SEARCH

SEARCH BY CITATION

Keywords:

  • c-fos;
  • junB;
  • protein kinase;
  • type I collagen;
  • parathyroid hormone/parathyroid hormone-related peptide receptor

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

In osteoblastic cells, transforming growth factor β1 (TGF-β1) has been found to regulate the expression of a variety of proto-oncogenes including c-fos, c-jun, and junB. The c-fos in particular has been implicated in the mitogenic effect of TGF-β1. Here, we examined the role of these early response genes in the regulation of osteoblast (OB) gene expression by two members of the TGF-β superfamily, TGF-β1 and bone morphogenetic protein 2 (BMP-2). In ROS 17/2.8 cells, TGF-β1 as well as BMP-2 up-regulated the expression of junB and c-fos messenger RNAs (mRNAs), and this increase was correlated in both cases with an increase in activator protein 1 (AP-1) DNA-binding activity involving JunB and c-Fos proteins. Protein kinase C (PKC)- and protein tyrosine kinase (PTK)-dependent pathways have been implicated in both TGF-β1 signaling and AP-1 gene regulation. Therefore, using the kinase inhibitors chelerythrine chloride and genistein, we showed that PKC and PTK activities, respectively, participated in TGF-β1- and BMP-2-induced increases in junB mRNA levels. Similarly, these kinase activities were involved in the stimulatory effect of BMP-2 on c-fos mRNA expression. Using a natural dominant negative for AP-1 transcriptional activity in ROS 17/2.8 cells, we then showed that AP-1 transcription factors mediated TGF-β1- and BMP-2-regulated expression of the (α1) collagen I gene as well as TGF-β1-regulated expression of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor. Our data emphasize the role of the AP-1 transcription factor in TGF-β1 and BMP-2 signaling and highlight the importance of this transcription factor family in the expression of OB genes.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

THE OSTEOGENIC FACTORS transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) play a critical role in bone physiology. They have been shown to modulate osteoblast (OB) proliferation and differentiation in vitro.(1) TGF-β1 has been reported to regulate expression of specific genes such as, osteocalcin,(2) osteopontin,(3) osteonectin, type I collagen, alkaline phosphatase,(4,5) and the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor.(6) Although the effects of BMP-2 on OB cells have been investigated less intensively, BMP-2 also has been found to regulate alkaline phosphatase expression and collagen synthesis, as well as PTH-dependent cyclic adenosine monophosphate (cAMP) production.(7,8) However, little is known concerning the molecular mechanisms involved in the regulation of these genes by either TGF-β1 or BMP-2.

TGF-β family members signal through transmembrane serine/threonine kinase receptors named “type I” and type II.”(9) The two types of receptors cooperate to transduce the signal to the intracellular compartment. Although this mechanism has been well described, the signaling events that lead downstream to the nucleus only recently have been clarified. The identification of a conserved family of proteins, now referred as “smad,” has been implicated in the TGF-β super family signaling pathway.(10) The smad proteins become phosphorylated in response to TGF-β1 or BMP-2 and have been shown to participate in DNA-binding complexes.(11) In addition to the smad pathway, the protein kinase C (PKC)- and mitogen-activated protein kinase (MAPK)-dependent pathways (12–16) also have been reported to participate in TGF-β1 signaling. One nuclear target of these transducing cascades is the transcription factor activator protein 1 (AP-1).(17) Among the different transcription factors that have been involved in the action of TGF-β1 on gene expression, AP-1 (like Fast-1(18) seems to play a particular role because it has been shown to cooperate with the smad proteins to mediate TGF-β-induced transcription.(19)

AP-1 complexes are composed of members of the Jun and Fos families and play a widespread role in the transduction of signals from the membrane to the nucleus. In response to a variety of extracellular stimuli, AP-1 complexes activate transcription of various target genes through their interaction with a consensus DNA sequence (TGAC/GTCA) called the AP-1 site. These target genes are involved in diverse cell responses including proliferation and differentiation.

AP-1 activities have been implicated in TGF-β1 responses in a variety of nonosteoblastic cells. Thus, the transcription of genes such as plasminogen activator inhibitor 1 (PAI-1(20)), α2(I) collagen,(21) collagenase-3,(22) clusterin,(23) interleukin-11,(24) and murine laminin α3A,(25) is modulated by TGF-β1 through AP-1 transcription factors. In OB cells, TGF-β1 also is known to regulate expression of members of the AP-1 family of transcription factors (26–29) and BMP-2 has been reported to induce c-fos in OB MC3T3-E1 cells.(30) Furthermore, in this cell type, AP-1 complexes participate in the regulation by TGF-β1 of osteocalcin,(31) retinoic acid receptor, retinoid X receptor,(32) and monocyte chemoattractant JE/monocyte chemoattractant protein 1 (JE/MCP1).(33) In this work, we examined the role of AP-1 family members in BMP-2- as well as in TGF-β1-induced gene expression in ROS 17/2.8 OB cells. We showed that both TGF-β1 and BMP-2 up-regulated messenger RNA (mRNA) levels encoding c-fos and junB, which correlated with an increase in AP-1 DNA-binding activity. In parallel, we examined the involvement of PKC- and PTK-dependent signaling pathways in these actions and found that these kinase activities may mediate the effects of TGF-β1 and BMP-2 on the mRNA expression of AP-1 family members. Finally, using a natural dominant negative for AP-1 transcriptional activity, we indicated the role of AP-1 transcription factors in TGF-β1- and BMP-2-regulated expression of the (α1) collagen I gene as well as in TGF-β1-regulated expression of the PTH/PTHrP receptor gene.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Reagents

Human recombinant TGF-β1 was purchased from Life Technologies, Inc. (Gaithersburg, MD, U.S.A.). Human recombinant BMP-2 was kindly provided by Genetics Institute (Cambridge, MA, U.S.A.). Chelerythrine chloride and genistein 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) were purchased from Sigma (St. Louis, MO, U.S.A.).

