ATF-2 cooperates with Smad3 to mediate TGF-β effects on chondrocyte maturation

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Experimental Cell Research 288 (2003) 198 –207

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ATF-2 cooperates with Smad3 to mediate TGF-␤ effects on chondrocyte maturation Andreia M. Ionescu, Edward M. Schwarz, Michael J. Zuscik, Hicham Drissi, J. Edward Puzas, Randy N. Rosier, and Regis J. O’Keefe* Center for Musculoskeletal Research, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, USA Received 24 January 2003

Abstract This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-␤. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-␤, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-␤-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-␤. However, in combination the effects were additive and similar to the inhibitory effects of TGF-␤ on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-␤ on colX, while together the blockade was complete. Similar effects were observed with another TGF-␤-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-␤ signaling. In contrast, although TGF-␤ does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-␤ and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-␤ signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-␤ or BMP-2. © 2003 Elsevier Science (USA). All rights reserved. Keywords: ATF-2; Smad3; TGF-␤; Chondrocyte biology; Chondrocyte differentiation; Chick sternal chondrocyte culture

Introduction Longitudinal skeletal growth is an exquisitely regulated process in which chondrocytes complete a program comprised of several steps: proliferation, differentiation, and hypertrophy. Hypertrophy is characterized by biochemical and transcriptional changes, including a 5- to 10-fold increase in cellular volume, increased alkaline phosphatase activity, and expression of type X collagen [1,2]. Chondrocyte differentiation and hypertrophy culminate in apoptosis and calcification of the matrix [3,4]. This sequence of events

* Corresponding author. Box 665, Department of Orthopaedics, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Fax: ⫹1-585-756-4727. E-mail address: [email protected] (R.J. O’Keefe).

is regulated by a large number of local and systemic molecules, some of which drive cellular maturation while others prevent cellular differentiation. Previous studies have characterized retinoic acid, thyroid hormone, and bone morphogenetic proteins (BMPs) as stimulators of terminal differentiation [5–7]. Insulin-like growth factor (IGF-I) and fibroblastic growth factor (FGF) regulate chondrocyte proliferation while transforming growth factor-␤ (TGF-␤) and parathyroid hormone-related peptide (PTHrP) inhibit maturation [8 –10]. Regarding TGF-␤ and PTHrP, transgenic mice with impaired signaling for these factors demonstrate premature differentiation of growth plate chondrocytes. Specifically, expression of a dominant negative truncated, kinase-defective TGF-␤ receptor in mouse skeletal tissue resulted in accelerated chondrocyte terminal differentiation [11]. Similarly, targeted dis-

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ruption of either PTHrP or its receptor accelerated chondrocyte maturation [12–14]. In contrast, mice overexpressing PTHrP have delayed chondrocyte terminal differentiation [15,16]. These findings suggest that TGF-␤ and PTHrP may act in a common signaling cascade to regulate endochondral bone formation. Using mouse metatarsal bone rudiments, Serra et al. [17] have shown that TGF-␤ stimulates PTHrP mRNA expression in the perichondrium and relies on PTHrP to inhibit hypertrophic differentiation. Similarly, we have demonstrated induction of PTHrP by TGF-␤ in epiphyseal/periarticular chondrocytes in monolayer cultures [18,19]. Thus, TGF-␤ signaling and its control of specific target genes, such as PTHrP, are critical for normal growth plate morphology and function. TGF-␤ signaling through the Smad transcription factors has been established in essentially all cell systems. TGF-␤ binding to the type II receptor recruits the type I receptor into a complex that results in phosphorylation of the transcription factors Smad2 and Smad3 [20]. In chondrocytes, we have previously shown that TGF-␤ signaling is dependent upon activation of Smad2 and Smad3 transcription factors. Overexpression of either Smad2 or Smad3 mimicked TGF-␤ treatment, while the dominant negatives were capable of diminishing TGF-␤ effects on cell differentiation [21]. Consistent with our findings, a mutant mouse homozygous for a targeted disruption of Smad3 exhibits a skeletal phenotype consistent with inhibition of TGF-␤ signaling, with progressive loss of articular cartilage resembling osteoarthritis and enhanced terminal differentiation of physeal chondrocytes [22]. In addition to the classical Smad pathway, TGF-␤ receptor activation has been shown to stimulate the mitogenactivated protein kinase (MAPK) family [23]. The three major arms of the family include the extracellular signalregulated kinase (ERK) and two stress-activated kinases, the c-Jun N-terminal kinase (JNK) and the p38 kinase. Downstream targets for the activated MAPKs are transcription factors from the CREB/ATF and AP-1 families. ERK activation leads to phosphorylation and activation of the transcription factors Elk1 and Elk2 [23], as well as the cAMP/PKA responsive transcription factor CREB (cAMP Response Element Binding protein) [24]. Activation of p38 kinase results in phosphorylation of the transcription factor ATF-2 [25], while JNK is capable of activating both c-jun and ATF-2 [26,27]. Not surprisingly, many promoters of the TGF-␤-regulated genes contain either AP-1 sites, such as PAI-1 [28], TIMP-1 [29], TGF-␤1 [30], c-Jun [31], and ␣2(I) collagen [32] or CREB/ATF binding sites such as fibronectin [33], cyclinD1 [34], and cyclinA [35]. The presence of multiple binding sites for different transcription factors allows for transcriptional cross-talk between Smads and the MAPKs during TGF-␤-induced gene expression. In fact, in the chondrogenic ATDC5 cell line, the Smad, ERK1/2, and p38 mitogen-activated protein kinase pathways cooperate to induce aggrecan expression in response to TGF-␤ [36]. Given the importance of TGF-␤ in regulat-

