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Endocrinology Vol. 145, No. 8 3696-3703
Copyright © 2004 by The Endocrine Society

Parathyroid Hormone Induces E4bp4 Messenger Ribonucleic Acid Expression Primarily through Cyclic Adenosine 3',5'-Monophosphate Signaling in Osteoblasts

Ibrahim C. Ozkurt, Flavia Q. Pirih and Sotirios Tetradis

Division of Diagnostic and Surgical Sciences and Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, California 90095-1668

Address all correspondence and requests for reprints to: Sotirios Tetradis, Division of Diagnostic and Surgical Sciences, Room 53-068 CHS, UCLA School of Dentistry, Los Angeles, California 90095-1668. E-mail: sotirist{at}dent.ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH binding to its receptor activates protein kinase A (PKA), protein kinase C (PKC), and calcium signaling to induce transcription of primary response genes in osteoblasts. Adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4BP4/NFIL3), a transcriptional repressor, is a PTH-induced primary response gene in primary mouse osteoblasts (MOBs). Here we investigate the signaling pathway(s) that lead to PTH induction of E4bp4 mRNA expression. Ten and 100 nM PTH induced maximum E4bp4 expression in MOBs. Forskolin (FSK), an adenylate cyclase inducer, 8-bromo-cAMP, a cAMP analog, and phorbol myristate acetate, a PKC activator, increased E4bp4 mRNA levels, whereas ionomycin, a calcium ionophore, had no effect. Pretreatment of cells with 30 µM H89, a PKA inhibitor, strongly inhibited PTH- and FSK-induced E4bp4 expression. In contrast, overnight pretreatment with 1 µM phorbol myristate acetate to down-regulate PKC signaling did not alter PTH and FSK effects. Moreover, PTH (3–34) that does not activate cAMP signaling did not increase E4bp4 expression. Prostaglandin E2, which signals through cAMP, increased E4bp4 mRNA at all doses, whereas prostaglandin F2{alpha} that primarily activates PKC and calcium signaling, induced E4bp4 only at high doses and fluprostenol that only activates PKC and calcium signaling, had no effect. Finally, 80 µg/kg PTH (1–34) ip injection induced E4bp4 mRNA expression at 1 h in mice. In contrast, 80 µg/kg PTH (3–34) had no effect. Our data suggest that PTH-induced E4bp4 mRNA expression is mediated primarily through cAMP-PKA signaling in vitro and in vivo. In conjunction with our previous report, we hypothesize that E4bp4 attenuates transcription of osteoblastic genes possessing E4bp4 promoter binding sites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH DIRECTLY TARGETS osteoblasts by binding to the PTH receptor, PTHR1. PTHR1, a member of the class II guanosine triphosphate-binding protein (G protein)-coupled receptor family, displays homology to calcitonin, secretin, and glucagon receptors (1). PTHR1 is coupled to three G proteins. Activation of Gs protein stimulates the formation of cAMP and initiates protein kinase A (PKA) signaling (2). Activated Gi inhibits this pathway (3). Gq stimulates phospholipase C activity and formation of 1,4,5-inositol triphosphate (IP3) and diacylglycerol (2). IP3 releases calcium from IP3-sensitive storage sites within the cell and diacylglycerol activates protein kinase C (PKC).

PTH strongly activates the cAMP-PKA pathway and less strongly PKC or intracellular calcium signaling cascades (4). Consistent with this observation, cAMP-PKA is the main pathway mediating PTH’s effects on osteoblastic gene expression (2, 5). Similar to in vitro data, in vivo studies point to cAMP-PKA as the main pathway mediating the PTH regulation of gene expression in bone (6, 7, 8, 9).

PKA, PKC, and calcium signaling cascades phosphorylate and activate transcription factors to induce transcription of several primary response genes (PRGs) (5, 10). Protein products of PRGs, in turn, carry out various cellular functions and ultimately alter the osteoblastic phenotype. A subgroup of PRGs encodes transcription factors that control expression of downstream gene targets and thus play a critical role in the propagation of regulated gene expression (11, 12).