Cell culture

Rat osteosarcoma ROS 17/2.8 cells(34) were cultured in Dulbecco's modified Eagle medium (DMEM)-F12 medium (Life Technologies, Inc.) supplemented with 10 mM HEPES and 10% fetal bovine serum (FBS; WISENT, Inc., St. Bruno, QC, Canada) in a humidified incubator, in 5% CO2, at 37°C. The cells were subcultured routinely every 3 days. For all experiments, cells were seeded at 15 × 103 cells/cm2 in 60-mm culture plates in medium containing 5% FBS and grown for 48 h. After removal of the culture medium, the cell layer was rinsed once with serum-free medium and fed with fresh medium containing 2% FBS, with the indicated reagent(s) or the corresponding vehicle.

Northern blot analysis

Total RNA was isolated using the TRIZOL reagent (according to the protocol of the manufacturer, Life Technologies, Inc.), fractionated on 1% agarose-formaldehyde gel (10 μg/lane), and transferred to a nylon membrane (Hybond N+; Amersham, Oakville, ON, Canada). Filters were prehybridized for 2 h at 65°C in hybridization buffer (HB; 0.3 M Na2HPO4, 0.2 M NaH2PO4, 7% sodium dodecyl sulfate [SDS], 1 mM EDTA, 1% bovine serum albumin [BSA], pH 7.0) and then hybridized overnight in the same buffer containing 1 × 106 cpm/ml of specific complementary DNA (cDNA). The junB(35) and c-jun(36) cDNAs were purchased from the American Type Culture Collection (ATTC; Rockville, MD, U.S.A.). The fos probe was a Bgl II/Sal I fragment of v-fos.(37) Rat type I collagen (α1R1) cDNA(38) was a generous gift of Dr. D. Rowe. PTH/PTHrP receptor cDNA(39) was kindly provided by Dr. John White. Filters were washed twice for 15 minutes in 2× SSC and 0.5% SDS at 65°C and then autoradiographed (X-OMAT AR film; Kodak, Montreal, PQ, Canada). As a standard for loading and normalization, filters were probed in parallel with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.(40) All signals were quantified by scanning densitometry and the final value was obtained by calculating the c-fos (or junB)/GAPDH ratio value. When mentioned or presented, the fold of stimulation was calculated as an increase over the respective control sample.

Statistics

The Student's t-test or analysis of variance (ANOVA) was used to determine statistical significance. The asterisk indicates that the value is statistically different from the value of the respective control (p < 0.05;p < 0.01;p < 0.001).

AP-1 electromobility shift assay

Preparation of nuclear extracts:

Cells (2 × 107) were washed twice with phosphate-buffered saline (PBS) containing 1 mM EDTA, scraped, pelleted, and resuspended in 400 μl of buffer A [10 mM Tris-HCl, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM ethylene glycol-bis(β-amino ethyl ether)-N,N,N′,N′-tetracetic acid (EGTA), 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] containing a protease inhibitor cocktail (for the inhibition of serine, cysteine and metalloproteases, Complete; Mini-Roche Diagnostics, Laval, PQ, Canada). After 15 minutes incubation on ice, 25 μl of 10% NP-40 were added and the cell suspension was mixed vigorously and centrifuged for 30 s at 18,300g. The resulting pellet was then resuspended in 50 μl of cold buffer C (20 mM Tris-HCl pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM PMSF) containing a protease inhibitor cocktail (Complete TM, Mini-Roche Diagnostics) and vigorously shaken for 1 h at 4°C for high salt extraction. Cellular debris was removed by centrifugation for 15 minutes at 4°C, and the supernatant fraction was stored at −70°C. Protein content was determined using Bradford protein assay (Bio Rad, Mississauga, ON, Canada).

Binding reaction:

Protein/DNA-binding reactions were performed in a final volume of 20 μl of binding buffer (10 mM Tris, pH 7.4, 100 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM EDTA, and 1 mM dithiothreitol) by combining X μg of nuclear protein extract and 1 μg of polydeoxyinosinic-deoxycytidylic acid [poly(dI-dC)] containing 1 × 105 cpm of [3H]-labeled probe for 20 minutes at room temperature. When indicated, 2 μl of JunB (or c-Fos) antibody (Santa-Cruz Biotechnology, Santa Cruz, CA, U.S.A.) were added to the binding mixture.