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ing chondrocyte maturation, we decided to investigate signaling pathways that may cooperate with the Smads to control the rate of chondrocyte maturation. Our manuscript explores TGF-␤ signaling through mitogen-activated protein kinases and focuses upon p38 kinase/ATF-2 signaling.

Materials and methods Chondrocyte cell culture Embryonic cephalic sternal chondrocytes (day 13) were prepared and cultured as described [37]. After isolation and primary culture for 5–7 days, floating cells were plated in secondary cultures at 2.5 ⫻ 105 cells/cm2 in DMEM containing 10% NuSerum IV (Collaborative Biomedical, Bedford, MA), 4 units/ml hyaluronidase, and 2 mM L-glutamate (Sigma Chemical, St. Louis, MO). After 6 days, upper sternal chondrocytes (USC) were harvested and plated in six-well plates for the transient transfection assay or 60-mm dishes for Western or Northern analysis or real-time PCR. Western blotting Chondrocytes were treated with 3 ng/ml TGF-␤ (Calbiochem, San Diego, CA) alone or with 10 ␮M SB203580 (Calbiochem) for different time points and subsequently washed with cold phosphate-buffered saline (PBS) and lysed on ice in Golden lysis buffer (GLB) [38] supplemented with protease inhibitor cocktail tablets (Boehringer Manheim), 1 mM sodium orthovanadate, 1 mM EGTA, 1 mM NaF, and 1 ␮M microcysteine. The protein concentration of the soluble material was estimated using Coomassie Plus Protein Assay kit (Pierce, Rockford, IL). Twenty-five micrograms of extracts was assayed by SDS–PAGE. After transfer to a nitrocellulose membrane, the blots were probed with the following antibodies: anti-Flag (Sigma), at a dilution of 0.4 ␮g/ml, or anti-ATF2 and anti-phosphorylated ATF2 antibody (New England Biolabs, Beverly, MA) at a dilution of 1:1000. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse polyclonal antibodies (Bio-Rad Laboratories, Hercules, CA) were used as secondary antibodies. The immune complexes were detected using ECL (Amersham, Arlington Heights, IL). Transfections and luciferase assay Three firefly luciferase reporter constructs were used in our transfection experiments: (1) CRE-Luc (PathDetect CRE cis-Reporting System; Stratagene, La Jolla CA) driven by a basic promoter (TATA box) plus four repeats of CRE; (2) Gal4-Luc and Gal4 dbd-CREB (PathDetect CREB trans-Reporting System; Stratagene) a reporter driven by five repeats of Gal4 binding element activated by a fusion protein between Gal4 DNA binding domain and CREB transactivation domain; and (3) p3TP-Lux [20]. Transfec-

