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Molecular Endocrinology, doi:10.1210/me.2007-0210
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Molecular Endocrinology 21 (11): 2713-2724
Copyright © 2007 by The Endocrine Society

Prostaglandin E2 Increases Transforming Growth Factor-ß Type III Receptor Expression through CCAAT Enhancer-Binding Protein {delta} in Osteoblasts

Thomas L. McCarthy, Tony H. Pham, Bianca I. Knoll and Michael Centrella

Department of Surgery and Section of Plastic Surgery, Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to Thomas L. McCarthy or Michael Centrella, 333 Cedar Street, MS 208041, New Haven, Connecticut 06520-8041. E-mail: thomas.mccarthy{at}yale.edu or michael.centrella{at}yale.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Variations in individual TGF-ß receptors (TßRs) may modify TGF-ß activity and significantly alter its effects on connective tissue growth or repair. Differences in the amount of TßR type III (TßRIII) relative to signal transducing TßRI occur on bone cells during differentiation or in response to other growth regulators. Here we investigated prostaglandin (PG) E2, a potent effector during trauma, inflammation, or mechanical load, on TßR expression in primary osteoblast-enriched cultures. PGE2 rapidly increased TßRIII mRNA and protein expression and enhanced TßRIII gene promoter activity through a discrete region within 0.4 kb of the transcription start site. PGE2 alters osteoblast function through multiple signal-inducing pathways. In this regard, protein kinase A (PKA) activators, PGE1 and forskolin, also enhanced gene expression through the TßRIII gene promoter, whereas protein kinase C activators, PGF2{alpha} and phorbol myristate acetate, did not. The stimulatory effect of PGE2 on TßRIII promoter activity was suppressed by a dominant negative PKA-regulatory subunit, but not by dominant negative protein kinase C. PGE2 specifically increased nuclear factor CCAAT enhancer-binding protein {delta} (C/EBP{delta}) binding to a half-binding site upstream of the basal TßRIII promoter region, and promoter activity was sensitive to C/EBP{delta} overexpression and to dominant-negative C/EBP{delta} competition. In parallel with their effect on TßRIII expression, activators of PKA decreased TGF-ß-induced activity. In summary, high levels of PGE2 that occur with inflammation or trauma may, through PKA-activated C/EBP{delta}, preferentially increase TßRIII expression and in this way delay TGF-ß-dependent activation of osteoblasts during the early stabilization phase of bone repair.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE TGF ß (1) FAMILY members interact with at least three distinct cell surface receptors (here termed TßRs) (1, 2, 3). TßRIII, also termed betaglycan, is a proteoglycan composed of a 100- to 120-kDa core protein and chondroitin sulfate and heparin chains, with an overall apparent molecular mass of about 250 kDa (4). It lacks a distinct intracellular signaling motif and may control the stability or ligand binding capacity of TßRII and have complex effects on signal generation through TßRI (5, 6, 7, 8). Earlier studies revealed variations in all three TßRs during osteoblast differentiation or in response to several osteotropic growth regulators (1, 7, 9, 10, 11, 12, 13, 14, 15). In these cases, differences in the distribution of TGF-ß among the TßRs can enhance, limit, or refocus its various effects. For example, TßRIII and TßRII levels decrease during expression of the osteoblast phenotype or in response to bone morphogenetic protein 2 (BMP-2), in combination with a decrease in the mitogenic potential of TGF-ß and an increase in its stimulatory effect on collagen synthesis (11, 12). In contrast, TßRIII levels increase and TßRI levels decrease in response to glucocorticoid, coincident with less TGF-ß activity (9, 10, 13). Some changes in TßR levels on osteoblasts are transcription dependent, whereas others relate to cell surface trafficking (7, 10, 11, 13, 15, 16, 17). Regardless, proportionately less TGF-ß binding to TßRI, the intracellular signal-transducing component of the TßR system, reduces its downstream effects. Whereas this can derive from less TßRI expression, it can also follow an increase in TßRIII, which when expressed transgenically at high levels, can diminish TGF-ß activity (8, 17).

TßRIII also interacts with other growth regulators, notably the fibroblast growth factor (18) and activin/inhibin systems (19, 20), suggesting more extensive effects than first appreciated. Changes in TßRIII expression are linked to cardiovascular developmental defects (21, 22), to transdifferentiation of hepatic stellate cells (23), and to pathological breast tumor progression (24, 25, 26), whereas mutations may be associated with prostate disease (27). TßRIII gene promoter sequences have been isolated and minimally characterized for basal and hormone-dependent control regions (17, 28), but virtually nothing is known about specific cis- or trans-acting elements associated with regulated TßRIII transcription.

Prostaglandin (PG) E2 levels increase in bone by mechanical loading, inflammation, and various cytokines and may account for the effects of several osteogenic growth factors (29, 30, 31). Prior evidence from primary cell cultures isolated from fetal rat bone showed variations in PGE2 activity that correlate with cell differentiation status and activation of the protein kinase A (PKA) and protein kinase C (PKC) signal transduction pathways (32, 33, 34). PGE2-dependent activation of PKA appears to balance, in part, its PKC-dependent stimulatory effects on proliferation and matrix protein synthesis in bone cells (32). Moreover, through PKA, PGE2 increases gene expression by transcription factors CCAAT enhancer-binding protein {delta} (C/EBP) (35, 36, 37), Runx2 (38), and Fra2 (39) in differentiating osteoblasts, predicting complex and highly focused downstream effects on various aspects of osteoblast function.

