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Endocrinology Vol. 138, No. 11 4672-4682
Copyright © 1997 by The Endocrine Society

ARTICLES

Transforming Growth Factor-ß1 Regulation of Prostaglandin G/H Synthase-2 Expression in Osteoblastic MC3T3-E1 Cells1

C. Pilbeam, Y. Rao, O. Voznesensky, H. Kawaguchi, C. Alander, L. Raisz and H. Herschman

Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030; and the Departments of Biological Chemistry and Molecular and Medical Pharmacology, University of California School of Medicine (H.H.), Los Angeles, California 90024

Address all correspondence and requests for reprints to: Dr. Carol C. Pilbeam, Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: pilbeam{at}nso.uchc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGFß) plays an important role in bone development and remodeling. TGFß stimulates PGE2 production, enhances interleukin-1-stimulated PGE2 production, and can stimulate PG-mediated bone resorption. We found that TGFß induced prostaglandin G/H synthase (PGHS-2) messenger RNA (mRNA) and PGE2 production in neonatal mouse calvarial cultures and in primary cells derived from these calvariae. We used MC3T3-E1 cells, an immortalized osteoblastic cell line derived from mouse calvariae, to examine the mechanism of PGHS-2 induction. PGHS-2 mRNA was rapidly induced by TGFß (10 ng/ml) in MC3T3-E1 cells; mRNA levels peaked at 4–8 h and were still elevated at 24 h. Induction of PGHS-2 protein and PGE2 production correlated with PGHS-2 mRNA levels. In contrast, TGFß had much less effect on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS-2 mRNA induction by TGFß was not enhanced by cycloheximide pretreatment, suggesting a requirement for new protein synthesis. To study transcriptional regulation, cells were stably transfected with a PGHS-2 promoter-luciferase reporter construct containing 371 bp of the 5'-flanking region and 70 bp of untranslated DNA from the PGHS-2 gene. TGFß-stimulated luciferase activity paralleled PGHS-2 mRNA induction. Stimulation of luciferase activity and PGHS-2 mRNA levels by other agonists, including interleukin-1, TGF{alpha}, forskolin, and phorbol 13-myristate 12-acetate, were enhanced by TGFß. A 90% drop in luciferase activity occurred with deletion of the region from -371 to -213 bp of the PGHS-2 promoter. The PG response to TGFß in MC3T3-E1 cells appears to be mediated primarily by transcriptional regulation of PGHS-2 expression through one or more cis-acting elements located between -371 and -213 bp in the 5'-flanking region of the PGHS-2 gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSFORMING growth factor-ß1 (TGFß1) is the prototypic member of a superfamily of polypeptide growth factors, including at least four distinct isoforms of TGFß, bone morphogenetic proteins, and activins (1). Members of this family act through serine-threonine kinase-specific receptors, possibly via Mad proteins (2) and/or mitogen-activated protein kinase signaling pathways (3). They have multiple effects on cellular proliferation and differentiation and play major roles in growth and development in many animal species (1, 4). TGFß1, hereafter called TGFß, may also play an important role in the regulation of inflammatory processes, as mice homozygous for the disrupted TGFß gene die about 3 weeks after birth from a wasting syndrome associated with inflammatory cell infiltration into many organs (5).

Bone matrix is a major repository for TGFß, and TGFß is thought to play an important role in bone development and remodeling (6, 7, 8). TGFß is synthesized by bone cells, is secreted as a latent complex, is stored in the extracellular matrix, and can be released by bone resorption (9, 10). Although TGFß has biphasic effects on osteoblastic proliferation, depending on cell density and differentiation state, as well as on TGFß concentration (11, 12, 13, 14), its effects on bone formation or type I collagen synthesis are generally stimulatory both in vitro and in vivo (8, 15, 16). However, overexpression of TGFß2 in osteoblasts in vivo causes bone loss (17). TGFß inhibits bone resorption in rat long bone cultures (18). In contrast, in mouse calvariae, TGFß can stimulate PG-dependent resorption (19). TGFß has biphasic effects on the production of osteoclast-like cells in mouse bone marrow cultures; low TGFß concentrations stimulate a PG-dependent increase in formation, whereas at high TGFß concentrations one observes a PG-independent inhibition (20). In addition to stimulating PG production in neonatal mouse calvariae, TGFß can stimulate PGE2 production in the clonal osteoblast-like MC3T3-E1 cell line (21) and enhance the stimulatory effects of other cytokines, such as interleukin-1 (IL-1), on PG production (22).

Prostaglandin G/H synthase (PGHS), a major enzyme regulating the conversion of arachidonic acid (AA) to PGs, has two isoforms. These two proteins, PGHS-1 and PGHS-2, are the products of distinct genes (23). PGHS-1 is usually constitutively expressed, but may also be regulated. In contrast, PGHS-2 is rapidly and transiently induced by mitogens and other ligands (23). The ligand-induced expression of PGHS-2 is necessary for mitogen-induced PGE2 production in fibroblasts and endotoxin-induced PGE2 production in macrophages (24, 25). PGHS-2 is the major enzyme regulating PG production in response to a variety of hormones and cytokines in MC3T3-E1 cells and neonatal mouse calvarial cultures (26, 27, 28, 29, 30, 31, 32, 33). Studies of the TGFß regulation of PGHS-2 expression have produced variable results, depending on cell type. Although TGFß alone has no effect on PGHS-2 expression in murine 3T3 fibroblasts or rat intestinal epithelial cells, it potentiates the induction of PGHS-2 expression by IL-1 and phorbol 13-myristate 12-acetate (PMA) (34, 35). In murine macrophages, TGBß alone has no effect on PGHS-2 expression, but inhibits the endotoxin induction of PGHS-2 expression transcriptionally (36).