Activator protein (AP)-1 family members c-jun and c-fos (13), regulator of G protein signaling-2 (RGS-2) (8, 14), Jun-terminal kinase (15), Nurr1 (16), Nur77 (17), IL-6 (18), leukemia inhibitory factor (18), ICER (19), and nuclear orphan receptor-1 (20) are among the PTH-induced PRGs. We have identified E4bp4 as a PTH-induced PRG in osteoblasts (21). E4BP4 is a basic leucine zipper (bZIP) transcription factor with a basic region similar to vitamin D-binding protein and a leucine zipper region similar to cAMP response element-binding protein and activating transcription factor-1 (22) and acts as transcriptional repressor in osteoblasts (21).

In this report, we determined the signaling pathway(s) leading to PTH-induced E4bp4 mRNA levels in primary mouse calvariae osteoblasts (MOBs) in vitro and investigated the PTH regulation of E4bp4 expression in vivo. We report that PTH induction of E4bp4 gene expression in MOB was mainly through cAMP-PKA pathway. PTH also induced E4bp4 expression in vivo, and this induction followed a parallel signaling pathway as in MOB cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Unless otherwise stated, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Fluprostenol and H89 were obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA).

Cell culture
MOB cells were collected and maintained in culture as previously described (16). All animals used in this study were killed according to protocol approved by UCLA Institutional Animal Care and Use Committee (ARC number 1998-175-12).

RNA extraction and Northern blot analysis
Total RNA extraction and Northern blot analysis was performed as previously described (21). For Northern blot analysis, 12 µg total RNA were used. To generate a full-length E4bp4 cDNA probe, pcDNA-3.1 vector carrying E4bp4 cDNA (21) was restriction digested, gel purified, and labeled with [32P]deoxy (d)-CTP (PerkinElmer, Atlanta, GA) by random primer labeling technique.

RNA extraction and semiquantitative RT-PCR (SQ-RT-PCR) for in vivo experiments
Three 1-month-old CD1 male mice (Charles River Laboratories, Wilmington, MA) per group were used. After treatment, animals were killed and calvariae were dissected. Tissues were kept in RNA Later (Ambion, Austin, TX) at 4 C overnight. The next day, RNA Later was aspirated and RNA extraction was carried out using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) and the protocol provided by the manufacturer. Three micrograms of total RNA were reverse transcribed, and 1.5 µl of RT product were PCR amplified in the presence of 1 µCi [32P]dCTP. Twenty-three PCR cycles for E4BP4 and 17 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message amplification were performed. The E4bp4 primers used amplified nucleotides +71 to +204 (sense primer, 5'-AGATGCTGCTGAACT-3'; antisense primer, 5'-GCGTCTTTCTTCTCGTCC-3'). The GAPDH primers used amplified nucleotides +476 to +577 (sense, 5'-ATTGTCAGCAATGCATCCTG-3'; antisense, 5'-ATGGACTGTGGTCATGAGCC-3'). The PCR product was sequenced to verify E4bp4 amplification (Davis Sequencing, Davis, CA).

Statistical analysis
ANOVA and Student’s t test were used to determine the statistical differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTH (1–100 nM) significantly induced E4bp4 mRNA expression
PTH rapidly and transiently increases E4bp4 mRNA expression in MOB cells, which peaks at 2 h and returns to control levels by 6–8 h (21). To determine the optimum dose for PTH induction of E4bp4 mRNA expression, MOB cells were treated with 0–100 nM PTH (Fig. 1AGo). Concentrations of 1–100 nM significantly induced E4bp4 mRNA levels (Fig. 1BGo). Ten and 100 nM PTH increased E4bp4 expression by 4.5-fold. Half-maximal dose was approximately 0.3 nM PTH.



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  		FIG. 1. PTH effect on E4bp4 mRNA levels. A, Confluent MOB cells were treated with 0.01–100 nM PTH for 2 h. E4bp4 and GAPDH mRNA levels were determined by Northern blot analyses. The ethidium bromide-stained 18 S and 28 S ribosomal RNA bands are shown. B, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from control; P < 0.05).