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tion efficiency was measured by co-transfection with a pRL vector from Promega and by determining the Renilla uniformis luciferase activity. Chondrocytes were transiently transfected using Superfect reagent following manufacturer’s instructions (Qiagen, Valencia, CA). Twelve hours after transfection, the cultures were changed to serum-free media for 6 h, followed by treatment with TGF-␤ (3 ng/ml) for 24 h. In some experiments, cotransfections were performed with Smad3 and Smad4 wild-type and dominant negative expression vectors (gift from Dr.R.Derynck) or with c-Jun and ATF-2 cDNA (gift from Dr.W.Min) or with a dominant negative ATF-2 cloned into the pRC-CMV vector (gift from Dr.C.Vinson [39]). In the cotransfection experiments 1 ␮g reporter and 1 ␮g expression vector (or empty vector for control) were cotransfected in a ratio of 1:3. Transfected cells were incubated with TGF-␤ for 24 h and proteins were extracted using 1⫻ Passive Lysis Buffer (Promega, Madison, WI). Luciferase activity was then measured in cell lysates using the Luciferase assay kit (Promega) and an Optocomp luminometer (MGM Instruments, Hamden, CT). Infection of chondrocytes cultures using retroviral systems Chick embryonic fibroblasts (CEFs) were grown in DMEM containing 10% fetal bovine serum, 0.2% fetal chick serum, and 1⫻ penicillin/streptomycin (Gibco) were transfected with the replication competent avian sarcoma virus RCASBP [40] and passed three times to allow viral replication. At confluence, growth media was changed to low serum (DMEM with 10% NuSerum). Viral supernatants were collected at 24-h intervals for 3 days and concentrated using a Centricon-80 Column (Millipore Corporation, Billerica, MA). At the time of secondary plating, chondrocytes were incubated with concentrated viral supernatant for 48 h. For dual-infection protocols we used retroviruses with two different envelopes (A and B) [40]. Cells were initially infected with viruses of one envelope for 48 h, washed with media, and then infected with viruses of the second envelope for an additional 48 h. We used both envelope forms (envA or envB) to express empty RCASBP for control, heat-resistant human alkaline phosphatase, and wild-type or dominant negative forms of ATF-2 and Smad3. Human alkaline phosphatase expression was detected following heating of the culture plates to 60°C for 30 min to eliminate endogenous alkaline phosphatase activity and using the NBT/BCIP staining as previously described [41].

(Stratagene) for 20 min at 68°C. Hybridization was done at 73°C for 1 h. The blot was washed twice for 15 min with 2⫻ SSC and 0.1% SDS, followed by a 30-min wash with 0.1⫻ SSC and 0.1% SDS. The blot was exposed to X-OMAT AR film (Kodak, Rochester, NY) for autoradiography. Real-time PCR Total RNA was extracted from cultures using the RNAeasy kit (Qiagen). RNA (1 ␮g) was reversed transcribed using Advantage RT-for-PCR kit from Clontech (Palo Alto, CA). Real-time PCR was performed using the RotorGene real-time DNA amplification system (Corbett Research, Sydney, Australia) and the fluorescent dye SYBR Green I to monitor DNA synthesis (SYBR Green PCR Master Mix; Applied Biosystems, Foster City, CA). The sequence of the forward type X collagen (colX) primer is 5⬘-ACATGCATTTACAAATATCGTTAC-3⬘ and the sequence of the reverse primer is 5⬘-AAAATAGTAGACGTTACCTTGACTC-3⬘. The sequence of the forward Indian hedgehog (Ihh) primer is 5⬘-CTGCTATTTGTGTGTGTGT-3⬘ and the sequence of the reverse primer is 5⬘-GTACAAGGCTCTGGTTTG-3⬘. GAPDH or ␤-actin were used for normalization of gene expression. The sequence of the forward GAPDH primer is 5⬘-TATGATGATATCAAGAGGGTAGT-3⬘ and the sequence of the reverse primer is 5⬘-TGTATCCAAACTCATTGTCATAC-3⬘. The PCR protocol included a 95°C denaturation step for 10 min followed by 35 cycles of 95°C denaturation (20 s), annealing (20 s), and 60°C extension (30 s). The annealing temperatures are as follows: GAPDH and IHH, 45°C, colX, 46°C. Detection of the fluorescent product was carried out at the end of the 60°C extension period. PCR products were subjected to a melting curve analysis and the data were analyzed and quantified with the RotorGene analysis software. Dynamic tube normalization and noise slope correction was used to remove background fluorescence. Statistics Statistical comparisons were made between the groups using the t test. Significance was considered present when the p value was less than 0.05 and is denoted in each of the figures.