We now show in MC3T3-E1 cells, an immortalized osteoblastic cell line derived from mouse calvariae (37), that TGFß-induced PGE2 production correlates with induced PGHS-2 messenger RNA (mRNA) and protein expression and that the induction of PGHS-2 expression is associated with increased luciferase activity in cells stably transfected with PGHS-2 promoter-luciferase reporter constructs. In addition, TGFß transcriptionally enhances the induction of PGHS-2 mRNA by IL-1 and multiple other agonists in these cells. Deletion analysis indicates that the cis-acting element(s) mediating the response to TGFß is located between -371 and -213 bp in the 5'-flanking region. To demonstrate the relevance of our observations in MC3T3-E1 cells for nonimmortalized cells, we show that TGFß induces PGHS-2 mRNA and PGE2 production in cultured neonatal mouse calvariae and primary osteoblastic cells derived from these calvariae.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human TGFß was provided by Genentech (South San Francisco, CA). Murine PGHS-2 complementary DNA (cDNA) has been described previously (38). DNA constructs consisting of -371 to +70 bp of the PGHS-2 gene, or 5'-deletions of this region, fused to a luciferase reporter gene in pXp-2 vector have been described previously (39, 40). Murine PGHS-1 cDNA was the gift of Drs. David DeWitt and William Smith (Michigan State University, East Lansing, MI). Murine cDNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified by PCR with a control amplimer set from Clontech (Palo Alto, CA). Rat osteocalcin cDNA was amplified by PCR (sense primer, 5'-CAGACACCATGAGGACCCTC-3'; antisense primer, 5'-AAAGCTGAAGCTGCCG-TTGG-3'). Type I collagen (COL1A1) cDNA has been described previously (41). Luciferase cDNA was amplified by PCR (sense primer, 5'-CGCTGGAGAGCAACGCAT-AAGGTTATG-3'; antisense primer, 5'-TAGTCTCAGTGAGCCCATATCCTTGTCG-3'). PGE2 antibody was purchased from Dr. Lawrence Levine (Waltham, MA). Recombinant human IL-1 (IL-1{alpha}) was provided by Dr. Peter Lomedico (Hoffman LaRoche, Nutley, NJ). Recombinant human TGF{alpha}, forskolin (FSK), PMA, PGE2, and cycloheximide (CHX) were purchased from Sigma Chemical Co. (St. Louis, MO). Polyclonal rabbit antimurine PGHS-2 antibody and NS-398 were purchased from Caymen Chemical Co. (Ann Arbor, MI). Other chemicals were obtained from Sigma.

Mouse calvarial culture
Seven-day-old CD-1 mice (Charles River Farms, Wilmington, MA) were killed following a protocol approved by the institutional animal care committee at the University of Connecticut Health Center. Half-calvariae were aseptically harvested, dissected free of suture tissue, and cultured in phenol red-free BGFb medium (Life Technologies, Grand Island, NY) containing 1 mg/ml BSA (Sigma) in 24-well dishes (Costar, Cambridge, MA) on a rocking platform in a humidified atmosphere of 5% CO2 at 37 C. Bones were cultured for 24 h before treatment with TGFß.

Primary osteoblastic cells
Whole calvariae were excised from 7-day-old CD-1 mice (Charles River Farms), rinsed in DMEM, and sequentially digested with 0.5 mg/ml crude collagenase P (Boehringer Mannheim, Indianapolis, IN) and 20% trypsin-EDTA. Five digests were performed, all for 10 min except the last one, which was for 30 min. Released cells were removed, the reaction was stopped with 10% FCS, and the solution was filtered through a Nitex membrane (Millipore Corp., Bedford, MA) to ensure a single cell suspension. Digests 4 and 5 were pooled and plated in six-well dishes (Costar, Cambridge, MA) at 20,000 cells/cm2 in the same medium used for MC3T3-E1 cells. Cells were grown until confluent (5 days) and then serum deprived for 24 h before treatment TGFß.

MC3T3-E1 cell culture
MC3T3-E1 cells were the gift of Dr. Yoshiyuki Hakeda (Meikai University School of Dentistry, Sakado, Saitama, Japan). Cells were plated in 6-well dishes (Costar) at a density of 5000 cells/cm2 and grown for 6 days in DMEM without phenol red (Sigma) containing 10% heat-inactivated FCS (Life Technologies, Grand Island, NY), penicillin (100 U/ml), and streptomycin (50 µg/ml) in a humidified atmosphere of 5% CO2 at 37 C. On the sixth day of culture, cells reached full confluence (~200,000 cells/cm2). At this time, cells were changed to serum-free medium with 0.1% BSA for 24 h before treatment with agonists and did not proliferate further. All treatment groups were controlled for test material vehicle, and the concentration of alcohol was 0.01% or less.