Nondenaturing polyacrylamide gel electrophoresis:

DNA-protein complexes were resolved by electrophoresis on 5% nondenaturing polyacrylamide gels in 0.5× TBE (0.089 M Tris/borate, 0.089 boric acid, and 0.002 M EDTA). After electrophoresis, gels were dried and exposed to XAR-5 film (Kodak) at −70°C. The AP-1 consensus oligonucleotide “TGACTCA” (Santa-Cruz Biotechnology) was radiolabeled with [γ32P] adenosine triphosphate (ATP) using polynucleotide kinase and used as a probe.

Cell transfections

The ΔFosB cDNA(41) (purchased from ATCC) was excised from the construct ΔFosB3s as an Xho I/Not I fragment and subcloned in the LacI repressible vector pOPRSVI (Stratagene, La Jolla, CA, U.S.A.). ΔFosB (pOPRSVI-ΔFosB) and Lac repressor (pCMVLacI) expression plasmids were transfected simultaneously into ROS 17/2.8 cells using the lipofectin reagent (Life Technologies). Stable clones were selected in culture medium containing 500 μg/ml G418 and 200 μg/ml hygromycin. Resistant clones were first screened by Northern blot for constitutive Lac repressor expression and inducible expression of ΔFosB on isopropyl-1-thio-β-D-galactoside (IPTG) stimulation. Three clones out of seven were positive for both ΔFosB and Lac repressor mRNA, and two of them (called ROPF3 and ROPF7) were used for further experiments. Incubation for 24-48 h in culture medium containing 5 mM IPTG was used to induce ΔFosB expression. ΔFosB protein expression was analyzed by immunoblotting (see the following section). During the incubation, IPTG (5 mM) was added every 24 h to maintain the stable expression of ΔFosB.

Immunoprecipitation and immunoblotting

ΔFosB-transfected cells (ROPF3 and ROPF7) were grown for 24 h and then stimulated or not stimulated (control) with IPTG (5 mM). After 48 h, cells were washed twice in ice-cold PBS, scraped off plates in cell lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 10% Glycerol, 1 mM PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstatin), and then sonicated for 20 s. Lysates were cleared by centrifugation at 12,000g for 15 minutes, and protein concentrations were determined using Micro BCA protein assay (Pierce, Rockford, IL, U.S.A.). Equal amounts of protein were rotated overnight at 4°C with 1 μg of anti-FosB (Santa-Cruz Biotechnology). After binding to protein A-Sepharose (Pharmacia Biotechnology, Uppsalla, Sweden), the immune complexes were washed three times with cell lysis before being resuspended in 25 μl of 2× SDS sample buffer (15% glycerol, 125 mM Tris/HCl, pH 6.8, 5 mM EDTA, 2% SDS, 0.1% bromophenol blue, and 1% 2-mercaptoethanol). Samples were resolved on 15% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane using a Bio-Rad Transblot apparatus (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline 0.05% Tween 20 (TBS-T) for 30 minutes and incubated for 2 h at room temperature with anti-FosB antibody (400 ng/ml; Santa Cruz Biotechnology) in TBS-T containing 5% nonfat dry milk. After washing with TBS-T (6× 5 minutes), the membrane was incubated for 45 minutes with horseradish peroxidase-conjugated secondary antibodies in TBS-T containing 5% nonfat dry milk. The membrane was washed (4× 5 minutes in TBS-T, 2× 5 minutes in TBS) before visualizing the immunoreactive bands by enhanced chemiluminescence (ECL; Amersham).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Effect of TGF-β1 and BMP-2 on c-fos and junB mRNA levels

We first examined the effects of TGF-β1 and BMP-2 on mRNA expression of members of the AP-1 family. Cells were incubated in the presence of vehicle (2% serum, as control), TGF-β1 (5 ng/ml), or BMP-2 (50 ng/ml) for 30-120 minutes. The experimental conditions were chosen according to our previous study.(42) Total RNA was isolated at the indicated times and subjected to Northern blot analysis for c-fos, junB (Fig. 1), and c-jun (data not shown) expression. As an indication of the basal level of expression of each gene, the time zero is shown (Fig. 1). Before treatment, weak constitutive expression of junB mRNA was observed whereas c-fos mRNA expression was not detectable. In control cells, addition of 2% serum (vehicle) resulted in a slight increase of junB mRNA and an induction of c-fos mRNA. The serum stimulatory effect on junB and c-fos mRNA expression was maximal after 30 minutes and returned to basal level after 90 minutes. In comparison, a greater increase of junB mRNA levels was observed with TGF-β1. This effect was at a maximum after 60 minutes of treatment and lasted for at least 120 minutes. In addition, TGF-β1 increased the expression level of c-fos mRNA but the effect was more transient. This effect reached maximum stimulation at 45 minutes of incubation, and after 120 minutes, the c-fos mRNA was no longer detectable. BMP-2 also increased junB and c-fos mRNA expression. BMP-2 stimulated junB mRNA expression with a slightly different time course than TGF-β1 because the effect was already observed after 30 minutes of incubation. Furthermore, the maximum stimulation was greater than with TGF-β1 and the stimulatory effect was still considerable after 120 minutes of treatment. The time courses of TGF-β1- and BMP-2-induced increases in c-fos mRNA levels were comparable. However, BMP-2 caused a greater increase in c-fos mRNA levels than did TGF-β1. In contrast, c-jun mRNA expression was not affected by either TGF-β1 or BMP-2 treatment in this cell line (data not shown).