Given important but variable roles for TGF-ß and PGE2 during connective tissue growth and repair, and their complex effects during the inflammatory response, in this study we addressed their possible interactions on differentiating bone cells. Our results reveal a rapid transcriptional effect by PGE2 on TßRIII expression through a defined gene promoter domain, perhaps to delay TGF-ß activity during an initial stabilization phase before cell replication and tissue repair.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PGE2 Increases TßRIII Expression
Cell surface TßRs have rapid turnover rates (16), predicting that they are sensitive targets for regulation. In this regard, 24-h treatment with PGE2 induced a 5.0-fold increase in TGF-ß binding to cell surface TßRIII, without similarly large or significant increases in binding to TßRII or TßRI. An increase in TGF-ß binding to TßRIII also occurred in response to PGE1 but not to PGF2{alpha}, suggesting that PGE2 caused PG receptor or downstream signal-restricted effects on TßRIII gene expression (Fig. 1Go, A and B). Exposure to PGE2 increased TßRIII mRNA levels by 5-fold within 6 h, and somewhat lower, albeit still 2-fold higher, levels of TßRIII mRNA remained after 24 h (Fig. 2AGo). PGE2 also enhanced the level of the high molecular mass TßRIII proteoglycan by 4.3 ± 1.2-fold within 22 h, and this effect was reduced by 46 h. Similarly, there was a rapid increase in a lower molecular mass immunoreactive band of about 100 kDa, consistent with intracellular TßRIII core protein (4) (left panel, Fig. 2BGo). Treatment with tunicamycin to suppress N-linked glycosylation of newly synthesized proteins and disrupt proteoglycan expression (40, 41) showed little or no effect on high molecular mass cell surface TßRIII within 6 h, but a greater than 90% loss by 24 h (supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), in agreement with the apparently different rates of TßRIII core protein and proteoglycan expression seen by Western blot analysis. Analogous to earlier studies in which TGF-ß itself reduces TßRIII gene promoter activity (28), 24-h treatment with TGF-ß1 reduced the amount of TßRIII protein and suppressed the increase induced by PGE2 (right panel, Fig. 2BGo). Together, these observations indicate that the stimulatory affect of PGE2 on ligand binding to TßRIII resulted from an increase in TßRIII gene expression. Lower amounts of TßRIII mRNA and protein seen at later times after maximal induction agree with earlier evidence for their approximate 20- to 22-h turnover rates (16).


Figure 1
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  		Fig. 1. PG-Dependent Effects on TGF-ß Binding

Confluent fetal rat osteoblasts were treated for 24 h with vehicle (0), or 1 µM PGE2 (E2), PGE1 (E1), or PGF2{alpha} (F2{alpha}), and then examined for [125I]TGF-ß1 binding. Cell extracts were fractionated by gel electrophoresis and autoradiography in panel A, and radioactive bands corresponding to TßRIII, TßRII, and TßRI were quantified from five studies by scanning densitometry in panel B. *, Significant increase in [125I]TGF-ß1 binding (P < 0.05). ODU, OD units.

 

Figure 2
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  		Fig. 2. PGE2-Dependent Effects on TßRIII Expression

Confluent fetal rat osteoblasts were treated for the times indicated with 1 µM PGE2. A, Total RNA was fractionated by gel electrophoresis, probed with radiolabeled rat TßRIII cDNA, and analyzed by autoradiography (upper left panel). Extraction efficiency and RNA integrity were verified by ethidium-stained 28S and 18S rRNA bands (lower left panel), and effects relative to 18S rRNA were determined in nine studies by scanning densitometry (right panel). *, Significant increase in TßRIII mRNA (P < 0.05). B, Cells were treated for the times shown with 1 µM PGE2 (left panel) or 120 pM TGF-ß1 and supplemented with 1 µM PGE2 for the last 4 h of culture (right panel). Total nuclear free cell extracts were fractionated by gel electrophoresis and analyzed by Western blot with antirat TßRIII antibody by chemiluminescence (as shown) and densitometry (as indicated in Results). ODU, OD units.

 
PGE2 Increases TßRIII Gene Promoter Activity
The TßRIII gene promoter comprises multiple but still undefined regulatory elements that enhance or suppress endogenous expression, or respond to other growth factors or hormones (17, 28). Consistent with its stimulatory effect on TßRIII mRNA expression, PGE2 enhanced reporter gene expression driven by 3.7 kb of DNA encompassing the TßRIII promoter (17). Analogous relative effects occurred with promoter fragments 3.7, 1.9, 1.8, and 0.4K (17), locating the active element to a 3'-proximal region (Fig. 3AGo). The stimulatory effect of PGE2 on TßRIII promoter activity was time and dose related (Fig. 3Go, B and C), replicated by PGE1, and less evident with PGF2{alpha} treatment (Fig. 3DGo).


Figure 3
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  		Fig. 3. PG-Dependent Effects on TßRIII Gene Promoter Activity

Fetal rat osteoblasts were transfected for 24 h with 75 ng/cm2 of the TßRIII promoter/reporter plasmid constructs shown. The cells were then treated with vehicle (0), PGE2 (E2), PGE1 (E1), or PGF2{alpha} (F2{alpha}), and assayed for reporter gene expression. When not indicated, PGs were tested at 1 µM (panels A, B, and D), or for 6 h (panels A, C, and D). *, Significant increase in TßRIII gene promoter activity (P < 0.05).