For long term continuous culture of MC3T3-E1 cells, cells were cultured in the same medium as that described above, and 50 µg/ml phosphoascorbate (Wako Pure Chemicals, Osaka, Japan) were added to the medium after the first week. On days 6, 13, and 20, a group of cultures was serum deprived in DMEM plus 0.1% BSA for 24 h and treated with TGFß (10 ng/ml) or vehicle for 4 h. RNA was extracted at the end of each week and saved for Northern analysis, which was performed on all samples at the same time.

DNA content
Cells grown in six-well dishes were washed with PBS, extracted with 10% trichloroacetic acid, and digested overnight with 0.5 M NaOH at 4 C. Aliquots were neutralized with 0.5 M acetic acid, and after the addition of diaminophenylindole (42), DNA content was determined fluorometrically (Fluorolite 1000, DynaTech Laboratories, Chantilly, VA). The DNA content was calculated as micrograms per well for six wells, and values are expressed as the mean ± SEM.

Stable transfection
The PGHS-2 promoter-luciferase fusion gene, containing 371 bp of the 5'-flanking sequence immediately proximal to the transcription start site and 70 bp of downstream untranslated DNA (-371/+70), and the series of shorter deletion constructs based on the -371/+70 construct have been described previously (39, 40). The promoterless luciferase vector was made by cutting the -371/+70 promoter-luciferase construct with BamHI and BglII, followed by ligation with T4 ligase (Life Technologies). Constructs were purified by CsCl banding and cotransfected with pSV2-neo into MC3T3-E1 cells, cultured as described above, to 50–80% confluence in six-well dishes. Cells in each well were rinsed twice with serum-free medium and incubated with 1 µg promoter-reporter DNA, 0.067 µg pSV2-neo DNA, and 8 µl Lipofectamine reagent (Life Technologies) in 1 ml serum-free medium without antibiotics. After 5 h of incubation, a second milliliter of medium with 20% FCS was added, and 19 h later, the medium was replaced with fresh complete medium. After 48 h, cells were split 1:10 into 100-mm dishes (Costar) and placed under selection with 400 µg/ml G418 for 2 weeks. Stable colonies (>200) were pooled to minimize effects secondary to variable integration sites. After selection, cells were grown in culture medium containing 200 µg/ml G418. To maintain uniform cell phenotype, the 371-bp construct and all shorter constructs were transfected at the same time in the same passage cells. Results were confirmed in two separate transfections.

Luciferase activity
Luciferase activity was measured in soluble cell extracts prepared with a kit from Promega (Madison, WI) using an automatic injection luminometer (Berthold Lumat, Wallac, Gaithersbury, MD). Activity was normalized to total proteins measured with a BCA protein assay kit (Pierce Chemical Co., Rockford, IL). For each experiment, three wells of cells were analyzed per treatment group. The 371-bp construct was included in all experiments with deletion constructs. All constructs were studied in three or more separate experiments.

Steady state RNA analysis
Three 35-mm wells of cells or six half-calvariae were pooled for RNA extraction by the method of Chomczynski and Sacchi (43). Briefly, cells or bones were homogenized in 4 M guanidinium thiocyanate, extracted with phenol/chloroform-isoamyl alcohol (24:1), RNA precipitated with isopropanol, and washed with 80% ethanol. After quantitation at 260 nm, 20 µg total RNA (20 µg/lane) were run on a 1% agarose-2.2 M formaldehyde gel, transferred to a nylon membrane (GeneScreen, New England Nuclear, Boston, MA) by positive pressure blotting (PosiBlotter, Stratagene, La Jolla, CA), and fixed to the membrane by UV irradiation (Stratolinker, Stratagene). After 3 h of prehybridization in a 50% formamide solution at 42 C, filters were hybridized overnight at 42 C in a similar solution with random primer [32P]deoxy-CTP (New England Nuclear, Wilmington, DE)-labeled cDNA probes. Filters were washed once in a 1 x SSC (standard saline citrate)-1% SDS solution at room temperature, once in 0.1 x SSC-0.1% SDS solution at 65 C, and then three more times in the latter solution at room temperature. After washing, the filter was exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -70 C. The signals were quantitated by densitometry (Bio-Rad Laboratories, Richmond, CA) or by analysis of scanned in images with Scan Analysis (Biosoft, Ferguson, MO). Filters were stripped with boiling 0.1% SDS-0.1 x SSC between hybridizations.