thumbnail image

Figure FIG. 1. Time course of TGF-β1 and BMP-2 effects on c-fos and junB mRNA levels. Cells were treated with vehicle (control, C), TGF-β1 (T, 5 ng/ml), or BMP-2 (B, 50 ng/ml). At the indicated time, total RNA was isolated and subjected to Northern blot analysis, as described in the Materials and Methods section. (A) A representative autoradiogram is shown. (B) The c-fos (or junB)/GAPDH mRNA ratio (average ± SD) from independent experiments (n = 3) is summarized in histograms. The asterisks indicate that the value is statistically different from the value of the respective control (*p < 0.05; **p < 0.01; ***p < 0.001).

Download figure to PowerPoint

Involvement of protein kinase activities in TGF-β1- and BMP-2-induced increases in c-fos and junB mRNA expression

In our previous study, we showed that PKC and protein tyrosine kinase (PTK) activities participated in TGF-β1 and BMP-2 up-regulation of (α1) collagen I gene expression in ROS 17/2.8 cells.(42) Moreover, these protein kinase activities have been shown to participate in the regulation of AP-1 gene transcription.(17) We therefore tested whether the increase of c-fos and junB gene expression elicited by either TGF-β1 or BMP-2 was dependent on PKC and PTK activities (Fig. 2). Cells were treated with TGF-β1 (5 ng/ml) or BMP-2 (50 ng/ml) in the presence of either vehicle (dimethylsulfoxide [DMSO]), chelerythrine chloride (5 μM, a specific PKC inhibitor), or genistein (15 μg/ml, a tyrosine kinase inhibitor). The effect of TGF-β1 on increasing junB mRNA levels was blocked by the presence of either genistein or chelerythrine chloride. However, neither inhibitor altered the effect of TGF-β1 on c-fos mRNA expression. In contrast, both genistein and chelerythrine chloride markedly reduced the BMP-2 stimulatory effects on both c-fos (approximately 70% and 50%, respectively) and junB (approximately 90% and 50%, respectively) mRNA expression. Taken together, these data indicate that stimulation of junB or c-fos mRNA by BMP-2 involves PTK- and PKC-dependent activities. Likewise, these kinase activities appear also to participate in the stimulatory effect of TGF-β1 on junB mRNA levels but they seem to have no role in its effect on c-fos expression.

thumbnail image

Figure FIG. 2. Effects of PKC and PTK inhibitors on the TGF-β1- and BMP-2-induced increases of c-fos and junB mRNA levels. Cells were pretreated with either vehicle (DMSO), chelerythrine chloride (ChCl, 10 μM), or genistein (Gen., 30 μg/ml) for 60 minutes and then incubated in the absence (C, control) or the presence of TGF-β1 (T, 5 ng/ml) or BMP-2 (B, 50 ng/ml) for another 60 minutes. Northern blot analysis of isolated total RNA was carried out as described in the Materials and Methods section. After normalization against GAPDH mRNA value, the fold stimulation over the control (control in the presence of either vehicle [DMSO], chelerythrine chloride [ChCl, 10 μM], or genistein [Gen., 30 μg/ml], respectively) was calculated. Results of independent experiments (n = 3) are summarized (average ± SD) in histograms below the autoradiogram of a typical experiment. The asterisks indicate that the value is statistically different from the value of the control (*p < 0.05; **p < 0.01; ***p < 0.001).

Download figure to PowerPoint

Effects of TGF-β1 and BMP-2 on AP-1 DNA-binding activity in ROS 17/2.8 cells

To assess whether the TGF-β1- and BMP-2-induced up-regulation of c-fos and junB mRNA resulted in an increase in AP-1 DNA-binding activity, electromobility shift assays (EMSAs) were performed. Cells were incubated in the presence of TGF-β1 (5 ng/ml) or BMP-2 (50 ng/ml) for 0, 2, and 4 h. Figure 3 shows that, in comparison with control, consensus AP-1 DNA-binding activity was augmented after 2 h of treatment by either TGF-β1 (Figs. 3A and 3B) or BMP-2 (Figs. 3C and 3D) and started to decrease after 4 h. Moreover, in the presence of either JunB or c-Fos antibody, the intensity of the single shifted band was partly abrogated in samples treated with TGF-β1 or BMP-2 for 2 h (Figs. 3B and 3D). Taken together, these data showed that TGF-β1 and BMP-2 increased not only gene expression of c-fos and junB but also the quantity of AP-1 complexes in these cells. Furthermore, the proteins JunB and c-Fos were shown to participate in the formation of these protein/DNA complexes.

thumbnail image

Figure FIG. 3. Effects of TGF-β1 and BMP-2 on AP-1 DNA-binding activity. Cells were treated with either (A) vehicle (control, 2% serum) or (B) TGF-β1 (5 ng/ml); (C and D) BMP-2 (50 ng/ml). At the indicated time, cells were harvested and EMSA was performed on nuclear extracts (see Materials and Methods section). (B and D) When indicated by the (+) sign, JunB or c-Fos antibody was added.