Results Northern blot analysis TGF-␤ activates ATF-2 in chondrocytes RNA was extracted from cultures using the RNAeasy kit (Qiagen). RNA (5 ␮g) was run on 1.2% agarose gel containing 17.5% formaldehyde. After transfer in 10⫻ SSC, the RNA was UV cross-linked to a Gene Screen Plus membrane (New England Nuclear, Boston MA). A synthetic type X oligonucleotide was end labeled as previously described [42]. Prehybridization was performed in QuickHyb solution

The activation of ATF-2 signaling was investigated in embryonic cephalic sternal chondrocytes over a 1-h time course following treatment with 3 ng/ml TGF-␤. Although ATF-2 protein levels remained constant, phosphorylated ATF-2 (pATF-2) levels were markedly increased as determined by Western blot (Fig. 1A). The increase in pATF-2

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Loss of function experiments further confirmed a cooperative effect between ATF-2 and Smad3 on the p3TP-Lux reporter. While TGF-␤ treatment stimulated the p3TP-Lux reporter 5.4-fold, basal promoter activity was reduced 62% and TGF-␤ effects were inhibited 49% in the presence of the p38 kinase antagonist SB203580 (Fig. 2B). These find-

Fig. 1. TGF-␤ induces activation of ATF-2 but not of CREB. Phosphorylation of ATF-2 was measured at different time points following TGF-␤ (3 ng/ml) treatment. Protein samples (25 ␮g per lane) were subjected to Western blot analysis using two different polyclonal rabbit IgG antibodies: one specific to the phosphorylated form of ATF-2 (phospho-ATF2) and another that recognizes both phosphorylated and unphosphorylated forms of ATF-2 (A). Phosphorylation of ATF-2 was measured at different time points following TGF-␤ (3 ng/ml) treatment in the presence or the absence of p38 inhibitor SB203580. USC were pretreated with 10 ␮M inhibitor for 45 min at 37°C, followed by TGF-␤ treatment (3 ng/ml) for different time points in the presence of the inhibitor. Protein samples were subjected to Western blot analysis using an antibody specific to the phosphorylated form of ATF-2 (phospho-ATF2) (B).

was detectable within 5 min, maximal by 15 min, and returned to baseline levels 1 h after TGF-␤ stimulation (Fig. 1A). To investigate the role of p38 signaling pathway on ATF-2 activation, cultures were treated with TGF-␤ in the presence and absence of SB203580, a pharmacological inhibitor of p38 kinase. Inhibition of p38 signaling resulted in an inhibition of ATF-2 phosphorylation by TGF-␤ (Fig. 1B). Thus, TGF-␤ signaling leads to rapid activation of the transcription factor ATF-2 by p38 kinase. ATF-2 cooperates with Smads in TGF-␤ signaling To determine whether activation of ATF-2 has functional importance we examined gain and loss of function experiments using both pharmacological and molecular methods in chondrocytes transfected with the Smad responsive reporter p3TP-Lux [21]. Chondrocytes were cotransfected with the reporter and different combinations of expression vectors for ATF-2 and Smad3/4 in a series of gain-offunction experiments (Fig. 2A). TGF-␤ treatment stimulated the p3TP-Lux reporter gene expression 5.2-fold. Alone, cotransfection with either wild-type ATF-2 (8.5fold) or c-Jun (13.1-fold) enhanced TGF-␤ effects, while in combination they caused even greater stimulation (17-fold). As expected, cotransfection with both Smad3 and Smad4 expression vectors also enhanced TGF-␤-mediated stimulation of the p3TP-Lux reporter (10.2-fold increase in luciferase activity). This response was further augmented by addition of c-Jun and ATF-2 in combination (Fig. 2A).