Western blot analysis
Cells were plated in 100-mm dishes (Costar) at a density of 5,000 cells/cm2, grown to confluence, and serum deprived for 24 h before treatment with TGFß. Cells were washed with PBS, harvested by centrifugation, and extracted with 0.5% Tween-20 in a 20 mM potassium phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonylfluoride, 1 mM EDTA, and 1 mM N-ethylmaleimide at 4 C for 30 min. This mixture was centrifuged at 14,000 x g for 30 min. The supernatant was dialyzed against N-ethylmaleimide without Tween-20 for 16 h, and an aliquot was mixed with diethylaminoethyl-cellulose (200 µl bed volume/mg protein) preequilibrated with the potassium phosphate buffer containing 0.05% Tween-20. Diethylaminoethyl-cellulose was precipitated by centrifugation, and protein in the supernatant was measured by the BCA protein assay kit (Pierce). Thirty micrograms of protein per treatment group were run on a SDS-polyacrylamide gel (10%) and transferred to a polyvinylidene difluoride membrane. Membranes were incubated with 1% nonfat dry milk at 4 C for 16 h to block nonspecific binding and then treated with a 1:2,000 dilution of polyclonal rabbit anti-PGHS-2 antiserum or nonimmune rabbit serum. Immunoreactive bands were stained using the Western exposure chemiluminescent detection kit (Clontech).

PGE2 assay
Medium was removed from cells or bones cultured as described above and used to measure PGE2 accumulation by RIA as described previously (44). In some instances, as indicated in the text, arachidonic acid (10 µM) was added during the last 10 min of culture to provide substrate for PG production. Data were expressed as the mean ± SEM.

Statistics
Means of groups were compared by ANOVA, and significance of differences was determined by post-hoc testing using Bonferroni’s method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of PGHS-2 mRNA by TGBß and IL-1 in MC3T3-E1 cells
We have previously shown that levels of PGHS-2 mRNA are low or undetectable in serum-free cultures of MC3T3-E1 cells, but can be induced by 2 h of incubation with TGFß (0.1–100 ng/ml) (26). Using 10 ng/ml TGFß, the dose that gave maximal effects in the previous study, we examined the time course for induction of PGHS-2 mRNA. Although a small increase in mRNA level was seen as early as 15–30 min after treatment with TGFß (Fig. 1Go), substantial induction of PGHS-2 mRNA occurred only after 2 h, with peak effects at 6–8 h (Fig. 2Go). In contrast, IL-1-induced PGHS-2 mRNA peaked much more rapidly (Fig. 1Go). The difference in time courses for PGHS-2 mRNA induction in response to IL-1 and TGFß suggest that these two ligands may induce PGHS-2 gene expression by differing mechanisms.



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  		Figure 1. Northern analysis of the induction of PGHS-2 mRNA at early time points and comparison of the effects of TGFß (10 ng/ml) and IL-1(10 ng/ml) in MC3T3-E1 cells. Total RNA was extracted and Northern analysis was performed as described in Materials and Methods. All lanes from both treatment groups were run on the same gel.

 


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  		Figure 2. A, Extended time course for the induction of PGHS-2 mRNA by TGFß (10 ng/ml) in MC3T3-E1 cells. RNA extraction and Northern analysis were performed as described in Materials and Methods. B, Comparison of PGHS-2 and PGHS-1 mRNA levels, normalized to GAPDH mRNA, determined by densitometry from the autoradiographic data in A. For PGHS-1 mRNA, the normalized values for TGFß-treated samples were divided by the normalized values for the control samples (TGFß-treated/control) at each time point.

 
In contrast to PGHS-2 gene expression, PGHS-1 mRNA is expressed in unstimulated MC3T3-E1 cells. PGHS-1 mRNA expression can be stimulated in these cells, although generally to a much lesser extent than PGHS-2 (26, 27, 29, 30). In a few experiments in MC3T3-E1 cells, TGFß stimulated PGHS-1 mRNA levels up to 5-fold at 4 h, with little change in GAPDH (Fig. 1Go). Generally, however, there was little induction of PGHS-1 mRNA levels (e.g. Fig. 2Go).

Induction of PGHS-2 protein and PGE2 production
Western blot analysis of MC3T3-E1 cells showed that TGFß-induced PGHS-2 protein was detected by 6 h (Fig. 3Go). TGFß also rapidly induced PGE2 production, beginning at about 3 h, with medium PGE2 levels peaking around 12 h (Fig. 4AGo). PGE2 production varied among experiments (compare Fig. 4Go with Table 1Go), but cumulative PGE2 in some experiments was 25–30 nM by 6 h. Although other agonists that we have studied, including PTH, basic fibroblast growth factor (bFGF), and IL-1, can induce high levels of PGHS-2 mRNA in MC3T3-E1 cells (25, 29, 31), TGFß is the only agonist that results in substantial PGE2 production in serum-free conditions. To give a better measure of enzyme activity, excess substrate for PGHS in the form of AA (10 µM) was added to cultures for the last 10 min (Fig. 4BGo). At 12 h, addition of AA increased PGE2 in control cultures from 0.3 ± 0.02 to 2.9 ± 0.3 nM, presumably reflecting the activity of PGHS-1. In the presence of AA, the increase in medium PGE2 with 10 relative to 1 ng/ml TGFß (Fig. 4BGo) was similar to the increase in peak densitometric intensity of PGHS-2 mRNA with 10 relative to 1 ng/ml TGFß measured in the same experiment (Fig. 4CGo).



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  		Figure 3. Induction of PGHS-2 protein by TGFß (10 ng/ml) in MC3T3-E1 cells. Protein extraction and Western analysis were performed as described in Materials and Methods.