 
PTH induced E4bp4 expression mainly through cAMP signaling
To study the signaling pathway(s) that lead to E4bp4 transcription by PTH, MOB cells were treated with agents that selectively activate PTHR1-coupled signaling pathways (Fig. 2Go, A and B). Forskolin (FSK), a direct activator of adenylate cyclase, and 8-bromo-cAMP (8Br-cAMP), a cAMP analog, were used to activate the cAMP pathway. Phorbol myristate acetate (PMA), a phorbol ester, was used to activate PKC signaling. Ionomycin, a calcium ionophore that elevates calcium influx from intracellular storage sites, was used to increase intracellular calcium concentration. FSK and 8Br-cAMP treatment increased E4bp4 mRNA expression 5- and 4-fold, respectively, comparable to the PTH induction (Fig. 2CGo). PMA treatment also significantly induced E4bp4 mRNA expression (1.4-fold) but to a lesser degree compared with PTH. Ionomycin did not alter E4bp4 gene expression.



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  		FIG. 2. FSK, 8Br-cAMP, PMA, and Ionomycin (Iono) effect on E4bp4 mRNA levels. A, Confluent MOB cells were treated with vehicle (cont), 1–10 nM PTH, 1–10 µM FSK, 0.1–1 µM PMA or 1 µM Iono for 2 h. E4bp4 and GAPDH mRNA levels were determined by Northern blot analyses. The ethidium bromide-stained 18 S and 28 S ribosomal RNA bands are shown. B, Confluent MOB cells were treated with vehicle (cont), 10 nM PTH or 1 mM 8Br-cAMP for 2 h. E4bp4 and GAPDH mRNA levels were determined by Northern blot analyses. C, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from control; P < 0.05).

 
To further investigate the role of cAMP-PKA signaling in PTH-induced E4bp4 expression, MOB cells were pretreated for 0.5 h with 30 µM H89, a PKA inhibitor, and then with 10 nM PTH, 10 µM FSK, or 1 µM PMA for 2 h (Fig. 3AGo). H89 pretreatment significantly blocked PTH- and FSK-induced E4bp4 mRNA expression (Fig. 3BGo).



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  		FIG. 3. Effect of H89 pretreatment on PTH induction of E4bp4 mRNA levels. A, Confluent MOB cells were pretreated with vehicle or 30 µm H89 for 0.5 h and then with vehicle (cont), 10 nM PTH, 10 µM FSK, or 1 µM PMA for 2 h. E4bp4 and GAPDH mRNA levels were determined by Northern blot analyses. The ethidium bromide-stained 18 S and 28 S ribosomal RNA bands are shown. B, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from (–) H89 control; P < 0.05).

 
To explore PKC pathway involvement in E4bp4 mRNA expression, MOB cells were pretreated with 1 µM PMA for 16 h. Prolonged PMA pretreatment down-regulates PKC signaling by depletion of available PKC isoforms (23). After PMA pretreatment, cells were treated with 10 nM PTH, 10 µM FSK, or 1 µM PMA for 2 h (Fig. 4AGo). PMA pretreatment did not affect basal, PTH-, or FSK-induced E4bp4 mRNA expression, but it attenuated freshly added PMA induced E4bp4 expression (Fig. 4BGo).



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  		FIG. 4. Effect of PMA pretreatment on PTH induction of E4bp4 mRNA levels. A, Confluent MOB cells were pretreated with vehicle or 1 µm PMA for 16 h and then with vehicle (cont), 10 nM PTH, 10 µM FSK, or 1 µM PMA for 2 h. E4bp4 and GAPDH mRNA levels were determined by Northern blot analyses. The ethidium bromide-stained 18 S and 28 S ribosomal RNA bands are shown. B, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from (–) PMA control; #, significantly different from (+) PMA control; x, significantly different from (+) PMA treatment; P < 0.05).

 
To further investigate cAMP regulation of E4bp4 mRNA induction, MOB cells were treated with PTH (3–34) for 2 h (Fig. 5AGo). PTH (3–34) lacks the ability to activate the cAMP pathway but still activates PKC and calcium signaling (10, 24). Despite a 10-fold higher dose (100 nM) than PTH (1–34), PTH (3–34) did not alter E4bp4 mRNA expression (Fig. 5BGo).



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  		FIG. 5. PTH (3–34) effect on E4bp4 mRNA levels. A, Confluent MOB cells were treated with vehicle (cont), 10 nM PTH (1–34), or 0.1–100 nM PTH (3–34) for 2 h. E4bp4 and GAPDH mRNA levels were determined by Northern blot analyses. The ethidium bromide-stained 18 S and 28 S ribosomal RNA bands are shown. B, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from control; P < 0.05).