 
Virtually the entire gene-inducing effect by PGE2 localized to a 194-bp region of the TßRIII gene promoter termed 0.2K/E, between promoter positions –687 and –493 at the 5'-end of fragment 0.4K (Fig. 4AGo). Fragment 0.2K/E displayed significantly lower basal activity due to the loss of constitutively active minimal promoter elements (17), but retained a biphasic time-dependent PGE2 response profile (Fig. 4BGo).


Figure 4
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  		Fig. 4. PGE2-Dependent Effects on Truncated TßRIII Gene Promoter Elements

Fetal rat osteoblasts were transfected for 24 h with 75 ng/cm2 of the TßRIII promoter/reporter plasmid constructs shown. The cells were then treated with vehicle (0) or 1 µM PGE2, and assayed for reporter gene expression. PGE2 was for 6 h in panel A, and for the times indicated in panel B. *, Significant increase in TßRIII gene promoter activity (P < 0.05).

 
PGE2 Increases TßRIII Expression through PKA
PGE2 differentially regulates bone cell activity through PKA- and PKC-generated events (32, 33, 34, 42). By and large, the PKA-related effects of PGE2 are shared by PGE1 and replicated by forskolin, and its PKC-related effects are shared by PGF2{alpha} and replicated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) (32, 33, 34). Treatment with forskolin reproduced the stimulatory effect of PGE2 on cell surface TßRIII (Fig. 5AGo) and TßRIII promoter activity (Fig. 5BGo). Also, forced expression of the PKA catalytic subunit (43) enhanced basal TßRIII promoter activity (gray bars, Fig. 6AGo), and forced expression of the PKA-regulatory subunit with a dominant-negative mutation (44), which cannot bind cAMP and release active catalytic subunit, suppressed its activity in PGE2-stimulated cells (black bars, Fig. 6AGo). By contrast, forced expression of constitutively active PKC{alpha} (45) did not increase basal TßRIII promoter activity (gray bars, Fig. 6BGo), and mutated, dominant-negative PKC{alpha} (45) did not inhibit the stimulatory effect of PGE2 (black bars, Fig. 6BGo). However, constitutively active PKC{alpha} effectively enhanced reporter plasmid SXN1C, driven by two Runx response elements derived from the TßRI gene promoter (gray bars, Fig. 6CGo), and dominant negative PKC{alpha} suppressed SXN1C activity induced by PGE2 (black bars, Fig. 6CGo), revealing that the PKC expression plasmids could function in other contexts. Consistent with TßRIII promoter activity, forced expression of the PKA catalytic subunit increased TßRIII mRNA within 6 h of induction (left panel, Fig. 6DGo), and forced expression of the dominant-negative PKA-regulatory subunit suppressed TßRIII mRNA in PGE2-treated cells (right panel, Fig. 6DGo). Moreover, amounts of staurosporine that significantly reduce PKC in isolated bone cells (32) did not suppress the stimulatory effect of PGE2 on the TßRIII promoter (Fig. 6EGo). These results are consistent with predominant, if not exclusive, activation through PKA.


Figure 5
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  		Fig. 5. PKA-Related Effects on TGF-ß Binding and TßRIII Gene Promoter Activity

A, Confluent fetal rat osteoblasts were treated for 24 h with vehicle (0), 1 µM PGE2 (E2), 10 µM forskolin (Fk), or 1 µM phorbol ester PMA (PE), and then examined for [125I]TGF-ß1 binding by gel electrophoresis and autoradiography. B, Cells were transfected for 24 h with 75 ng/cm2 of the TßRIII promoter/reporter plasmid 0.2K/E. The cells were then treated for 6 h with vehicle (0), 1 µM E2, 10 µM Fk, or 1 µM PE, and examined for reporter gene expression. *, Significant increase in TßRIII gene promoter activity in panel C (P < 0.05).

 

Figure 6
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  		Fig. 6. PKA-Restricted Effects on TßRIII Gene Promoter Activity and mRNA Expression

Panel A, Fetal rat osteoblasts were cotransfected for 24 h with 75 ng/cm2 of TßRIII promoter/reporter plasmid 0.2K/E in combination with 25 ng/cm2 of vector (0), or expression plasmids encoding PKA-catalytic subunit (Ct), PKA-regulatory subunit (Rg), or mutated dominant-negative PKA-regulatory subunit (Rgµ). Panel B, Cells were cotransfected for 24 h with 0.2K/E in combination with 25 ng/cm2 of vector (0), constitutively active PKC{alpha} (C), or mutated dominant-negative PKC{alpha} (Dn). Panel C, Cells were cotransfected for 24 h with plasmid SXN1C in combination with 25 ng/cm2 of vector (0), constitutively active PKC{alpha} (C), or mutated dominant-negative PKC{alpha} (Dn). Panel D, Cells were transfected for 24 h with PKA-catalytic subunit (Ct), PKA-regulatory subunit (Rg), or mutated dominant-negative PKA-regulatory subunit (Rgµ) as in panel A. In panels A–C the cells were then treated for 6 h with vehicle (control) or 1 µM PGE2. Panel E, Cells were transfected for 24 h with 0.2K/E and pretreated for 15 min with the amounts of staurosporine shown before supplementing for 6 h with vehicle (control) or 1 µM PGE2. In panels A, B, C, and E, cell extracts were examined for reporter gene expression. In panel D, total RNA extracts were examined for TßRIII mRNA and ethidium stained for rRNA as in Fig. 2AGo. *, Significant increase by PKA Ct or PKC{alpha}; **, significant decrease in the stimulatory effect of PGE2 (P < 0.05).