 


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  		Figure 4. Comparison of the induction of PGE2 production and the induction of PGHS-2 mRNA by different concentrations of TGFß (1 and 10 ng/ml) in MC3T3-E1 cells. A, Cumulative medium PGE2 levels were measured by RIA. Each symbol represents the mean and SEM for three samples. B, Cultures were given exogenous arachidonic acid (10 µM) 10 min before medium was taken for RIA. Each symbol represents the mean and SEM for three samples. C, Induction of PGHS-2 mRNA, expressed as the densitometric intensity normalized to GAPDH mRNA, in the same experiment. Total RNA was extracted and Northern analysis was performed as described in Materials and Methods.

 

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  		Table 1. Inhibition of TGFß (10 ng/ml)-stimulated PGE2 production by the nonsteroidal antiinflammatory drug, NS-398 (0.1 µM), at 6 h in MC3T3-E1 cells

 
To assess the contribution of cellular proliferation to medium PGE2 levels, DNA content was measured. Although cellular proliferation was expected to be low because cells were serum deprived for 24 h and then treated with serum-free medium, TGFß might have potent mitogenic effects. However, the DNA content in cultures treated for 24 h with TGFß (10 ng/ml), 58.9 ± 1.8 µg, was not significantly different from that in control cultures, 53.1 ± 2.5 µg.

NS-398, a nonsteroidal antiinflammatory drug that is a selective inhibitor of PGHS-2 (25, 45, 46), completely blocked TGFß-induced PGE2 production (Table 1Go). The abrogation of PGE2 production by NS-398 (0.1 µM) in these cells is consistent with induced PGHS-2 being the major source of TGFß-induced PGE2 production under these conditions.

Effect of CHX on TGFß-induced PGHS-2 expression
Induction of PGHS-2 mRNA expression in MC3T3-E1 cells by TGFß peaked later than induction by IL-1 (10 ng/ml; Fig. 1Go). The time course for IL-1 effects is similar to that for other PGHS-2 agonists in these cells, including serum (26), bFGF (31), PTH (33), and TGF{alpha} (our unpublished data). In all of these cases, CHX substantially enhances the ligand-induced accumulation of PGHS-2 mRNA. An example of this enhancement is shown in Fig. 5Go, where cells were treated with PMA (1 µM) and CHX (10 µg/ml). PGHS-2 mRNA induction was detected at 1.5 h in PMA-treated cells, then disappeared. In the presence of CHX, however, there was dramatic superinduction of PMA-stimulated PGHS-2 mRNA accumulation. In contrast to the marked CHX enhancement of the PMA effects, the peak PGHS-2 mRNA response to CHX plus TGFß did not differ from the response to CHX alone. Because CHX alone induces PGHS-2 mRNA, strict interpretation of combined effects is not possible, but comparison with CHX effects on other agonists suggests that new protein synthesis might be required for the maximal effect of TGFß on PGHS-2 mRNA accumulation in MC3T3-E1 cells.



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  		Figure 5. Effects of CHX (10 µg/ml) on the induction of PGHS-2 mRNA by TGFß (10 ng/ml) and PMA (1 µM) in MC3T3-E1 cells. CHX was added 30 min before other treatments. Two different autoradiograph exposures for PGHS-2 mRNA are shown to permit better comparison of band intensity over the time course. Total RNA was extracted and Northern analysis was performed as described in Materials and Methods.

 
Stimulation of PGHS-2 promoter activity
To study transcriptional regulation of the PGHS-2 promoter regions, MC3T3-E1 cells were stably transfected with a chimeric reporter construct in which 371 bp of the 5'-flanking region of the PGHS-2 gene immediately proximal to the PGHS-2 transcription start site plus 70 bp of the downstream untranslated DNA are fused to a luciferase reporter gene. The TGFß induction of luciferase mRNA expression peaked at 5 h. Stimulation of luciferase activity by TGFß is compared with stimulation by other agonists in Fig. 6Go. TGFß increased luciferase activity by 6-fold at about 6 h. Consistent with the differences seen on Northern analysis, the peak induction of luciferase activity in response to TGFß occurred later than the peak induction of the other agonists. In addition, there was a small shoulder in the TGFß-stimulated curve for luciferase activity at about 3 h, suggesting that two separate TGFß-mediated effects may be involved in the induction of PGHS-2 gene expression.



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  		Figure 6. Comparison of the stimulation by other agonists with the stimulation by TGFß (10 ng/ml) of luciferase activity in MC3T3-E1 cells stably transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter. All results are from the same experiment. Treatments were IL-1 (10 ng/ml), PMA (1 µM), FSK (10 µM), and TGF{alpha} (30 ng/ml). Luciferase activity, expressed as counts per sec (cps) and normalized to protein, was measured as described in Materials and Methods. Values are normalized to the the control value at time zero. Each symbol represents the mean and SEM of three samples.

