The Cyclin D1 and Cyclin A Genes Are Targets of Activated PTH/PTHrP Receptors in Jansens Metaphyseal Chondrodysplasia
Frank Beier and
Phyllis LuValle1
Canadian Institutes of Health Research Group in Skeletal Development and Remodeling (F.B.), Department of Physiology, University of Western Ontario, London, Ontario, Canada N6A 5C1; and Department of Biochemistry and Molecular Biology (P.L.), University of Calgary, Calgary, Alberta, Canada T2N 4N1
Address all correspondence and requests for reprints to: Frank Beier, Department of Physiology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1. E-mail: fbeier{at}uwo.ca.
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ABSTRACT
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Jansens metaphyseal chondrodysplasia (JMC) is an autosomal dominant disorder characterized by short-limbed dwarfism, delayed ossification, and hypercalcemia. Activating mutations in the PTH/PTHrP receptor have been identified as the molecular cause of this disorder. Although these mutations have been shown to increase cAMP accumulation, little is known about possible target genes of the downstream signaling pathways that may contribute to the pathogenesis of the disease. Here we demonstrate that JMC mutations of the PTH/PTHrP receptor induce activation of the cyclin D1 and cyclin A promoters in primary mouse chondrocytes and rat chondrosarcoma cells. Induction of cyclin D1 expression is required for stimulation of E2F-dependent transcription by mutant receptors. Activation of the cyclin D1 and cyclin A promoters requires a functional cAMP response element in both genes. Inhibition of protein kinase A or the transcription factor cAMP response element binding protein blocks the stimulation of both promoters by mutant receptors, whereas inhibition of activating transcription factor 2, c-Fos, or c-Jun has only minor effects. In summary, our data suggest that stimulation of cell cycle gene expression and cell cycle progression by mutant PTH/PTHrP receptors contribute to the pathogenesis of JMC.
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INTRODUCTION
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JANSENS METAPHYSEAL CHONDRODYSPLASIA (JMC) is a rare autosomal dominant disorder characterized by short-limbed dwarfism, delayed ossification, and hypercalcemia (1, 2, 3). Three different activating mutations (H223R, T410P, I458R) in the PTH/PTHrP receptor have been identified as the molecular cause of this disorder (4, 5, 6). Overexpression of the mutant receptors in COS-7 cells showed that these mutations caused increased basal synthesis of cAMP, suggesting that increased activity of protein kinase A and its downstream signaling pathways contributes to the pathogenesis of the disease (6, 7).
Recent work from a number of laboratories has begun to identify the function of the PTH/PTHrP receptor in skeletal development. Genetic disruption of the PTH/PTHrP receptor gene in mice revealed a prominent role for the gene in the control of chondrocyte differentiation, most likely in a feedback loop with the secreted signaling molecule indian hedgehog (8, 9). Interruption of PTHrP signaling causes premature differentiation of chondrocytes, leading to skeletal deformities and perinatal lethality (10). In contrast, overexpression of the PTH/PTHrP receptor containing a JMC mutation results in abnormal skeletal development due to a delay in hypertrophic differentiation of chondrocytes (11). In summary, these data suggest that the PTH/PTHrP receptor regulates skeletal development by stimulating proliferation and/or inhibiting hypertrophic differentiation of chondrocytes and ossification.
We have recently identified the cyclin D1 gene as a target of PTHrP in chondrocytes (12). Cyclin D1 is a positive regulator of progression through the G1 phase of the cell cycle and is induced by many mitogenic stimuli. It regulates cell cycle progression through association with and activation of cyclin-dependent kinases 4 and 6 (13, 14, 15). The activated cyclin/cyclin-dependent kinase complexes phosphorylate the retinoblastoma protein pRb and its close relatives, the p107 and p130 proteins. Hyperphosphorylation of these proteins causes their dissociation from E2F transcription factors, thus enabling E2F factors to activate the transcription of target genes necessary for DNA replication and progression through the cell cycle. Enhanced transcription from promoters containing E2F binding sites is therefore closely coupled to cyclin D1-induced cell cycle progression.