 
C/EBP{delta} Binds to and Regulates the TßRIII Gene Promoter
EMSA with five overlapping oligonucleotides (Table 1Go) that span the entire PGE2-sensitive 194-bp region of the TßRIII gene promoter showed constitutive DNA binding within several nuclear protein complexes. The 3'-terminal probe 542/490 contains a strong GC box [5'-GAGGAGGGGC-3'] at positions –527 to –518 where endogenous constitutive Sp1 and Sp3 binding, as defined by EMSA with specific antibodies and point mutations, is entirely depleted by mutation (17). However, PGE2 had no effect on Sp1 or Sp3 binding within these complexes. Further sequence analysis of this region with MatInspector (Genomatix Software, GmbH) located a putative C/EBP binding site [5'-TGGAGCAAG-3'] at positions –573 to –565 within the centrally located probe 598/541 (17), but again, no PGE2-dependent change in nuclear factor binding was evident with this probe. Of all five probes derived from this PGE2-sensitive region of the TßRIII promoter, only the 5'-terminal probe, 693/637, produced a dramatic PGE2-inducible gel shift, where a protein/DNA doublet increased 4.4 ± 0.3-fold after 4 h of treatment (Fig. 7AGo). Overlapping sequence and analogous lower migrating protein-DNA complexes common to probes 693/637 and 650/594 indicated that PGE2-induced protein binding occurred between positions –693 and –650. Sequence screening revealed consensus core binding sequences for nuclear factor-{kappa}B (NF-{kappa}B) [5'-GGGATCCC-3'] at –667 to –660 and activator protein 4 (AP-4) [5'-CAGCTG-3] at –659 to –654, and a near match for a half-binding site for the C/EBPs [3'-GTCGACAAT-5'] at –651 to –659. Preincubation with anti-C/EBP{delta} antibody completely eliminated PGE2-induced complex formation by this doublet, but antibody to the leucine zipper nuclear factors Fra-2 and cAMP-response element-binding protein (CREB), also induced by PKA (39, 46), had no effect (Fig. 7BGo). Competition with unlabeled homologous oligonucleotide 693/637 reduced all nuclear factor-binding complexes, whereas unlabeled oligonucleotide with mutations in the C/EBP half-binding site suppressed all but the C/EBP{delta} antibody-sensitive complex. Analogous effects occurred with unlabeled oligonucleotides 668/637 and 668/637µ derived from a region that more closely defines the C/EBP{delta} antibody-sensitive complex (Fig. 7CGo).


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  		Table 1. Oligonucleotides Used in EMSA Studies

 

Figure 7
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  		Fig. 7. C/EBP{delta} Binding to the PGE2-Sensitive Region of the TßRIII Gene

A–C, Confluent fetal rat osteoblasts were treated for 4 h with vehicle (0) or 1 µM PGE2 as indicated, and nuclear extracts were tested by EMSA with 32P-labeled and unlabeled oligonucleotide probes derived from the PGE2-sensitive region of the TßRIII gene promoter. Nuclear extracts were preincubated in panel B with no addition (0), antibody (Ab) to nuclear factor AP-4, the DNA binding p65 Rel A subunit of NF-{kappa}B (p65), C/EBP{delta}, Fra-2, CREB, or nonimmune IgG; or in panel C with 100-fold excess of unlabeled competitor (Comp) oligonucleotides encoding native or mutated (µ) TßRIII promoter sequences, as detailed in Table 1Go.