 
Autoamplification by PGE2
PGs can induce PGHS-2 mRNA expression in these cells (29). Because TGFß induces PGE2 production, it is possible that PGE2 produced in response to TGFß stimulates, at later times, PGHS-2 expression. To evaluate this possibility, cells were treated with TGFß (10 ng/ml) in the presence and absence of NS-398 (0.1 µM) to block PGE2 production. Peak TGFß-stimulated luciferase activity at 6 h was decreased about 25% by NS-398, but the overall pattern, suggestive of two separate inductive effects, was unchanged (Fig. 7Go).



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  		Figure 7. Effects of NS-398 (0.1 µM) on TGFß (10 ng/ml)-stimulated luciferase activity in MC3T3-E1 cells stably transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter. Luciferase activity, expressed as counts per sec (cps) and normalized to protein, was determined as described in Materials and Methods. Each symbol represents the mean and SEM of three samples.

 
TGFß enhances PGHS-2 mRNA accumulation by other inducers
TGFß (1 ng/ml) enhanced the induction of PGHS-2 mRNA expression at 2 h in MC3T3-E1 cells by TGF{alpha} (30 ng/ml), FSK (10 µM), and PMA (1 µM; Fig. 8Go) and by IL-1 (10 ng/ml) (data not shown). The time course for stimulation of luciferase activity by the combination of TGFß (1 ng/ml) and TGF{alpha} (30 ng/ml) is shown in Fig. 9Go. Peak luciferase activity in response to the combination occurred at 2–3 h, the time of maximum luciferase activity in cultures treated with TGF{alpha} alone (Fig. 6Go). A similar pattern was found for TGFß enhancement of IL-1 stimulation of luciferase activity (data not shown). Luciferase activities at 3 h obtained for various combinations of agonists with TGFß (1 ng/ml) are compared in Fig. 10Go. In all cases, the activity of the combination was more than the sum of individual activities.



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  		Figure 8. Enhancement by TGFß (1 ng/ml) of the induction of PGHS-2 mRNA by various agonists in MC3T3-E1 cells. The agonists used were PMA (1 µM), FSK (10 µM), and TGF{alpha} (30 ng/ml). Cells were treated for 2 h. Total RNA was extracted and Northern analysis was performed as described in Materials and Methods.

 


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  		Figure 9. Time course for the enhancement by TGFß (1 ng/ml) of TGF{alpha} (30 ng/ml)-stimulated luciferase activity in MC3T3-E1 cells stably transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter. Luciferase activity, expressed as counts per sec (cps) and normalized to protein, was determined as described in Materials and Methods. Each symbol represents the mean and SEM of three samples.

 


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  		Figure 10. Enhancement by TGFß (1 ng/ml) of luciferase activity stimulated by various agonists in MC3T3-E1 cells stably transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter. Treatments were vehicle [control (CON)], IL-1 (10 ng/ml), TGF{alpha} (30 ng/ml), PMA (1 µM), and FSK (10 µM). Cells were treated for 3 h. Luciferase activity, expressed as counts per sec (cps) and normalized to protein, was determined as described in Materials and Methods. Values are normalized to the untreated control value. Each bar represents the mean and SEM of three samples.

 
Mapping of the 5'-flanking region of the PGHS-2 gene for TGFß response elements
To determine regions within the 371-bp 5'-flanking region mediating the response to TGFß, MC3T3-E1 cells were stably transfected with a series of 5'-deletion constructs. The TGFß (1 ng/ml)-stimulated increase in luciferase activity measured at 4.5 h for each construct, expressed as fold induction for that construct, is shown in Fig. 11Go. Each deletion construct was studied in one to four experiments (each with n = 3), and all experiments included the -371/+70 bp construct. Similar results were obtained in an independently transfected set of cells. The mean induction in cells carrying the -371/+70 bp promoter-luciferase construct was 5-fold. Stimulation was not seen in cells carrying only the promoterless vector (Luc). There was a 30% reduction in TGFß-stimulated luciferase activity with deletion to -300/+70 bp and a 90% reduction with deletion to -213/+70 bp. In contrast, for other agonists, such as IL-1 (47), serum (data not shown), and TGF{alpha} (data not shown), a major portion of the luciferase response was maintained until the -150/-41 bp region was deleted.



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  		Figure 11. Effect of sequential deletion of the -371/+70 PGHS-2 DNA on the ability of TGFß (1 ng/ml) to stimulate luciferase activity in MC3T3-E1 cells stably transfected with these deletions fused to a luciferase reporter. Fold induction was calculated relative to the corresponding control group for each construct, and controls were normalized to 1 for all groups. Cells were treated for 4.5 h. The vector that contains no PGHS-2 DNA is designated Luc. Luciferase activity, normalized to protein, was determined as described in Materials and Methods. Each bar represents the mean and SEM of 3–12 samples.

 
Osteoblastic cell phenotype and TGBß induction of PGHS-2 mRNA
MC3T3-E1 cells are immortalized osteoblastic cells derived from neonatal mouse calvariae. The process of immortalization may have altered their responses to exogenous ligands. However, we found that agonists that induce PGHS-2 expression in MC3T3-E1 cells, including bFGF, PTH, PGE2, and IL-1, also induce PGHS-2 in neonatal mouse calvarial cultures (28, 30, 32, 33). Similarly, in this study, TGFß (10 ng/ml) treatment induced PGHS-2 mRNA accumulation in cultured neonatal calvariae (Fig. 12AGo). TGFß had little effect on PGHS-1 mRNA; the ratio of treated to control PGHS-1 mRNA normalized to GAPDH was 1.2 at 8 h (data not shown). TGFß also increased medium PGE2, from both endogenous substrate and exogenous arachidonic acid (Fig. 12BGo).