Fig. 2. ATF-2 cooperates with Smad3 to control transcription of the p3TP-Lux reporter gene. USC were transfected with 1 ␮g luciferase reporter p3TP-Lux and 1 ␮g of different combinations of empty vector, ATF2, c-Jun, Smad3, and Smad4. Twelve hours after transfection, cells were washed, followed by 24 h treatment with or without TGF-␤ (3 ng/ml). The relative luciferase activities (means ⫾ SEM; n ⫽ 3) are presented. *Statistical significance at p ⱕ 0.05 compared to control samples transfected with empty vector. **Statistical significance at p ⱕ 0.05 compared to the Smad3/Smad4-transfected samples (A). USC were transfected with 1 ␮g p3TP-Lux luciferase reporter, pretreated with 10 ␮M inhibitor or vehicle for 45 min at 37°C, followed by stimulation with TGF-␤ (3 ng/ml) for 24 h. The cells were then assayed for luciferase activity. *Statistical significance at p ⱕ 0.05 compared with the untreated control (B). USC were transfected with 1 ␮g luciferase reporter p3TP-Lux and 1 ␮g of different combinations of empty vector or dominant negative ATF-2 with Smad3/Smad4. Twelve hours after transfection, cells were washed, followed by 24 h treatment with or without TGF-␤ (3 ng/ml). The relative luciferase activities (means ⫾ SEM; n ⫽ 3) are presented. *Statistical significance at p ⱕ 0.05 compared to control samples transfected with empty vector. **Statistical significance at p ⱕ 0.05 compared with their respective controls (B).

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ings were supported in experiments using a dominant negative ATF-2 (A-ATF2) cotransfected with p3TP-Lux alone or in the presence of Smad3 or the Smad3/4 combination. A-ATF2 resulted in a 63% reduction in basal luciferase levels and a 54% inhibition in TGF-␤ stimulation of the reporter. A-ATF2 also completely blocked the Smad3 and Smad3/4 induction of the p3TP-Lux reporter in both the presence and the absence of TGF-␤ treatment (Fig. 2C). Collectively, these results indicate that ATF-2 acts cooperatively with Smads to mediated TGF-␤ signaling at the p3TP-Lux reporter. Establishing methods for dual transgene expression in chondrocytes Determination of the cooperative interaction of ATF-2 and Smad3 in the regulation of TGF-␤-responsive genes requires a high level of expression of both gene products in the cell cultures. While viral infection methods facilitate a high level of transgene expression, it is limited by the fact that dual infection is typically not feasible [43]. We next demonstrated that a high level of simultaneous overexpression of two different genes could be obtained through use of a dual infection method using chicken replication competent retroviruses expressing unique envelope proteins, RCASBP(envelopeA) and RCASBP(envelopeB), which bind different receptors on the surface of the cells. Initial experiments showed that both retroviruses were similar in their ability to infect chondrocytes and to result in transgene expression. Both A-ATF2-RCASBP(envA) and A-ATF2-RCASBP(envB) resulted in similar levels of protein expression and were comparable to expression levels observed in cells infected with Smad3-RCASBP(envA) (Fig. 3A). These findings show that envA and envB viruses have an equal capacity to infect the cultures and lead to transgene expression. To establish methods whereby coinfection with these viruses can lead to expression of both transgenes, chondrocytes were infected with control RCAS virus expressing either envelope A or B followed by infection 48 h later with RCAS virus expressing the other envelope along with the alkaline phosphatase transgene. Alkaline phosphatase activity was assessed 48 h later. Infection with control RCASBP(B) followed by infection with alkaline phosphatase-RCASBP(A) resulted in minimal NBT/BCIP staining for alkaline phosphatase activity compared to control cultures infected with only alkaline phosphatase-RCASBP(A) (Fig. 3B). In contrast, prior infection with empty RCASBP(A) followed by infection with alkaline phosphatase-RCASBP(B) results in alkaline phosphatase staining that is comparable with control cultures infected with only alkaline phosphatase-RCASBP(B) (Fig. 3C). Efficient dual protein expression requires consecutive infection with RCASBP(A) followed by RCASBP(B). Finally, we investigated whether the second viral infection affects transgene expression from the primary viral infection. Western blot was performed with total cell extracts from chon-