 
Prostaglandins (PGs) induced E4bp4 expression mainly through cAMP signaling
PGs, similar to PTH, mediate gene transcription through G protein-coupled receptors in osteoblasts. To study whether E4bp4 mRNA is induced with other ligands that activate cAMP signaling or whether it is specific for PTH signaling, we used the natural or synthetic PGs (i.e. PGE2, PGF2{alpha}, and fluprostenol). MOB cells were treated with 0.1–10 µM PGE2, 0.1–100 µM PGF2{alpha}, 1 and 10 µM fluprostenol, and 10 nM PTH (Fig. 6Go, A and B). PGE2 significantly induced E4bp4 mRNA expression at all doses (Fig. 6BGo). Maximum E4bp4 mRNA expression with PGE2 was comparable to PTH. Ten and 100 µM PGF2{alpha} also significantly induced E4bp4 mRNA expression but to a lesser degree than PTH and comparable PGE2 doses. However, lower doses of PGF2{alpha}, had no effect on E4bp4 mRNA levels (Fig. 6BGo). One and 10 µM fluprostenol did not have any effect on E4bpP4 mRNA levels (Fig. 6BGo).



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  		FIG. 6. PGE2, PGF2{alpha}, and Fluprostenol (Flup) effect on E4bp4 mRNA levels. A, Confluent MOB cells were treated with vehicle (cont), 10 nM PTH, 0.1–10 µM PGE2, 0.1–100 µM PGF2{alpha} or 1–10 µM Flup for 2 h. E4bp4 and GAPDH mRNA levels were determined by Northern blot analyses. The ethidium bromide-stained 18 S and 28 S ribosomal RNA bands are shown. B, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from control; P < 0.05).

 
PTH induced E4bp4 expression through cAMP signaling in vivo
To test whether anabolic regiments of PTH can induce E4bp4 expression in bone in vivo, 1-month-old male CD1 mice were injected ip with 80 µg/kg PTH (1–34) for 0–4 h. E4bp4 mRNA levels peaked after 1 h PTH treatment and declined thereafter (Fig. 7Go, A and B). The fold induction was comparable between in vivo and in vitro experiments (~4- to 5-fold). We also tested whether in vivo PTH-induced E4bp4 signaling in calvariae matched the induction of E4bp4 in MOB cells in vitro. Mice were injected with 20–80 µg/kg PTH (3–34) and with 80 µg/kg PTH (1–34). PTH (3–34) did not induce E4bp4 expression in contrast to PTH (1–34) (Fig. 7Go, C and D).



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  		FIG. 7. A, PTH (1–34) effect on E4bp4 mRNA levels in vivo. One-month-old CD1 male mice were injected ip with 80 µg/kg PTH (1–34) for 0–4 h and calvariae were dissected. E4bp4 and GAPDH mRNA levels were determined by SQ-RT-PCR. B, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from control; P < 0.05). C, PTH (3–34) effect on E4bp4 mRNA levels in vivo. One-month-old CD1 male mice were injected ip with vehicle (cont), 80 µg/kg PTH (1–34), or 20–80 µg/kg PTH (3–34) for 1 h. E4bp4 and GAPDH mRNA levels were determined by SQ-RT-PCR. D, Plot of mean ± SEM expressed as percentage of maximum PTH induction normalized to GAPDH expression (*, significantly different from control; P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Preosteoblastic/osteoblastic cells express high levels of PTHR1 and are the direct targets of PTH in bone (25, 26, 27). PTH has significant and diverse effects on osteoblastic function that greatly depend on the dose and intermittent vs. continuous administration of the hormone (28, 29, 30, 31). Many of PTH’s effects are due to regulated gene expression through transcriptional or posttranscriptional mechanisms (5, 30). PTH-induced genes that act as transcription factors are central in the propagation of regulated osteoblastic gene expression (32, 33, 34). We have recently published that the transcriptional inhibitor E4BP4 is a PTH-induced PRG in osteoblasts (21).