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  		Figure 12. Induction of PGHS mRNA and PGE2 production by TGFß in cultured neonatal mouse calvariae. Calvariae were cultured for 24 h, then treated with TGFß (10 ng/ml) for 8 h. A, Total RNA was extracted and Northern analysis was performed as described in Materials and Methods. B, Medium PGE2 levels were measured by RIA. Values are expressed as the mean ± SEM for six samples. - AA, Bars represent cumulative medium PGE2 produced from endogenous substrate. + AA, After treatment with TGFß, calvariae were cultured for 10 min in fresh medium with AA (10 µM) to assess enzyme activity. **, P < 0.01; *, P < 0.05 (significantly different from the control).

 
As confirmation that the effect is demonstrated by osteoblastic cells, we examined the osteoblastic-enriched population (pooled populations 4 and 5) of primary cells obtained by sequential enzymatic digestion of neonatal mouse calvariae. TGFß (10 ng/ml) induced PGHS-2 mRNA expression, but did not stimulate PGHS-1 mRNA expression (Fig. 13AGo). TGFß also increased medium PGE2 accumulation (Fig. 13BGo). DNA content did not differ significantly between control and TGFß-treated cultures (18.4 ± 1.2 and 20.1 ± 3.3 µg, respectively) at the end of the experiment.



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  		Figure 13. Induction of PGHS-2 mRNA and PGE2 production by TGFß in cultured murine primary osteoblastic cells. Cells were sequentially digested from neonatal mouse calvariae, as described in Materials and Methods. Populations 4 and 5 were pooled, cultured to confluence, and serum deprived for 24 h before treatment with TGFß (10 ng/ml) for 7 h. A, Total RNA was extracted and Northern analysis was performed as described in Materials and Methods. B, Medium PGE2 levels were measured by RIA, and values are expressed as the mean ± SEM (n = 6). **, P < 0.01 (significantly different from the control).

 
MC3T3-E1 cells can vary greatly in phenotype, depending on culture conditions and passage number, from proliferative preosteoblasts to mature bone nodule-forming osteoblasts (37, 48). To examine the effects of MC3T3-E1 phenotype on the PGHS-2 response to TGFß, we cultured cells continuously for 3 weeks, assessing the PGHS-2 mRNA response to TGFß and the mRNA expression of type I collagen and osteocalcin at the end of each week (Fig. 14Go). Type I collagen mRNA was highly expressed in cells harvested at the end of all 3 weeks. Osteocalcin, a marker of more differentiated osteoblasts (49), was expressed only in those MC3T3-E1 cultures harvested after 2 and 3 weeks. Cells harvested at the end of 1 week, which did not express osteocalcin mRNA, mimicked the conditions used in our other studies. The TGFß induction of PGHS-2 mRNA was seen in all 3 weeks, although the induction was decreased at the end of week 3. Hence, these data suggest that TGFß can induce PGHS-2 gene expression in both the early, or proliferative, stages and the more differentiated stages of MC3T3-E1 cells in culture.



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  		Figure 14. Induction of PGHS-2 mRNA in MC3T3-E1 cells cultured continuously for 1–3 weeks; comparison with markers of osteoblastic differentiation, osteocalcin (OC) and type I collagen (COL1A1). At the end of each week, a group of cells was serum deprived for 24 h, then treated with TGFß (10 ng/ml) for 4 h. Total RNA was extracted and stored until Northern analysis could be performed for all samples simultaneously, as described in Materials and Methods. All samples were electrophoresed on the same gel.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Unlike previous studies with other cell types, in which TGFß alone had no effect on PGHS-2 expression (34, 35), we found that TGFß can induce PGHS-2 mRNA and PGE2 production in neonatal mouse calvarial cultures, primary murine osteoblastic cells, and MC3T3-E1 cells. In MC3T3-E1 cells, the time course for induction was delayed relative to that of other agonists for which the PGHS-2 response is characteristic of a primary response gene (26, 31, 33). Maximal induction of mRNA with TGFß (10 ng/ml) occurred at 6–8 h, and mRNA levels remained elevated at 24 h. The delay in response to TGFß may be due to a requirement for new protein synthesis for maximal PGHS-2 response, as suggested by the effects of CHX.

PGHS-2 mRNA expression and luciferase enzyme activity in MC3T3-E1 cells transfected with a PGHS-2 promoter-luciferase reporter gene were increased similarly by TGFß treatment, indicating that the PGHS-2 response to TGFß is at least in part transcriptional. The time course for luciferase stimulation demonstrated a shoulder, suggesting that the overall response may be a combination of two sequential effects. The TGFß-induced increase in luciferase expression was reduced 90% as a result of deletion of the region between -371 to -213 bp of the PGHS-2 promoter, suggesting the presence of cis-acting regulatory sequences in this region.