Maximal transcription of the cyclin D1 gene in chondrocytes requires a functional cAMP response element (CRE) (16). Normal transcription of the cyclin A gene, which controls progression through later stages of the cell cycle, also requires a CRE (17). We have shown that the transcription factors activating transcription factor 2 (ATF-2) and CRE-binding protein (CREB) regulate the activity of the cyclin D1 CRE in chondrocytes (12, 16). Because CREB is a direct target of protein kinase A (18) and a downstream target of PTH/PTHrP signaling (19), we asked whether JMC mutations could activate cyclin D1 and cyclin A transcription through CREB and possibly ATF-2.
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RESULTS
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JMC Mutations Activate CRE- and E2F-Dependent Transcription
Expression constructs for wild-type and mutant PTH/PTHrP receptors were transfected into primary mouse chondrocytes (Fig. 1A
) or rat chondrosarcoma (RCS) cells (Fig. 2A
) together with the reporter plasmid pCRE-Luc, encoding the firefly luciferase gene under the control of a promoter containing several CRE elements. Whereas the wild-type receptor did not activate CRE-dependent transcription significantly in the presence of 1% fetal bovine serum (FBS), all three receptor mutations (H223R, T410P, and I458R) caused a strong increase in luciferase activity. Because transcription from the cyclin D1 and cyclin A promoters in chondrocytes is controlled by CRE elements, we next examined whether the mutant receptors could activate these promoters in primary chondrocytes (Fig. 1
, B and C) and RCS cells (Fig. 2
, B and C). All three mutated receptors stimulated the activity of the cyclin D1 and cyclin A promoters strongly, in contrast to the wild-type receptor.

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Figure 1. Gene Induction by Mutant PTH/PTHrP Receptors in Primary Chondrocytes
The reporter plasmids pCRE Luc (A), -1745CD1Luc [containing a 1745-nucleotide fragment of the cyclin D1 promoter (B)], p707CycALuc [containing a 707-nucleotide fragment of the cyclin A promoter (C)], and pE2F-TA-Luc (D) were transfected into primary mouse chondrocytes together with pRlSV40 (encoding the Renilla luciferase gene under control of the SV40 promoter) and either empty expression vector as control or expression vectors for wild-type or H223R, T410P, or I458R mutant PTH/PTHrP receptors. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity shown (*, P > 0.03 when wild type was compared with vector control).
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Figure 2. Gene Induction by Mutant PTH/PTHrP Receptors in RCS Cells
The reporter plasmids pCRE Luc (A), -1745CD1Luc (B), p707CycALuc (C), and pE2F-TA-Luc (D) were transfected into RCS cells together with pRlSV40 and either empty expression vector as control or expression vectors for wild type or H223R, T410P, or I458R mutant PTH/PTHrP receptors. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity shown (* P > 0.2 when wild type was compared with vector control). E, pCRE Luc, -1745CD1Luc, p707CycALuc, and pE2F-TA-Luc were transfected RCS cells together with pRlSV40 and cultured in the presence of 1% FBS and with vehicle or 10-8 M PTHrP for 48 h. Cells were then harvested, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. F, pCRE Luc was transfected into RCS cells either alone or with empty expression vector or expression vectors for wild-type or mutant PTHrP receptors. After transfection, medium was changed to medium containing 1% FBS containing PTHrP (10-8 M). After 48 h, cells were harvested, and firefly luciferase activity was measured and standardized to Renilla luciferase activity.
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Cyclin D1 and cyclin A control cell cycle progression through the activation of E2F-dependent transcription. We therefore tested the effects of overexpression of mutated PTH/PTHrP receptors on the activity of a promoter containing several E2F sites (Figs. 1D
and 2D
). All three mutated receptors induced E2F-dependent transcription, whereas the wild-type receptor was not able to do so. These data suggest that JMD mutations in the PTH/PTHrP receptor control chondrocyte cell cycle progression directly through induction of cyclin D1 and cyclin A transcription and indirectly through activation of E2F-dependent transcription. Because all the results shown in Figs. 1
and 2
suggested similar mechanisms of PTH/PTHrP receptor action in primary chondrocytes and RCS cells, all subsequent experiments were performed in RCS cells. In addition, some of the subsequent experiments were performed with the T410P mutant only, because all three mutants displayed very similar regulation of the promoters used in this study.