 
The presence of a binding doublet suggested the possibility of a heterodimer within this binding complex, but the effect of PGE2 appeared largely C/EBP{delta} restricted because little or no change in complex formation occurred with anti-C/EBPß antibody (Fig. 8AGo). PGE2 rapidly increased the amount of nuclear C/EBP{delta}, which migrates at Mr 36 kDa (47, 48, 49), from very low levels in untreated cells. Analogous to changes in TßRIII expression, this effect was biphasic, with less C/EBP{delta} evident by 24 h (Fig. 8BGo). Moreover, overexpression of C/EBP{delta}, but not C/EBPß, significantly enhanced TßRIII promoter activity (Fig. 8CGo). The stimulatory effect of PGE2 was also greater in C/EBP{delta}-overexpressing cells (Fig. 8DGo), whereas overexpression of a dominant-negative C/EBP{delta} fragment encoding leucine zipper dimerization and DNA-binding domains but no gene transactivation domain (38, 47) significantly limited the effect of PGE2 on TßRIII promoter activity (Fig. 8EGo). Coexpression of native or truncated C/EBP{delta} in combination with TßRIII promoter fragment 0.4K, which contains this region in addition to minimal TßRIII promoter elements, gave analogous results (supplemental Fig. 2 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), whereas overexpression of dominant-negative Fra-2 had no inhibitory effect. Therefore, endogenous C/EBP{delta} in PGE2-activated cells specifically and preferentially can bind one of two C/EBP-binding sites within this regulatory region of the TßRIII gene promoter and enhance TßRIII gene promoter activity. EMSA studies with a more promiscuous C/EBP binding probe termed "HS3D" from the IGF-I gene promoter (Table 1Go), which is sensitive to C/EBPß or C/EBP{delta} (47), also showed a biphasic binding profile in response to PGE2 that paralleled C/EBP{delta} expression and TßRIII gene promoter activity (Fig. 8FGo), precluding the possibility that C/EBPß might associate at a later time. Indeed, we have never seen high levels of C/EBP binding to DNA when endogenous C/EBP levels and C/EBP-sensitive gene promoter activity diminish, but acknowledge that this might occur in other contexts. EMSA with nuclear extract from PGE2-treated cells transfected to express C/EBPß or C/EBP{delta}, in combination with C/EBPß or C/EBP{delta}-specific antibody, revealed that each factor effectively associates with the C/EBP response element in HS3D (Fig. 8GGo). Analogous to its effect on the TßRIII promoter, overexpression of C/EBP{delta} alone enhanced IGF-I gene promoter activity, whereas overexpression of either C/EBPß or C/EBP{delta} increased IGF-I gene promoter activity in response to PGE2 (Fig. 8HGo). Therefore, C/EBPß, when it is expressed, can accumulate in the nucleus with PGE2 activation and can function through an alternate C/EBP-binding element, in contrast to the relatively restricted effect of C/EBP{delta} on TßRIII gene promoter activity in these cells.


Figure 8
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  		Fig. 8. C/EBP{delta}-Dependent Control of TßRIII Gene Expression

A, EMSA was performed as in Fig. 7Go with TßRIII probe TR3–693/637 using nuclear extracts preincubated with no addition (0), antibody (Ab) to nuclear factor C/EBP{delta}, C/EBPß, or nonimmune IgG. B, Cell extracts were fractionated by gel electrophoresis and analyzed by Western blot with anti-C/EBP{delta} antibody by chemiluminescence. C–E, Cells were cotransfected for 24 h with 75 ng/cm2 of TßRIII promoter/reporter plasmid 0.2K/E in combination with vector (0) or expression plasmids encoding C/EBP{delta} ({delta}), C/EBPß (ß), or truncated dominant-negative C/EBP{delta} (DN C/EBP{delta}), at the amounts shown in panels C and E, or 25 ng/cm2 in panel D. In panels D and E, cells were then treated for 6 h with vehicle (control) or 1 µM PGE2 as indicated. F, EMSA was performed with an alternate C/EBP-binding probe HS3D (Table 1Go) using nuclear extracts from cells treated with PGE2 for the times indicated. G, EMSA was performed with probe HS3D using nuclear extract from cells transfected for 24 h with expression plasmids encoding C/EBP{delta} ({delta}) or C/EBPß (ß) at 25 ng/cm2 and treated for 4 h with PGE2. Extracts were preincubated with nonimmune IgG or antibody to nuclear factor C/EBPß or C/EBP{delta} as indicated. H, Cells were cotransfected for 24 h with 75 ng/cm2 of IGF-I gene promoter/reporter plasmid 1711b in combination with vector (0) or expression plasmids encoding C/EBP{delta} or C/EBPß, and treated with vehicle or PGE2 as in panel D. *, Significant increase by C/EBP{delta} overexpression in panels C, D, and H; **, significant increase in the stimulatory effect of PGE2 in panels D and H; *, significant decrease in the stimulatory effect of PGE2 in panel E (P < 0.05).

 
PGE2 Suppresses TGF-ß Activity
TGF-ß stimulates proliferation by cells that express a differentiating osteoblast phenotype and, by and large, cells with proportionately high levels of TßRIII are less sensitive to TGF-ß induction (11, 50, 51). Consistent with this and its stimulatory effect on TßRIII expression, PGE2 (black symbols, Fig. 9AGo) significantly reduced the mitogenic effect of TGF-ß alone (gray symbols, Fig. 9AGo) at lower concentrations, on the ascending limb of its biphasic induction profile. Suppression by PGE2 was dose related (Fig. 9BGo) and replicated by PGE1 but not by PGF2{alpha} (Fig. 9CGo). Earlier studies showed that PGE2 and PGE1 could modestly reduce basal DNA synthesis after 48 h of treatment (32, 47), but this was not significant relative to the larger effects in TGF-ß-treated cells. A decrease in TGF-ß activity also occurred with forskolin but not with PMA (Fig. 9DGo), consistent with changes in TßRIII expression.