The very low levels of measurable PGE2 production in unstimulated MC3T3-E1 cells despite the expression of PGHS-1 mRNA are consistent with previous studies that suggested that PGHS-1 cannot use endogenous arachidonic acid (24, 25). TGFß stimulated PGE2 production in MC3T3-E1 cells from both endogenous and exogenous arachidonic acid, and TGFß-induced PG amplified the TGFß-induction of PGHS-2. TGFß-stimulated PGE2 production correlated with TGFß-induced PGHS-2 mRNA and protein expression. In addition, the bulk of TGFß-induced PGE2 production was blocked by the PGHS-2-selective NSAID, NS-398. These data are consistent with those from other studies in MC3T3-E1 cells and many other cells, suggesting that PGHS-2 is the major mediator of cytokine, growth factor, and hormone regulation of PG production (26, 27, 31, 33).

Deletion mapping of the murine PGHS-2 promoter indicates that a major response region for TGFß lies between -371 and -214 bp. A TGFß-responsive region has also been recently reported between -454 and -288 bp in the 5'-flanking region of the human PGHS-2 gene (50). Within the -371/-214 bp region in the murine promoter is a consensus Sp-1 site sequence at -239/-234 bp (5'-AGGGCGG-3') and a possible AP-1 site (3'-TGAGTCT-5') at -277/-272 bp. Both types of sites have been implicated in TGFß responsiveness in other studies. For example, TGFß increases type I collagen gene expression in liver Ito cells in part through an AP-1 site (51), and the autoinduction of TGFß is mediated via an AP-1 complex in a human adenocarcinoma cell line (52). TGFß stimulation of {alpha}2(I) collagen gene expression in human fetal fibroblasts is mediated through an element containing an Sp-1-binding site (53). A novel TGFß response element (5'-CACAGGCCAGAC-3') has been identified in the promoter for the germ-line Iga constant region gene (54). Sequences at -290/-284 bp (5'-GCCAGAC-3') and -254/-259 bp (5'-CACAGG-3') in the PGHS-2 promoter are identical to regions of this response element. Site-directed mutagenesis will be required to determine, which, if any, of these sequences modulates PGHS-2 gene expression in MC3T3-E1 cells in response to TGFß.

The pleiotropic effects of TGFß are probably due in part to its interactions with other factors, and understanding the physiologic actions of this ligand will probably require an understanding of these interactions (4). In some cases, TGFß alone has no measurable effect (34, 35), and the major action of TGFß is to modulate the effects of another factor. TGFß has both positive and negative interactions with growth factors, such as TGF{alpha} and epidermal growth factor (55, 56), and with hormones, such as 1,25-dihydroxyvitamin D (57). In addition to its direct actions, TGFß mediates biological responses indirectly, by regulating the production of other growth factors (58). In the current study, TGFß potentiated the induction of PGHS-2 mRNA accumulation and the stimulation of PGHS-2 promoter activity in MC3T3-E1 cells by agonists that act via a wide variety of pathways, e.g. IL-1, TGF{alpha}, FSK, and PMA. The ability of TGFß to enhance the induction of PGHS-2 mRNA by other agonists suggests that TGFß may act on the PGHS-2 promoter at one or more sites that are independent from sites required by these other agonists. Our preliminary studies on luciferase activity in MC3T3-E1 cells, transfected with the same series of deletion constructs as that used in this study and stimulated by IL-1 (47) and by FSK, serum, and TGF{alpha} (unpublished data), suggest that major response elements for these other agonists lie between -150 and -40 bp. In contrast, the enhancement of luciferase expression from the PGHS-2 promoter-luciferase reporter by TGFß requires the region between -371 and -213 bp. TGFß can regulate both receptor binding (59) and signaling pathways (60). However, the variety of signaling pathways represented by the agents studied here as well as the requirement for a distal region of the PGHS-2 promoter for the response to TGFß suggest that the mechanism for TGFß enhancement of PGHS-2 gene expression in MC3T3-E1 cells may involve TGFß-mediated interaction of trans-acting factors.

TGFß is an important regulator of bone turnover. It seems likely that TGFß-stimulated PG production mediates some effects of TGFß on bone, as reported for TGFß-stimulated resorption in mouse calvariae (19). It is also possible that some of the differences in the effects of TGFß on osteoblastic cells, observed as a function of varying TGFß concentrations (8) or as a function of the presence or absence of serum (11), might be secondary to differences in TGFß-stimulated PG production. PGs themselves have biphasic effects on bone formation and resorption (61), and hence, TGFß-induced PGs might explain some of the biphasic effects of TGFß (11, 12, 13, 14). A role for TGFß-stimulated PG would seem most likely at high concentrations of TGFß. For example, the unexpected finding of bone loss in mice overexpressing TGFß2 in bone (17) might be due to stimulation of PG production. However, because TGFß can enhance the induction of PGHS-2 expression by multiple other agonists, even low concentrations of TGFß, in the presence of such agonists, might induce physiologically significant PG levels.


   Footnotes
 
1 This work was supported by NIH Awards AR-18063 and AR-38933 (to L.G.R.), DK-48361 (to C.P.), and GM-24797 (to H.H.). Back

Received January 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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