cell cultures following PTHrP treatment. Short-term experiments were performed using transiently transfected chondrocytes (2 days), while longer term experiments (7 days) were performed with chondrocytes infected with a replication competent retrovirus that permitted sustained expression in a larger percentage of cells. In the absence of PTHrP, ACREB, and AFOS had minimal effects in both the transiently transfected and retrovirally infected cultures (Fig.5, A and B). However, in the presence of PTHrP, transient transfection of ACREB and AFOS constructs stimulated type X collagen (colX) mRNA expression, 1.6- and 1.3-fold, respectively (Fig. 5 A), compared with vector-transfected control chondrocytes. These signaling pathways appeared to be synergistic, since co-transfection of both ACREB and AFOS resulted in even greater colX mRNA expression (5.2-fold). Effects of the dominant negative signaling molecules were much greater when the constructs were introduced by retroviral infection. Alone, both ACREB and AFOS infection resulted in a larger induction of colX in PTHrP-treated cultures compared with minimal increases in retroviral infected control samples (Fig. 5 B). In PTHrP-treated cultures, transient transfection with both ACREB and AFOS resulted in only small increases in alkaline phosphatase activity (data not shown). Much larger effects on alkaline phosphatase activity were observed when the cultures were infected with retroviral constructs, consistent with the findings observed with the other maturation marker, type X collagen. In the PTHrP-treated cultures, ACREB doubled and AFOS tripled alkaline phosphatase activity (Fig.6) compared with smaller increases in untreated samples (1.3- and 1.7-fold increases). Collectively, these findings demonstrate that PTHrP effects on maturation are mediated by both CREB and AP-1 signaling. Furthermore, while the findings suggest basal AP-1 and CREB signaling, these transcription factors are much more relevant in the presence of PTHrP. To evaluate the effects of PTHrP on proliferation, sternal chondrocytes were treated with PTHrP and analyzed by fluorescence-activated cell sorter 24 h latter (Table I). PTHrP treatment resulted in more than a 2-fold increase in the number of cells entering the cell cycle and undergoing DNA synthesis (S + G2 + M). Since cyclin D1 is a required for t