Next, we compared the stimulation of promoter activities by mutant PTH/PTHrP receptors to stimulation by PTHrP (Fig. 2E
). Because no PTH/PTHrP receptor expression plasmids were cotransfected in these experiments, promoter stimulation was conferred by the endogenous wild-type receptors. Basal activity of all four promoters was similar to the cotransfection experiments (compare with Fig. 2
, AD). Activation of endogenous wild-type receptors by PTHrP led to a 20- to 35-fold activation of the four promoters (Fig. 2E
); in contrast, promoter activation by overexpressed mutant receptors was maximal 12-fold in all cases (Fig. 2
, AD).
It is not known whether JMD mutations change the expression or stability of the PTH/PTHrP receptor and therefore receptor expression levels in the transfected cells. To address this issue, we transfected RCS cells with wild-type or mutant receptors and the CRE reporter gene, and subsequently stimulated the cells with PTHrP. Overexpression of all four different receptors caused an approximately 4-fold higher induction of CRE-dependent transcription by PTHrP when compared with untransfected or vector-transfected cells (Fig. 2F
). Because wild-type and mutant receptors should display similar activity in the presence of ligand, these data suggest that they are expressed at equal levels in the transfected cells.
Cyclin D1 Induction Is Required for Stimulation of E2F-Dependent Transcription by Mutant PTH/PTHrP Receptors
We used antisense oligonucleotides to determine whether cyclin D1 is required for induction of E2F-dependent transcription by mutant PTH/PTHrP receptors. In the presence of cyclin D1 antisense oligonucleotides, induction of pE2F-TA-Luc by mutant receptors was reduced by 33% (compared with control oligonucleotides; Fig. 3A
). The cyclin A promoter is a target of E2F transcription factors itself. Induction of cyclin A promoter activity by mutant receptors was reduced by 53% in the presence of cyclin D1 antisense oligonucleotides (Fig. 3B
). Similarly, cyclin D1 antisense oligonucleotides reduced activation of pE2F-TA-Luc and the cyclin A promoter by PTHrP in RCS cells expressing only the wild-type receptor by 35 and 32%, respectively, but had no effects on the activities of the cyclin D1 and CRE-dependent promoters (Fig. 3C
).

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Figure 3. Cyclin D1 Is Required for Induction of E2F-Dependent Gene Expression by Mutant Receptors
PE2F-TA-Luc (A) or p707CycALuc (B) was transfected into RCS cells together with pRlSV40 and empty expression vector or expression vectors for the T410P mutant PTH/PTHrP receptor. Cells were incubated without oligonucleotides or with 10 µM of control oligonucleotides or cyclin D1 antisense oligonucleotides. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. C, pCRE Luc, -1745CD1Luc, p707CycALuc, and pE2F-TA-Luc were transfected RCS cells together with pRlSV40 and cultured in the presence of 1% FBS and vehicle or 10-8 M PTHrP for 48 h. PTHrP-treated cells received 10 µM of control (con) or cyclin D1 antisense (as) oligonucleotides. Cells were then harvested, and firefly luciferase activity was measured and standardized to Renilla luciferase activity.
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Activation of Cyclin D1 and Cyclin A Transcription by Mutant PTH/PTHrP Receptors Requires CRE Sites
We next examined the role of the AP-1 and CRE sites in the stimulation of the cyclin D promoter by mutant PTH/PTHrP receptors. Mutation of the AP-1 site at position -963 or deletion of promoter sequences upstream of position -66 caused a 28% reduction in promoter induction by the T410P mutation. Mutation of the CRE in the -66 nucleotide promoter, however, completely abolished the responses to overexpression of the receptor (Fig. 4A
). Mutation of the CRE in the context of the full-length promoter or simultaneous mutation of both the CRE and the AP-1 sites resulted in a loss of responsiveness. These data suggest that the CRE is the major determinant of cyclin D1 promoter induction by mutant PTH/PTHrP receptors, with a minor contribution by the AP-1 site.