PTH binding to PTHR1 on osteoblast activates a series of G protein-linked signaling cascades, which, in turn, induce PKA, PKC, and calcium pathways. cAMP-PKA signaling appears to be the dominant pathway for the actions of PTH on osteoblasts (2, 5), although PKC and calcium signaling have been associated with increased cell proliferation and DNA synthesis (35, 36). PTH-activated cAMP-PKA is the key signaling pathway for most of the primary response genes including the AP-1 family members, c-fos and c-jun (5, 13), Jun-terminal kinase (15), RGS-2 (8, 14), Nurr1 (16), Nur77 (17), IL-6 (18), leukemia inhibitory factor (18), inducible cAMP early repressor (ICER) (19), and neuron-derived orphan receptor-1 (NOR-1) (20).

Here we provide evidence that supports cAMP signaling as the main pathway mediating PTH-induced E4bp4 gene expression in MOB cells. Direct activation of the cAMP pathway with FSK and 8-bromo-cAMP strongly induced E4bp4 mRNA levels (Fig. 2Go). Although involvement of PKC signaling was considered, because PKC activation with PMA also caused a weak but significant induction of E4bp4 mRNA (Fig. 2Go), this was not supported by further experiments (Figs. 4–6GoGoGo).

The significance of cAMP-PKA signaling in PTH-regulated E4bp4 mRNA levels was observed by the use of specific inhibitors. Pretreatment of MOB cells with the PKA inhibitor, H89, significantly inhibited the ability of PTH or FSK to induce E4bp4 gene expression (Fig. 3Go). However, H89 was not able to completely block the PTH and FSK effect. This could be due to partial PKA inhibition at the H89 used (30 µM). However, non-PKA-mediated cAMP effects have been reported (37, 38), thus suggesting that the partial H89 inhibition of E4bp4 expression might be due to other cAMP signaling pathways. A 16-h pretreatment of cells with PMA that inhibits PKC signaling (23) did not have a significant effect on the PTH or FSK induction of E4bp4 mRNA levels (Fig. 4Go). Finally, the PTH analog PTH (3–34) that does not activate cAMP signaling but retains the ability to signal through PKC and calcium pathways (24, 39), did not have any effect on the expression of E4bp4 mRNA levels, contrary to PTH (1–34) (Fig. 5Go). These studies collectively point to the cAMP-PKA signaling as the main pathway mediating the PTH regulation of E4bp4 gene expression.

To study whether E4bp4 mRNA induction in osteoblasts is exclusive to PTH, and also to further investigate regulation of E4bp4 mRNA expression through other than PTHR1-G protein-coupled receptor signaling, we treated MOB cells with PGE2, PGF2{alpha} and fluprostenol. These PGs mediate their effects by binding and activating the G protein-coupled PG receptors on the cell membrane. Although PGE2 recognizes and activates the EP1, EP2, EP3, and EP4 receptors, PGF2{alpha} and fluprostenol activate the FP receptor (40, 41, 42). The EP1 and FP receptors preferentially stimulate phospholipase C and activate PKC and calcium signaling. EP2 and EP4 receptors stimulate adenylate cyclase and activate the cAMP pathway. Finally, EP3 receptors couple to Gi and inhibit cAMP signaling (43). PGE2, similar to PTH, strongly induced E4bp4 mRNA expression at all doses used (Fig. 6Go). High-dose PGF2{alpha} induced E4bp4 mRNA expression, whereas fluprostenol did not have any effect (Fig. 6Go). Because fluprostenol is a specific FP receptor activator, whereas PGF2{alpha} at high doses can cross-react and bind EP receptors to activate the cAMP-PKA pathway (44, 45, 46), the PGF2{alpha} induction of E4bp4 mRNA levels is probably not mediated through FP receptor but rather through EP receptor signaling.

E4bp4 expression is induced by phytohemagglutinin in T cells (47), thapsigargin in aortic smooth muscle cells (48), glucocorticoids in mouse fibroblasts (49), IL-3, IL-4, oncogenic RAS mutants or GM-CSF treatment in pro-B lymphocytes (50, 51, 52, 53), and overexpression of the tumor suppressor PTEN in cancer cells (54). Contrary to the signaling that mediates PTH induction of E4bp4 in osteoblasts, intracellular calcium and PKC signaling play a critical role in regulation of E4bp4 by thapsigargin in vascular smooth muscle cells (48) and by phorbol esters in MLA144 and S-LB-1 T-cell lines (47). Interestingly, in our experiments, activation of PKC or intracellular calcium signaling in MOB cells, either directly by various agonists or through activation of the FP receptor by fluprostenol, did not significantly induce E4bp4 mRNA levels.