Figure 9
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  		Fig. 9. PG-Dependent Effects on TGF-ß Activity

Confluent fetal rat osteoblasts were serum deprived and treated for 24 h with vehicle (0; gray circles in panel A), PGE2 (E2; black circles in panel A), or with PGE2 (E2), PGE1 (E1), PGF2{alpha} (F2{alpha}), forskolin (Fk), or the phorbol ester PMA (PE) in panels B, C and D, and then challenged for 24 h with TGF-ß1 (T). DNA synthesis was measured by pulse labeling with [3H]thymidine during the last 2 h of culture, and by assaying for acid-insoluble incorporation. When not indicated, compounds were tested at maximally effective concentrations of 1 µM for PGs and PMA, 10 µM for Fk, and 12 pM for TGF-ß1. TGF-ß1 significantly increased DNA and collagen synthesis at all concentrations shown. *, Significant inhibition of TGF-ß1 activity (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
All TGF-ß-sensitive cells require signals generated through TßRI. This varies in intensity or in focus through changes in the expression of other components of the TGF-ß response system, including TßRII and TßRIII (3). Some differences relate to specific TGF-ß isoforms, some to the phenotype of the responding cell and others to native or hormone-induced changes in individual TßRs. Loss of functional TßRI limits the activity of all three TGF-ß isoforms, whereas loss of TßRIII specifically prevents TGF-ß2 binding to TßRII, but not to TßRII-B found on osteoblasts (3, 52). Indeed, we found analogous effects by TGF-ß1 and TGF-ß2 in osteoblasts after BMP-2 pretreatment, which suppresses TßRIII expression (Ref. 11 and our unpublished results). The total amount of cell surface TßRIII, and consequently the ratio of TßRIII to TßRI, can vary with differentiation or cell phenotype and parallel differences in TGF-ß sensitivity. For example, low levels of TßRIII that occur on differentiated rat osteosarcoma cells, or on normal osteoblasts in response to BMP-2, coincide with less cell surface TßRII and a transition in the nature of TGF-ß function (7, 11, 12, 17). Curiously, unlike the effect of BMP-2 that simultaneously decreases TßRIII and enhances TßRI expression, TßRIII antisense expression severely limits TGF-ß binding at all three receptors (supplemental Fig. 3, published as supplemental data on The Endocrine Society’s Journals web site at http://mend.endojournals.org), precluding studies to assess the effect of PGE2 on TGF-ß activity in the absence of only TßRIII. Nevertheless, transgenic overexpression of TßRIII significantly limits TGF-ß activity (8, 17), confirming that changes in TßRIII gene expression can affect the TGF-ß system independently of an endogenous signaling motif.

Here we show that PGE2 induces a PKA-dependent increase in TßRIII expression through C/EBP{delta}. This occurs without decreases in TGF-ß binding to cell surface TßRII or TßRI but coincides with a decrease in TGF-ß activity. We earlier found that glucocorticoids also increase TßRIII expression on osteoblasts. However, this happened in combination with a significant decrease in TßRI expression, which itself compromises TGF-ß activity (9, 13, 15). Based on the distribution of TGF-ß among three interacting cell surface components (1, 53), effects on TGF-ß activity could be more evident at low TGF-ß concentrations at which activation of signal-transducing TßRs would be incomplete. Indeed, the inhibitory effect of PGE2 on DNA synthesis was more pronounced in response to low, nonsaturating amounts of TGF-ß. Therefore, the stimulatory effect of PGE2 on TßRIII expression coincides with results predicted by native TßRIII expression patterns (1, 11), by transgenic TßRIII gene overexpression (8, 17), and by mechanical strain (14), where an increase in TßRIII expression can have a significant effect on TGF-ß activity. Importantly, many studies described local TGF-ß expression and activity during various phases of bone repair (54), but what little is known about TßRs has focused on TßRI and TßRII (55), and virtually nothing is known about inflammatory mediators on local TßR expression. The possible physiological importance of PGE2 in this context derives from evidence that early exposure to some cyclooxygenase inhibitors that inhibit PG synthesis from arachidonic acid can suppress fracture healing in vivo (56, 57, 58). Regarding our results, a PGE2-dependent delay in osteoblast activation by the local TGF-ß that accumulates after fracture, in part through increased TßRIII, may permit initial tissue stabilization and early events required before later stages of bone healing where TGF-ß increases bone cell replication and new matrix synthesis. The stimulatory effect of PGE2 on TßRIII expression may then become self-limiting, because TGF-ß itself can down-regulate TßRIII promoter activity (28) and, in some instances, suppress C/EBP{delta}-dependent gene expression and activity (59, 60). Nonetheless, in other situations, C/EBPs may have supportive roles with regard to TGF-ß function or prevention of metabolic disease, suggesting tissue- or context-restricted functions (61, 62, 63). It is important to emphasize that PGE2 can have multiple direct or indirect effects unrelated to TGF-ß. In this regard, we earlier showed that PGE2 can increase basal protein synthesis by osteoblasts preconditioned by transient exposure to glucocorticoid, revealing events through other growth factor-dependent or -regulatory pathways (47). To date, however, we have not observed a situation in which a proportional increase in TßRIII has failed to decrease some aspect of TGF-ß activity in rat bone (1, 9, 11, 12, 14, 17) or kidney-derived cells (8), in contrast to our own observations in rat aortic vascular cells (5) or L6 myoblasts (8).

The biological significance of changes in TßRIII expression is very apparent in other contexts. For example, loss of functional TßRIII limits epithelial to mesenchymal cell transformation during heart development through changes in TGF-ß2 sensitivity (21, 22). TßRIII levels also increase on glucocorticoid-treated hepatic stellate cells and decrease during their transdifferentiation to myofibroblasts (23). Other evidence reveals that loss of TßRIII correlates with higher grade and more invasive human breast cancers, and that these effects are reversed in vivo in mice by transgenic reexpression of TßRIII (24, 25, 26).