Download figure to PowerPoint

Influence of the expression of a dominant negative for AP-1 transcriptional activity on TGF-β1 and BMP-2 effects on (α1) collagen I mRNA levels

We recently showed that both TGF-β1 and BMP-2 transcriptionally up-regulated the expression of (α1) collagen I gene in ROS 17/2.8 cells.(42) These actions are dependent on de novo protein synthesis suggesting an indirect mechanism. Potential candidates for intermediaries of these effects of TGF-β1 and BMP-2 appeared to be AP-1 transcription factors, which previously have been shown to participate in the regulation of collagen I expression.(43) The stimulatory effects of TGF-β1and BMP-2 on c-fos and junB mRNA levels we showed in the current studies supported this hypothesis. Therefore, to address this issue, we established stable cell lines in which the expression of a natural dominant negative for AP-1 transcriptional activity ΔFosB(41) could be induced in the presence of IPTG. ΔFosB is a truncated form of FosB missing the C-terminal 101 amino acids of FosB. This protein forms a heterodimer with each of the Jun proteins but does not activate a promoter with an AP-1 site. Consequently, ΔFosB has been found to inhibit the transcriptional activities of Jun and Fos. Figure 4 shows the IPTG-regulated expression of the natural mutant ΔFosB in ROS 17/2.8 cells. Transfected cells (ROPF7 and ROPF3) were incubated without or with IPTG (5 mM) for 24 h, before adding TGF-β1 (5 ng/ml) or BMP-2 (50 ng/ml) for another 48 h. As shown in Fig. 5, induction of ΔFosB expression blocked the effects of TGF-β1 and BMP-2 on (α1) collagen I mRNA levels revealing that AP-1 transcriptional activities were required for these actions.

thumbnail image

Figure FIG. 4. Immunoblot of ΔFosB expression in ROS 17/2.8-derived cells (ROPF3 and ROPF7). The indicated cell lines were uninduced or induced with IPTG for 48 h. Cell lysates were immunoprecipitated with anti-FosB antibody (Materials and Methods section) and the immune complexes were subjected to SDS-PAGE, transferred to PVDF membrane, and immunoblotted for ΔFosB expression. No ΔFosB expression was detectable in ROS 17/2.8 parental cells or in uninduced ROPF3 and ROPF7 cell lines.

Download figure to PowerPoint

thumbnail image

Figure FIG. 5. Effect of dominant negative ΔFosB on TGF-β1- and BMP-2-induced increase of (α1) collagen I mRNA levels. ROPF7 and ROPF3 cells pretreated without (−) or with (+) IPTG (5 mM) for 24 h were incubated in the presence (or not) of a fresh dose of IPTG (5 mM) and either vehicle (C, 2% serum), TGF-β1 (T, 5 ng/ml), or BMP-2 (B, 50 ng/ml) for another 48 h. Northern blot analysis was performed as described in the Materials and Methods section. (A) A representative autoradiogram obtained with ROPF7 cells is shown. (B) After normalization against GAPDH mRNA, the fold stimulation over the control was calculated. Results (average ± SD) from the two cell lines (ROPF7, n = 3; ROPF3, n = 3) are pooled together and summarized in the histogram. The asterisks indicate that the value is statistically different from the value of the control(1) (*p < 0.05; **p < 0.01). Control in the absence or the presence of IPTG (5 mM), respectively.

Download figure to PowerPoint

Effect of ΔFosB on the TGF-β1-induced increase of PTH/PTHrP receptor mRNA expression

Up-regulation of PTH/PTHrP receptor (PTH-R) mRNA expression by TGF-β1 has been reported previously in ROS 17/2.8 cells.(6) In our experimental conditions, we confirmed this observation and tested whether the expression of the mutant ΔFosB also could influence the effect of TGF-β1 on PTH-R mRNA levels in these cells. After 48 h, PTH-R mRNA expression was stimulated (approximately 4-fold) by TGF-β1 (5 ng/ml) in the two ΔFosB expressing cell lines ROPF3 and ROPF7 in the absence of IPTG (Fig. 6). However, induction of ΔFosB expression by IPTG markedly reduced the effect of TGF-β1 on PTH-R mRNA expression. This indicates that AP-1 transcriptional activity is at least partially involved in TGF-β1-induced up-regulation of PTH-R in these cell lines. In contrast, BMP-2 had no effect on PTH-R mRNA expression in these cell lines (data not shown).

thumbnail image

Figure FIG. 6. ΔFosB expression altered TGF-β1 up-regulation of PTH/PTHrP receptor (PTH-R). ROPF3 and ROPF7 cells pretreated without (−) or with (+) IPTG (5 mM) for 24 h were incubated in the presence (or not) of a fresh dose of IPTG (5 mM) and either vehicle or TGF-β1 (5 ng/ml) for another 48 h. Northern blot analysis was performed as described in the Materials and Methods section. (A) A representative autoradiogram obtained with ROPF7 cells is shown. (B) After normalization against GAPDH mRNA, the fold stimulation over the control was calculated. Results (average ± SD) from the two cell lines (ROPF7, n = 3; ROPF3, n = 3) are pooled together and summarized in the histogram. The asterisks indicate that the value is statistically different from the value of the control (***p < 0.001). Control in the absence or the presence of IPTG (5 mM), respectively.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