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Figure 4. CRE Sites Are Required for Induction of Cyclin Gene Expression by Mutant Receptors
A: The cyclin D1 promoter plasmids -1745CD1Luc, -963Cd1Luc, -963AP1 mutCD1Luc, -66CD1Luc, -66CREmutCD1Luc, -1745CREmutCD1Luc, or -1745CRE/AP1 mutCD1Luc were transfected into RCS cells together with pRlSV40 and empty expression vector or an expression vector for the T410P mutant PTH/PTHrP receptor. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. The induction of promoter activity by the mutant receptor relative to empty expression vector is shown (*, P > 0.1 when 1745CREmutCD1Luc was compared with -1745CRE/AP1 mutCD1Luc). B, The cyclin A promoter plasmids p707CycALuc and p707McycALuc were transfected into RCS cells together with pRlSV40 and empty expression vector or an expression vector for the T410P mutant PTH/PTHrP receptor. Cells were incubated with 10 µM of control oligonucleotides (black bars) or cyclin D1 antisense oligonucleotides (open bars). Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. The induction of promoter activity by the mutant receptor relative to empty expression vector is shown.
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Similar experiments were performed with the cyclin A promoter. Mutation of the CRE site in the promoter caused a 60% reduction in the transcriptional responses to the T410P mutant receptor (compare the black bars in Fig. 4B
). Induction of the CRE mutant by the T410P mutant was completely abolished by cyclin D1 antisense oligonucleotides (open bars in Fig. 4B
). These data suggest that the mutant receptors activate cyclin A transcription through two independent pathways: one directly through the CRE site and the other involving a cyclin D1-dependent pathway.
Activation of Cyclin D1 and Cyclin A Transcription by Mutant PTH/PTHrP Receptors Requires Protein Kinase A Signaling
Jansens mutations in the PTH/PTHrP receptor have been shown to stimulate the production of cAMP and presumably the activity of protein kinase A (PKA) (4, 6, 7). We used the PKA inhibitor H89 to examine the role of PKA in activation of cyclin D1 and cyclin A transcription by the T410P mutant (Fig. 5A
). H89 treatment reduced responses of both promoters to the receptor mutant to minimal levels. In contrast, staurosporine (an inhibitor of the phospholipase C/protein kinase C pathway) did not affect transcriptional activation by mutant receptors. H89 also inhibited the activation of cyclin D1 transcription by exogenous PTHrP through the wild-type receptor, although not as efficiently as observed for the mutant receptors (Fig. 5B
). Staurosporine had no effect on transcriptional induction by PTHrP, suggesting that additional (PKA- and protein kinase C-independent) pathways contribute to the activation of gene expression by the wild-type PTH/PTHrP receptor. Neither H89 nor staurosporine affected the induction of cyclin D1 transcription by TGFß treatment, which activates the cyclin D1 promoter independently of the PKA target CREB (12). Finally, overexpression of PKA in RCS cells induced both the cyclin D1 and cyclin A promoters (Fig. 5C
). These data confirm the role of the PKA pathway in promoter induction by wild-type and mutant PTH/PTHrP receptors.

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Figure 5. PKA Signaling Is Required for Induction of Gene Expression by Mutant Receptors
A, The reporter plasmids -1745CD1Luc and p707CycALuc were transfected into RCS cells together with pRlSV40 and empty expression vector or an expression vector for the T410P mutant PTH/PTHrP receptor. Cells were incubated in the presence of 10 µM H89, 10-8 M staurosporine, or dimethylsulfoxide (control). Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. The induction of promoter activity by the mutant receptor relative to empty expression vector is shown. B, -1745CD1Luc was transfected into RCS cells together with pRlSV40. Cells were stimulated with PTHrP (10-8 M) or TGFß (1 ng/ml) and incubated in the presence of 10 µM H89, 10-8 M staurosporine, a combination of both, or dimethylsulfoxide (control). Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. C, -1745CD1Luc and p707CycALuc were transfected into RCS cells together with pRlSV40 and empty expression vector or an expression vector for PKA. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity.