Consistent with our results, previous studies in cultured human ovarian granulose-luteal cells first revealed PGE2-dependent increases in TßRIII mRNA expression that were replicated by dibutyryl-cAMP but not by phorbol ester, predicting a PKA-dependent event (64, 65). Our new evidence showing the stimulatory effect of PGE2 on TßRIII gene promoter activity, and abrogation with mutated PKA-regulatory subunit, establishes the importance of the PKA pathway with reasonable certainty. Moreover, dissection of the TßRIII gene promoter allowed us to resolve the specific cis- and trans-acting transcriptional elements associated with this event. Although the PGE2-sensitive region of the TßRIII promoter includes several possible response elements, only a C/EBP{delta}-DNA binding complex increased dramatically with PGE2 treatment. We previously showed a rapid PGE2-induced translocation of C/EBP{delta} in osteoblasts, which increases IGF-I gene expression (37, 47, 48). Curiously, transgenically expressed C/EBPß can also bind and drive gene expression through a C/EBP site in the IGF-I promoter, in contrast to the TßRIII promoter that preferentially binds and responds to C/EBP{delta}. This difference may derive from still unresolved variations between the individual C/EBP-binding sequences, their orientation, or their flanking domains.

PGE2 also increases new C/EBP{delta} expression (these studies and Ref. 47). However, the stimulatory effects of PGE2 on TßRIII mRNA and gene promoter activity decline at later times, predicting counter control, perhaps through still unknown response elements or autologous regulators of C/EBP{delta} translocation or activity. Indeed, other situations that control TßRIII expression could be related to changes in C/EBP{delta} synthesis or activity. For example, glucocorticoid also rapidly induces new C/EBP{delta} expression in many cells including osteoblasts (47, 66, 67, 68), perhaps accounting for its stimulatory effect on TßRIII expression in bone and liver cells (9, 10, 12, 13, 23). Studies in other tissues will further define the importance of C/EBP{delta} on TßRIII expression and TGF-ß activity during organ development, mechanical load, trauma, and inflammatory disease, and on other important growth factor systems that are known to be influenced by changes in the expression of TßRIII.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells
Primary osteoblast-enriched cultures were isolated from parietal bones of 22-d-old Sprague Dawley rat fetuses (Charles River Laboratories, Inc., Wilmington, MA), as approved by the Yale Institutional Animal Care and Use Committee. Bone sutures were dissected and cells were released by five sequential collagenase digestions. Cells pooled from the last three digestions express features of differentiating osteoblasts, including high levels of nuclear factor Runx2, PTH receptor, type I collagen synthesis, and alkaline phosphatase. They also exhibit an increase in osteocalcin expression in response to vitamin D3, differential sensitivity to TGF-ß, BMP-2, and various PGs, and form mineralized nodules under conditions that promote long-term differentiation in vitro. Cells were plated at 4000/cm2 in DMEM supplemented with 10% fetal bovine serum and 100 µg/ml ascorbic acid and grown for 6 d before transfection or treatment, which were performed in serum free medium (11, 32, 50, 69, 70, 71, 72, 73).

TGF-ß Binding
Cells were incubated with 150 pM [125I]TGF-ß1 (4000 Ci/mmol) for 3 h at 4 C in serum-free medium containing 4 mg/ml BSA. Unbound radioligand was removed by multiple washings, and bound TGF-ß was covalently cross-linked with 0.2 mM disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL). Cells were extracted, equal amounts of cell extracts were fractionated on a denaturing 5–15% gradient polyacrylamide SDS gel, and TßR binding patterns were examined by autoradiography and densitometry (11).

mRNA Analysis
Total RNA was extracted with acid-guanidine-monothiocyanate, precipitated with isopropyl alcohol, and dissolved in sterile water. TßRIII mRNA was assessed by fractionation on a 1.5% agarose/2.2 M formaldehyde gel, blotting on charged nylon, and hybridization with 32P-labeled cDNA encompassing the 3.9-kb coding region of the rat TßRIII gene (4). rRNA was assessed by ethidium staining of a parallel gel. Radiolabeled products were examined by autoradiography and densitometry (11, 13).

Western Immunoblots
Equal amounts of protein were fractionated on a denaturing 5–15% gradient polyacrylamide-SDS gel and electroblotted onto polyvinylidine difluoride membranes (NEN Life Science Products, Boston, MA) along with prestained molecular weight markers. Blots were blocked in 5% fat-free powdered milk, probed with a 1:1000 dilution of anti-TßRIII primary antibody (kind gift of Dr. Fernando Lopez-Casillas), or anti-C/EBP{delta} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and reactive bands were visualized with a 1:2,500 dilution of goat antirabbit IgG secondary antibody linked to horseradish peroxidase (The Jackson Laboratory, Bar Harbor, ME) and chemiluminescence (Western Lightning; PerkinElmer Life Sciences, Wellesley, MA).