AP-1 transcription factors have been implicated in the regulation of skeletal development.(44) The presence of AP-1 sites in the promoters of many bone-specific genes such as osteocalcin, alkaline phosphatase, and type I collagen(45) suggests an important relationship between the expression of AP-1 family members and the expression of the OB phenotype. In fact, variations in basal expression of Fos and Jun family members have been shown over the course of OB differentiation.(44) In addition, TGF-β1 and BMP-2, which are two potent regulators of OB function, have been shown to modulate the expression of AP-1 family members, (29–33) suggesting that this particular family of transcription factors could be involved in TGF-β1 or BMP-2 signaling in OB cells. Our present results provide evidence for a critical role of AP-1 transcriptional activity in TGF-β1-induced increases in gene expression of the OB markers (α1) collagen I and PTH-R. Furthermore, our data show in a direct manner the participation of AP-1 transcriptional activity in the stimulation of gene expression by BMP-2, as previously proposed by Chalaux et al.(46)

In ROS 17/2.8 cells, we showed that TGF-β1 and BMP-2 increased c-fos and junB mRNA levels with comparable time courses. Both factors induce a very transient expression of c-fos. Such transitory induction of c-fos expression is typical of these genes and is likely because of the down-regulation of c-fos gene expression by the gene product itself.(47) In comparison, the effects of TGF-β1 and BMP-2 on junB expression are of longer duration. Time courses of TGF-β1-induced increases in c-fos and junB, similar to what we saw in ROS 17/2.8 cells, also have been observed in normal human OBs (hOBs).(29) However, in the human cells, TGF-β1 decreased c-jun mRNA levels while no effect was observed on this transcript in ROS 17/2.8 cells (data not shown). The effect of BMP-2 on junB mRNA levels was of considerable magnitude, which suggests that JunB could play a very significant role in BMP-2 signaling. In agreement with this hypothesis, JunB has been suggested to participate in the inhibition of myogenic differentiation induced by BMP-2 in myogenic C2C12 cells.(46)

The AP-1 transcription factor complex has been identified as a target of the PKC and MAPK signaling pathways. (47–49) Moreover, these signaling cascades also have been involved in TGF-β1(12–15, 42) actions on gene expression. Here, we show that PKC- and genistein-sensitive PTK-dependent activities participate in the stimulation of junB expression by both TGF-β1 and BMP-2. Likewise, these kinase activities were involved in BMP-2 but not TGF-β1 induction of c-fos mRNA expression, suggesting that TGF-β1- and BMP-2-induced c-fos expression are not mediated through identical kinase cascades. Although chelerythrine chloride and genistein exhibited no effect on TGF-β1-induced c-fos expression, this, of course, does not completely exclude the participation of any PKC- or PTK-dependent activities in this mechanism. Furthermore, the protein kinase A (PKA)-dependent signaling pathway, which contributes to the activation of the cAMP response element-binding protein (CREB) and which can be activated by TGF-β1,(50) may be involved in c-fos gene regulation. Overall, the c-fos promoter is complex, and a variety of response elements have been identified,(17) indicating that several signaling pathways can lead to c-fos gene transcription.

TGF-β1- and BMP-2-induced increases of c-fos and junB mRNA levels were correlated with an increase in AP-1 DNA-binding activity in which the proteins JunB and c-Fos are involved. These data further supported the hypothesis that AP-1 transcriptional activity might participate in TGF-β1- or BMP-2-induced responses in ROS 17/2.8 cells. Our hypothesis was then verified using cell lines expressing a natural dominant negative for AP-1 transcriptional activity ΔFosB.(41) Expression of ΔFosB abolished the effect of both TGF-β1 and BMP-2 on (α1) collagen I gene mRNA levels, which revealed that the action of both TGF-β1 and BMP-2 on the expression of this transcript are dependent on AP-1 transcriptional activity. AP-1 has been shown to mediate diverse TGF-β1 responses in various cell types. (20–28) Therefore, we tested whether this transcription factor complex also could be involved in the regulation by TGF-β1 of another gene expressed by the OB. TGF-β1 has been reported to up-regulate PTH/PTHrP receptor mRNA levels in ROS 17/2.8 cells.(6) In our studies, we confirmed this observation and showed that expression of the natural mutant ΔFosB decreased the effect of TGF-β1 on PTH/PTHrP receptor mRNA expression by 50%, indicating that AP-1 transcriptional activity is involved in this mechanism. However, further studies of the rat PTH-R promoter regions are needed to determine the precise role of AP-1 transcriptional activity in the regulation of the PTH-R gene by TGF-β1.