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CREB Confers Activation of Cyclin D1 and Cyclin A Transcription by Mutant PTH/PTHrP Receptors
The transcription factor CREB is activated by PKA and is a target of PTH/PTHrP receptor signaling. We have shown that CREB and the related factor ATF-2 are necessary for maximal transcription of the cyclin D1 and cyclin A genes in chondrocytes. We cotransfected the cyclin D1 promoter vector, the cyclin A promoter vector, or the E2F reporter with the T410P mutant receptor expression vector and expression vectors for dominant-negative CREB, ATF-2, c-Fos, and c-Jun to examine the role of these transcription factors in promoter induction by mutant PTH/PTHrP receptors. Dominant-negative CREB severely inhibited the induction of both promoters and the E2F reporter by the mutant receptor, whereas dominant-negative ATF-2, c-Fos, or c-Jun caused only mild inhibition of promoter induction (Fig. 6
).

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Figure 6. CREB, But Not ATF-2, c-Fos, or c-Jun, Is Required for Induction of Gene Expression by Mutant Receptors
The reporter plasmids -1745CD1Luc, p707CycALuc, and pE2F-TA-Luc were transfected into RCS cells together with pRlSV40, empty expression vector or an expression vector for the T410P mutant PTH/PTHrP receptor, and empty expression vector or expression vectors for dominant-negative forms of CREB, ATF-2, c-Fos, or c-Jun. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. The induction of promoter activity by mutant receptors relative to empty expression vector is shown.
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We used Gal4 fusion proteins to directly test whether CREB is activated by mutant PTH/PTHrP receptors. As shown in Fig. 7
, overexpression of receptor mutants induced a strong increase in CREB activity, whereas the wild-type receptor caused a weaker, but significant, activation of CREB (Fig. 7A
). In contrast, a Gal4-ATF-2 fusion was activated to a much lesser extent by mutant receptors and not at all by the wild-type receptor (Fig. 7B
).

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Figure 7. Mutant PTH/PTHrP Receptors Activate CREB
The reporter plasmid pRFLuc was cotransfected with pRlSV40, expression vectors for Gal4-CREB (A) or Gal4-ATF-2 (B), and empty expression vector or expression vectors for wild-type or H223R, T410P, or I458R mutant PTH/PTHrP receptors. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. Activation of CREB by the wild-type receptor was significantly different from both vector-transfected controls and mutant receptors (*, P < 0.0005 when wild type was compared with mutants or vector control).
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DISCUSSION
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We have demonstrated that JMC mutations in the PTH/PTHrP receptor stimulate the transcription of the cyclin D1 and cyclin A genes, two positive regulators of cell cycle progression. In addition, mutant receptors also stimulate E2F-dependent transcription. In contrast, the wild-type receptor did not affect cell cycle gene expression in these experiments, suggesting that under our experimental conditions (1% FBS) the concentrations of PTH and PTHrP are not sufficient to activate gene expression. However, the wild-type receptor was able to stimulate CRE-dependent transcription in response to exogenous PTHrP (data not shown), proving that the construct encodes a functional receptor. Furthermore, overexpression of the wild-type receptor stimulated the activity of CREB in the Gal4 assay. However, this activation does not appear sufficient for transcriptional activation of downstream promoters such as the cyclin D1 and cyclin A promoters. The reasons for this discrepancy are not known; one possibility is that activation of the endogenous promoters (but not the Gal4-controlled promoter) requires factors other than activation of CREB. Because E2F acts downstream of cyclin D1 in the control of cell cycle progression, it is not surprising that inhibition of cyclin D1 expression inhibited the activation of E2F activity by JMC mutations. This inhibition, however, was not complete, suggesting that other factors contribute to the activation of E2F by mutant PTH/PTHrP receptors. Our data suggest that cyclin A could be one of these factors, but we cannot exclude a contribution of additional proteins such as the D2 or D3 cyclins. However, it is also possible that the used antisense oligonucleotides do not block cyclin D1 protein expression completely, thereby allowing some E2F activation through cyclin D1-dependent mechanisms.