Although we used primary osteoblasts for our in vitro experiments, we further investigated the E4bp4 gene expression by PTH in vivo. For these studies, we performed ip injections of 80 µg/kg PTH, a dose and method of administration that demonstrates anabolic effects on bone metabolism (55, 56, 57). PTH very rapidly increased E4bp4 mRNA in calvariae with maximum induction approximately 0.5–1 h after injection (Fig. 7Go, A and B). This is a faster response compared with in vitro PTH induction of E4bp4 gene expression, which peaks around 2–3 h (21). PTH injections may result in more transient hormone levels compared with the rather continuous PTH treatment during the in vitro experiments.

Parallel to our in vitro findings, cAMP signaling appears to mediate the E4bp4 induction in vivo because PTH (3–34) did not have any effect on E4bp4 expression (Fig. 7Go, C and D). cAMP-PKA signaling has been hypothesized as the main pathway in the in vivo PTH induction of RGS-2 (8), the disintegrin and metalloprotease with thrombospondin type-1 domains-1 (ADAMTS-1) (7), the ubiquitin-specific protease UBP41 (6), as well as for the inhibition of osteoprotegerin (9) in rat femoral metaphyses.

E4BP4 is a bZIP transcription factor that binds DNA as dimer to regulate transcription (22, 58). Other bZIP transcription factors induced by in vivo PTH treatment in bone include c-jun, junB, c-fos, and fra-2 (59, 60). These AP-1 members activate transcription of downstream target genes and have been hypothesized to propagate the PTH-regulated osteoblastic gene expression (61, 62). Furthermore, PTH treatment leads to phosphorylation and activation of cAMP response element-binding protein/cAMP response element modulator family members (63, 64). PTH also inhibits several genes (2) through de novo protein synthesis. Interestingly, E4BP4 acts as a transcriptional repressor in osteoblasts (21), suggesting that E4BP4 could target genes that are transcriptionally repressed by PTH. However, it is conceivable that E4BP4 antagonizes the transcriptional activation of other PTH-induced bZIP members, thus acting as a feedback mechanism to reinstate PTH-induced transcription to basal levels. A potential example of such a mechanism is the regulation of cyclooxygenase-2 (cox-2) gene expression. PTH rapidly and transiently induces cox-2 promoter activity and gene transcription through cAMP-PKA signaling (65). PTH also induces E4BP4, which inhibits cox-2 promoter activity (21) suggesting that, through cAMP signaling, PTH regulates the transcription of this gene using E4BP4 as a feedback regulator.

In summary, our data support cAMP-PKA signaling as the major pathway mediating PTH induction of E4bp4 gene expression in MOB cells in vitro and mouse calvariae in vivo. Moreover, PGE2, a cAMP-PKA signaling activator, caused a comparable E4bp4 mRNA induction to PTH. In conjunction with our previous data (21), we hypothesize that E4BP4 might be included in feedback regulatory mechanisms or in direct inhibition of osteoblastic genes possessing E4bp4 promoter binding sites.


   Acknowledgments
 
We thank Dr. Jeanne Nervina and Dr. Clara Magyar for insightful comments during review of the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health/National Institute of Dental and Craniofacial Research Grant R01-DE13316. I.C.O. was the recipient of a fellowship from the Ministry of National Education, Turkey.

Abbreviations: AP, Activator protein; 8Br-cAMP, 8-bromo-cAMP; bZIP, basic leucine zipper; cox-2, cyclooxygenase-2; E4BP4, adenovirus E4 promoter-binding protein; EP receptors, specific receptors for PGE2; FP receptors, specific receptors for PGF2{alpha}; FSK, forskolin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP3, 1,4,5-inositol triphosphate; MOB, mouse osteoblasts; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol myristate acetate; PRG, primary response genes; PTHR1, PTH receptor; RGS-2, regulator of G protein signaling-2; SQ, semiquantitative RT-PCR.

Received October 24, 2003.

Accepted for publication April 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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