Our present data therefore show that AP-1 family members are key components in the regulation of OB genes by TGF-β1 and BMP-2 and emphasize the importance of this transcription factor family in signaling by the TGF-β family.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  • 1
    Centrella M, Horowitz MC, Wozney JM, McCarthy TL 1994 Transforming growth factor-β gene family members and bone. Endocr Rev 15:2739.
  • 2
    Noda M 1989 Transcriptional regulation of osteocalcin production by transforming growth factor-β in rat osteoblast-like cells. Endocrinology 124:612617.
  • 3
    Noda M, Yoon K, Prince CW, Butler WT, Rodan GA 1988 Transcriptional regulation of osteopontin production in rat osteosarcoma cells by type β transforming growth factor. J Biol Chem 263:1391613921.
  • 4
    Noda M, Rodan GA 1986 Type-β transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells. Biochem Biophys Res Commun 140:5665.
  • 5
    Noda M, Rodan GA 1987 Type-β transforming growth factor (TGFβ) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells. J Cell Physiol 133:426437.
  • 6
    McCauley LK, Beecher CA, Melton ME, Werkmeister JR, Jüppner H, Abou-Samra A-B, Segre GV, Rosol TJ 1994 Transforming growth factor-β1 regulates steady-state PTH/PTHrP receptor mRNA levels and PTHrP binding in ROS 17/28 osteosarcoma cells. Mol Cell Endocrinol 101:3313368.
  • 7
    Takuwa Y, Ohse C, Wang EA, Wozney JM, Yamashita K 1991 Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3. Biochem Biophys Res Commun 174:96101.
  • 8
    Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, Khan AJ, Suda T, Yoshiki S 1991 Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol 113:681687.
  • 9
    Massagué J 1996 TGFβ signalling, receptors, transducers and mad proteins. Cell 85:947950.
  • 10
    Massagué J, Hata A, Liu F 1997 TGF-β signalling through the smad pathway. Trends Cell Biol 7:187192.
  • 11
    Yingling JM, Datto MB, Wong C, Frederick JP, Liberati NT, Wang X-F 1997 Tumor suppressor Smad4 is a transforming growth factor β-inducible DNA binding protein. Mol Cell Biol 17:70197028..
  • 12
    Halstead J, Kemp K, Ignotz RI 1995 Evidence for involvement of phosphatidylcholine-phospholipase C and protein kinase C in transforming growth factor-β signalling. J Biol Chem 270:1360013603.
  • 13
    Mucsi I, Skorecki KL, Goldberg HJ 1996 Extracellular signal-regulated kinase and the small GTP-binding protein, Rac, contribute to the effects of transforming growth factor-β1 on gene expression. J Biol Chem 271:1656716572.
  • 14
    Afti A, Djelloul S, Chastre E, Davis R, Gespach C 1997 Evidence for a role of Rho-like GTPases and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) in transforming growth factor β-mediated signalling. J Biol Chem 272:14291432.
  • 15
    Reimann T, Hempel U, Krautwald S, Axmann A 1997 Transforming growth factor-β1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells. FEBS Lett 403:5760.
  • 16
    Frey RS, Mulder KM 1997 Involvement of extracellular signal-regulated kinase 2 and stress-activated protein kinase/jun N-terminal kinase activation by transforming growth factor β in the negative growth control of breast cancer cells. Cancer Res 57:628633.
  • 17
    Whitmarsh AJ, Davis RJ 1996 Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74:589607.
  • 18
    Chen X, Rubock MJ, Whitman M 1996 A transcriptional partner for MAD proteins in TGF-β signalling. Nature 383:691696.
  • 19
    Zhang Y, Feng XH, Derynck R 1998 Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature 394:909913.
  • 20
    Keeton MR, Curriden SA, Van Zonnevel A-J, Loskutoff DJ 1991 Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor β. J Biol Chem 266:2304823052.
  • 21
    Chung K-Y, Agarwal A, Uitto J, Mauviel A 1996 An AP-1 binding sequence is essential for regulation of the human α2(I) collagen (COL1A2) promoter activity by transforming growth factor-β. J Biol Chem 271:32723278.
  • 22
    Urìa AJ, Jimenènez MG, Balbin M, Freije JMP, Lòpez-Otìn C 1998 Differential effects of transforming growth factor-β on the expression of collagenase-1 and collagenase-3 in human fibroblasts. J Biol Chem 273:97699777.
  • 23
    Jin G, Howe PH 1997 Regulation of clusterin gene expression by transforming growth factor β. J Biol Chem 272:2662026626.
  • 24
    Tang W, Yang L, Yang Y-C, Leng SX, Elias JA 1998 Transforming growth factor-β stimulates interleukin-11 transcription via complex activating protein-1-dependent pathways. J Biol Chem 273:55065513.
  • 25
    Virolle T, Monthouel M-N, Djabari Z, Ortonne J-P, Meneguzzi G, Aberdam D 1998 Three activator protein-1-binding sites bound by the Fra-2JunD complex cooperate for the regulation of murine laminin α3A (lama3A) promoter activity by transforming growth factor-β. J Biol Chem 273:1731817325.
  • 26
    Lafon C, Mazars P, Guerrin M, Barboule N, Charcosse J-Y, Valette A 1995 Early gene responses associated with transforming growth factor-β1 growth inhibition and autoinduction in MCF-7 breast adenocarcinoma cells. Biochim Biophys Acta 1266:288295.