Ligand-activated PTH/PTHrP receptors activate at least two different signaling pathways, adenylate cyclase/PKA and phospholipase C/protein kinase C (20). Inhibition of PKA or its downstream target CREB reduced the effects of JMC mutations on cyclin D1, cyclin A, and E2F-dependent promoters to minimal levels. In contrast, inhibition of the phospholipase C/protein kinase C pathway by staurosporine did not affect promoter induction by the mutant receptors. The three mutations used here have been shown to have different efficiencies in the activation of phospholipase C (6), but have very similar effects on cell cycle gene expression in our experiments, strengthening our conclusion that these effects are independent of phospholipase C. Our data support the described stimulatory effect of the mutations on cAMP production (4, 6, 7) and suggest that JMD mutations activate cell cycle progression through the PKA/CREB pathway. In contrast, activation of cell cycle gene expression by treatment of untransfected cells with exogenous PTHrP could not be blocked completely with H89 and not at all by staurosporine, suggesting the involvement of a third pathway (or an alternative receptor) that confers weak stimulation of cell cycle gene expression.
Activation of endogenous PTH/PTHrP receptors by PTHrP caused a stronger induction of all promoters than overexpression of mutant receptors. Several possibilities could explain this observation. First, the point mutations might not induce complete activation of the receptors. Second, it is likely that activation of additional pathways as described above (which are not blocked by H89 or staurosporine) contributes to the stronger promoter activation by activated wild-type receptors. Third, it is possible that the expression levels of mutant receptors on the cell surface of transfected cells is lower than that of endogenous wild-type receptors. Further experiments will be necessary to distinguish between these possibilities.
Induction of cyclin D1 transcription by mutant receptors is conferred mainly by the CRE site, with a minor contribution of the AP-1 site. CRE activation is likely conferred by activation of CREB, which binds to the cyclin D1 CRE in chondrocyte nuclear extracts (16). Activation of the AP-1 site could be direct, because CREB has been shown to be able to bind to AP-1 sites (21). Alternatively, this effect could be mediated by the induction of AP-1 protein expression in response to PTH/PTHrP receptor signaling, as described by others (19, 22). However, the results of the mutations in the full-length promoter suggest that loss of the CRE, whereas the AP-1 site is intact, leads to a complete loss of cyclin D1 promoter induction by mutant PTH/PTHrP receptors. These data suggest that the cyclin D1 AP-1 site is only functional in the presence of the CRE under these conditions. In contrast, the AP-1 site confers stimulation of the cyclin D1 promoter by exogenous PTHrP and TGFß even in the absence of a CRE (12). These data again suggest that activation of wild-type PTH/PTHrP receptors by exogenous ligand stimulates additional pathways that are not involved in signaling from the activated mutants used in this study.
The CRE is also necessary for activation of the cyclin A promoter by JMC mutations. However, inhibition of cyclin D1 expression also reduces transcriptional induction of the cyclin A gene by the receptor mutations in a CRE-independent fashion. This effect is likely mediated by E2F factors because the cyclin A gene is a known target of E2F transcription factors (23). Our data suggest that signaling from mutant PTH/PTHrP receptors activates cyclin A transcription through two independent pathways, a CREB-CRE and a cyclin D1-E2F pathway.
We suggest that CREB plays a crucial role in the response of cell cycle genes to activated mutant PTH/PTHrP receptors (data in this manuscript) and to PTHrP stimulation of wild-type RCS cells (12). Long et al. (24) recently showed that the number of chondrocytes containing CREB phosphorylated on serine 133 (which indicates activation of CREB) is not changed in PTHrP-deficient mice. However, it is likely that CREB phosphorylation in vivo is induced not only by PTHrP, but by a larger number of growth factors and hormones. Loss of PTHrP could therefore result in a lower amount of phosphorylated CREB per cell (which in general cannot be visualized by immunohistochemistry), but not in the number of cells staining positively for phosphorylated CREB. Long et al. also describe a reduction in chondrocyte proliferation as a result of inhibition of CREB activity in chondrocytes in vivo, which is in agreement with the model we propose. A requirement for CREB in PTHrP signaling in chondrocytes has also been described by us (12) and others (19).
In summary, we have shown that JMC mutations in the PTH/PTHrP receptor activate the expression of the cyclin D1 and cyclin A genes, as well as E2F-dependent gene expression. In vivo, the highest levels of PTH/PTHrP receptor expression are found at the transition from proliferating to hypertrophic chondrocytes (22), suggesting that one of the main roles of the receptor is the control of the exact timing of cell cycle exit and onset of differentiation. We therefore suggest that activation of the PTH/PTHrP receptor, either through its ligand PTHrP under physiological conditions (12, 19) or through activating mutations in JMC, causes a delay in cell cycle exit of chondrocytes through up-regulation of cyclin D1 and cyclin A expression.