Transfections
TßRIII gene promoter activity was assessed with fragments cloned from the rat TßRIII gene and inserted into firefly luciferase reporter plasmid pGL2-Basic (17). Promoter fragment 0.2K/E was subcloned from fragment 0.4K by digestion with EagI, and fragment 0.2B was subcloned from fragment 0.4K by digestion with BssHII, and reinsertion into reporter plasmid pGL2-Basic. To assess the roles of PKA, PKC, and the C/EBPs on TßRIII gene promoter activity, cells were cotransfected with expression plasmids encoding catalytic, regulatory, constitutively active, or dominant negative subunits of PKA (38, 39, 42, 43, 44, 74) or PKC (45), or wild-type or dominant-negative C/EBPs (35, 47). To control for PKC activity, cells were cotransfected with expression plasmids encoding constitutively active or dominant-negative subunits of PKC in combination with reporter plasmid SXN1C driven by two Runx response elements derived from the TßRI gene promoter (13). To control for C/EBPß overexpression, cells were transfected with reporter 1711b containing a 1.7-kb fragment of the rat IGF-I gene promoter cloned upstream of firefly luciferase (47). Plasmids were pretitrated for optimal expression efficiency and transfected with reagent TransIT LT1 (Mirus Corp., Madison, WI). Cells at 70% confluence were exposed to plasmid DNA for 24 h in medium containing 5% fetal bovine serum, treated for various times as indicated in the figures, in serum-free medium. The cells were rinsed and lysed, and nuclear-free supernatants were analyzed for reporter activity and corrected for protein content. Transfection efficiency was assessed in parallel with positive and negative reporter plasmids (70, 75).

EMSA
Double-strand oligonucleotide probes contained in TßRIII promoter fragment 0.2K/E (Table 1Go) or IGF-I promoter fragment HS3D were labeled with [32P]dCTP and Klenow fragment of Escherichia coli DNA polymerase I, and gel purified. Extracted nuclear protein (3 µg) from control or PGE2-induced cells was preincubated with no addition, antisera to AP-4 (kind gift of Dr. Richard B. Gaynor) NF-{kappa}B, C/EBP{delta}, C/EBPß, Fra-2, CREB, nonimmune IgG (Santa Cruz Biotechnology, Inc.), or unlabeled oligonucleotide (Table 1Go), and then supplemented with 32P-labeled probe. Protein-bound DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel and examined by autoradiography (13, 70).

DNA Synthesis
DNA synthesis rates were measured by labeling with 5 µCi/ml [methyl-3H]thymidine (80 Ci/mmol) during the last 2 h of culture, lysing the cells in 0.1 M sodium dodecyl sulfate, 0.1 N NaOH, collecting the precipitate formed with 10% trichloroacetic acid, and scintillation counting (11, 50).

Statistical Analysis
Differences were assessed by one-way ANOVA with Tukey post hoc analysis using SigmaStat software (Jandel Corp., San Rafael, CA) from eight or more replicate samples and two or more independent cell preparations. Protein and mRNA levels were from at least two studies and, when expressed graphically, from three or more studies. A significant difference was assumed by a P value of <0.05.


   ACKNOWLEDGMENTS
 
We thank Drs. Joan Massague (Sloan Kettering Memorial Institute) and Fernando Lopez-Casillas (Universidad Nacional Autonoma de Mexico) for cDNA encoding rat TßRIII and anti-TßRIII antibody; Dr. G. Stanley McKnight (University of Washington) for expression constructs encoding native and mutant PKA subunits; Dr. Peter J. Parker (London Research Institute) for expression constructs encoding constitutively active and dominant-negative PKC subunits; and Dr. Richard B. Gaynor (University of Texas Southwestern) for anti-AP-4 antibody.


   FOOTNOTES
 
These studies were supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Award AR 39201.

The authors have nothing to disclose.

First Published Online July 17, 2007

Abbreviations: AP-4, Activator protein 4; BMP, bone morphogenetic protein; C/EBP{delta}, CCAAT enhancer-binding protein {delta}; CREB, cAMP-response element-binding protein; NF-{kappa}B, nuclear factor-{kappa}B; PG, prostaglandin; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TßR, TGF-ß receptor.

Received for publication April 24, 2007. Accepted for publication July 13, 2007.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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  70. Ji C, Casinghino S, Chang DJ, Chen Y, Javed A, Ito Y, Hiebert SW, Lian JB, Stein GS, McCarthy TL, Centrella M 1998 CBFa(AML/PEBP2)-related elements in the TGF-ß type I receptor promoter and expression with osteoblast differentiation. J Cell Biochem 69:353–363[CrossRef][Medline]
  71. Carpenter TO, Moltz KC, Ellis B, Andreoli M, McCarthy TL, Centrella M, Bryan D, Gundberg CM 1998 Osteocalcin production in primary osteoblast cultures derived from normal and Hyp mice. Endocrinology 139:35–43[Abstract/Free Full Text]
  72. Centrella M, Casinghino S, Gundberg C, McCarthy TL, Wozney J, Rosen V 1996 Changes in bone morphogenetic protein sensitivity relative to differentiation in fetal rat bone cell cultures. Ann NY Acad Sci 785:224–226[Medline]
  73. Ji C, Casinghino S, McCarthy TL, Centrella M 1997 Multiple and essential Sp1 binding sites in the promoter for transforming growth factor-ß type I receptor. J Biol Chem 272:21260–21267[Abstract/Free Full Text]
  74. Correll LA, Woodford TA, Corbin JD, Mellon PL, McKnight GS 1989 Functional characterization of cAMP-binding mutations in type I protein kinase. J Biol Chem 264:16672–16678[Abstract/Free Full Text]
  75. Ji C, Casinghino S, McCarthy TL, Centrella M 1996 Cloning, characterization, and expression of the transforming growth factor-ß type I receptor promoter in fetal rat bone cells. J Cell Biochem 63:478–490[CrossRef][Medline]




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