  • 27
    Zentella A, Weis FMB, Ralp DA, Laiho M, Massaguè J 1991 Early gene responses to transforming growth factor-β in cells lacking growth-suppressive RB function. Mol Cell Biol 11:49524958.
  • 28
    Mauviel A, Chung K-Y, Agarwal A, Tamai K, Uitto J 1996 Cell-specific induction of distinct oncogenes of the Jun family is responsible for differential regulation of collagenase gene expression by transforming growth factor-β in fibroblasts and keratinocytes. J Biol Chem 271:1091710923.
  • 29
    Subramaniam M, Oursler MJ, Rasmusen K, Rigg B, Spelsberg TC 1995 TGF-β regulation of nuclear proto-oncogenes and TGF-β gene expression in normal human osteoblast-like cells. J Cell Biochem 57:5261.
  • 30
    Ohta S, Hiraki Y, Shigeno C, Suzuki F, Kasai R, Ikeda T, Kohno H, Lee K, Kikuchi H, Konishi J, Bentz H, Rosen DM, Yamamuro T 1992 Bone morphogenetic proteins (BMP-2 and BMP-3) induce the late phase expression of the proto-oncogene c-fos in murine osteoblastic MC3T3-E1 cells. FEBS Lett 314:356360.
  • 31
    Banerjee C, Stein JL, Van Wijnen AJ, Frenkel B, Lian JB, Stein SG 1996 Transforming growth factor-β1 responsiveness of the rat osteocalcin gene is mediated by an activator protein-1 binding site. Endocrinology 137:19912000.
  • 32
    Chen Y, Takeshita A, Ozaki K, Kitano S, Hanazawa S 1996 Transcriptional regulation by transforming growth factor β of the expression of retinoic acid and retinoid X receptor genes in osteoblastic cells is mediated through AP-1. J Biol Chem 271:3160231606.
  • 33
    Takeshita A, Chen Y, Wanatabe A, Kitano S, Hanazawa S 1995 TGF-β induces expression of monocyte chemoattractant JE/monocyte chemoattractant protein 1 via transcriptional factor AP-1 induced by protein kinase in osteoblastic cells. J Immunol 155:419426.
  • 34
    Majeska JR, Rodan SB, Rodan GA 1980 Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. Endocrinology 107:14941503.
  • 35
    Ryder K, Lau LF, Nathans D 1988 A gene activated by growth factors is related to the oncogene v-jun. Proc Natl Acad Sci USA 85:14871491.
  • 36
    Ryder K, Nathans D 1988 Induction of protooncogene c-jun by serum growth factors. Proc Natl Acad Sci USA 85:84648467.
  • 37
    Van Beveren C, Van Straaten F, Curran T, Muller R, Verma IM 1983 Analysis of FBJ-MuSV provirus and c-fos (mouse) gene reveals that viral and cellular fos gene products have different carboxy termini. Cell 32:12411255.
  • 38
    Genovese C, Rowe D, Kream B 1984 Construction of DNA sequences complementary to rat α1 and α2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23:62106216.
  • 39
    Abou-Samra AB, Juppner H, Force T, Freeman MW, Kong XF, Schipani E, Urena P, Richards J, Bonventre JV, Potts JT, Kronenberg HM, Segre GV 1992 Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells, a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 89:273322736.
  • 40
    Tso JY, Sun X-H, Kao T-H, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs, genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:24852502.
  • 41
    Nakabeppu Y, Nathans D 1991 A naturally occurring truncated form of FosB that inhibits Fos/Jun transcriptional activity. Cell 64:751759.
  • 42
    Palcy S, Goltzman D 1999 Protein kinase signalling pathways involved in the up-regulation of the rat α1(I) collagen gene by transforming growth factor β1 and bone morphogenetic protein 2 in osteoblastic cells. Biochem J 343:2127.
  • 43
    Katai H, Stephenson JD, Simkevich CP, Thompson JP, Raghow R 1992 An AP-1-like motif in the first intron of human proα1(I) collagen gene is a critical determinant of its transcriptional activity. Mol Cell Biochem 118:119129.
  • 44
    McCabe LR, Kockx M, Lian J, Stein J, Stein G 1995 Selective expression of fos-and jun-related genes during osteoblast proliferation and differentiation. Exp Cell Res 218:255262.
  • 45
    Owen TA, Bortell R, Yocum SA, Smock SI, Zhang M, Abate C, Shalhoub V, Aronin N, Wright KL, Van Wijnen AJ, Stein JL, Curran T, Lian JB, Stein GS 1990 Coordinate occupancy of AP-1 sites in the vitamin D-responsive and CCAAT box elements by Fos-Jun in the osteocalcin gene, model for phenotype suppression of transcription. Proc Natl Acad Sci USA 87:99909994.
  • 46
    Chalaux E, Lòpez-Rovira T, Rosa JL, Bartrons R, Ventura F 1998 JunB is involved in the inhibition of myogenic differentiation by bone morphogenetic protein-2. J Biol Chem 273:537543.
  • 47
    Angel P, Karin M 1991 The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:129157.
  • 48
    Alblas J, Slager-Davidov R, Steenbergh PH, Sussenbach JS, Van der Burg B 1998 The role of MAP kinase in TPA-mediated cell cycle arrest of human breast cancer cells. Oncogene 16:131139.
  • 49
    Karin M 1995 The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem 270:1648316486.
  • 50
    Wang L, Zhu Y, Sharma K 1998 Transforming growth factor-beta1 stimulates protein kinase A in mesangial cells. J Biol Chem 273:85228527.