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MATERIALS AND METHODS
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Materials
Human cyclin D1 promoter plasmids -1745CD1Luc, -963CD1Luc, -963AP1 mutCD1Luc, -66CD1Luc, -66CREmutCD1Luc, -1745CREmutCD1Luc, and -1745CRE,AP1 mutCD1Luc (12, 16, 25, 26) and rat cyclin A promoter plasmids p707CycALuc and p707McycALuc (27, 28) have been described and were generously provided by R. G. Pestell (Albert Einstein College of Medicine, New York, NY) and K. Oda (Science University of Tokyo, Tokyo, Japan), respectively. The PKA expression vector, the CRE reporter plasmid pCRELuc, the Gal4-responsive reporter vector pRFLuc, and the plasmids encoding CREB and ATF-2 Gal4 fusions were from Stratagene (La Jolla, CA). The E2F-responsive plasmid pE2F-TA-Luc was from CLONTECH Laboratories, Inc. (Palo Alto, CA). Expression plasmids for wild-type and mutant PTH/PTHrP receptor were kindly provided by H. Juppner (6, 7). The construction of the expression vector for dominant-negative ATF-2 has been described (16). Expression vectors for dominant-negative c-Fos, c-Jun, and CREB were generously provided by C. Vinson (29). Staurosporine and H89 were purchased from Calbiochem (La Jolla, CA), and PTHrP and TGFß were purchased from Sigma (St. Louis, MO).
Cell Culture, Transfections, and Luciferase Assays
Isolation of primary mouse chondrocytes and culture of primary chondrocytes and RCS cells (30) were performed as described previously (16, 17, 31, 32). Transfections were performed as described (16, 17, 32) using Lipofectin (Life Technologies, Inc., Gaithersburg, MD), with the exception that cells were cultured in the presence of 1% FBS for 48 h after transfections. Cells were cotransfected with 1.0 µg of promoter plasmid, 0.15 µg of pRlSV40 (encoding the Renilla luciferase gene under control of the SV40 promoter for standardization), and 0.1 µg of empty expression vector or expression vectors for wild-type or mutant PTH/PTHrP receptors. Cells were harvested 48 h after transfection, and firefly luciferase activity was measured and standardized to Renilla luciferase activity. The average and SDs of three experiments, each performed in triplicate, are shown. Statistical analyses were performed using ANOVA. Rat cyclin D1 antisense or control oligonucleotides (10 µM) (33) were included in the culture medium where indicated; 10-8 M staurosporine, 10 µM H89, 10-8 M PTHrP, or 1 ng/ml TGFß were included where indicated.
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ACKNOWLEDGMENTS
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We are grateful to B. de Crombrugghe and V. Lefebvre (both from the M. D. Anderson Cancer Center, Houston, TX) for RCS cells; C. Vinson (National Cancer Institute, Bethesda, MD) for the dominant-negative CREB, c-Fos, and c-Jun expression vectors; H. Juppner for PTH/PTHrP receptor expression plasmids; and R. G. Pestell and K.Oda for cyclin D1 and cyclin A reporter plasmids.
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FOOTNOTES
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This work was supported by funds from the Alberta Heritage Foundation for Medical Research, the Canadian Institutes of Health Research, the Arthritis Society, and the Canada Research Chair Foundation (to F.B.), and by grants from the Canadian Institute of Health Research, the Alberta Cancer Foundation, the Alberta Heritage Foundation for Medical Research, and the Arthritis Society (to P.L.).
1 Current address: Department of Orthopedics, University of Florida at Gainesville, Gainesville, Florida. E-mail: luvalpa{at}ortho.ufl.edu. 
Abbreviations: AP-1, Activator protein 1; ATF-2, activating transcription factor 2; CRE, cAMP response element; CREB, CRE-binding protein; FBS, fetal bovine serum; JMC, Jansens metaphyseal chondrodysplasia; PKA, protein kinase A; RCS, rat chondrosarcoma.
Received for publication June 15, 2001.
Accepted for publication May 13, 2002.
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