Fig. 3. Dual transgene expression in chondrocytes. USC were infected with A-ATF2 RCASBP(A) (AAA), A-ATF2 RCASBP(B) (AAB), and Smad3 RCASBP(A) (S3) for 48 h, cells were lysed, and the protein extracts were used in a Western blot with an anti-Flag antibody (A). USC were infected with RCASBP(B) (c(B)) or nothing for 48 h followed by infection with alkaline phosphatase RCASBP(A) (AP(A)) for the next 48 h. Subsequently, staining for alkaline phosphatase with NBT/BCIP was performed (B). USC were infected with RCASBP(A) (c(A)) or nothing for 48 h followed by infection with alkaline phosphatase RCASBP(B) (AP(B)) for the next 48 h. Subsequently, staining for alkaline phosphatase with NBT/ BCIP was performed (C). USC were infected with Smad3 RCASBP(A) (S3(A)) for 48 h followed by infection with RCASBP(B) (c(B)) or nothing for 48 h. Subsequently, Western blot with anti-Flag antibody was performed (D).

drocytes incubated with Smad3-RCASBP(A) alone or in combination with RCASBP(B). We found that addition of RCASBP(B) did not substantially perturbe the levels of Smad3 protein (Fig. 3D). TGF-␤ inhibits colX expression through both Smad3 and ATF-2 signaling Treatment of chondrocytes with TGF-␤ inhibits maturation as illustrated by a decrease in colX. The effect is minimal at 24 h, but the inhibition of colX is progressive after that point, with maximal effects observed following 96 h of treatment (Fig. 4A). To determine whether Smad3 and ATF-2 act together to regulate TGF-␤-mediated gene expression in chondrocytes, we infected the cells with RCAS viruses expressing domi-

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cooperation between ATF-2 and Smad3 as mediators of TGF-␤ effects in chondrocytes (Fig. 4C). TGF-␤ induces PTHrP expression through both Smad3 and ATF-2 signaling PTHrP plays a major role in chondrocyte growth and differentiation. Since PTHrP is also a TGF-␤-responsive gene in cartilage cells, we examined whether Smad3 and ATF-2 also act cooperatively in its regulation [19]. TGF-␤ treatment caused a 44% increase in PTHrP at 24 h and a maximal 2.7-fold increase at 96 h (Fig. 5A). Similarly, control TGF-␤ cultures infected with empty RCASBP(A)

Fig. 4. ATF-2 cooperates with Smads during TGF-␤ inhibition of chondrocyte maturation and downregulation of colX. USC were treated with TGF-␤ (3 ng/ml) for different time points, the RNA was harvested, and changes in colX were measured by Northern blot of mRNA. The ethidium bromide-stained 18s rRNA was used as a loading control (A). USC were incubated for 2 days with viral supernatant of RCASBP(A) followed by another 2 days incubation with RCASBP(B). The RCASBP viruses used were either empty or carrying different combinations of dominant negatives (B) or different combination of cdnas (C). Subsequently, USC were treated with TGF-␤ (3 ng/ml) for 4 days, the RNA was harvested, and changes in colX were measured by Northern blot of mRNA. The ethidium bromide-stained 18s rRNA was used as a loading control.

nant negative ATF-2 (A-ATF2) or Smad3 (Smad3⌬C) either alone or in combination. Both A-ATF2 and Smad3⌬ partially blocked the inhibitory effect of TGF-␤ on colX expression. However, simultaneous infection with both dominant negatives completely blocked the TGF-␤ effect on colX expression, demonstrating cooperation between the two transcription factors (Fig. 4B). We further assessed the cooperative effects of ATF-2 and Smad3 by performing gain-of-function experiments using RCAS virus expressing wild-type Smad3 and ATF-2. Fig. 4C shows that both wild-type ATF-2 and Smad3 equally inhibited colX expression. The effect was smaller than the inhibitory effect observed in TGF-␤-treated cultures. However, dual expression of both wild-type ATF-2 and Smad3 had a larger inhibitory effect on colX and mimicked the effect of TGF-␤ addition to the cultures. Thus, both gain- and loss-of-function experiments demonstrate

Fig. 5. ATF-2 cooperates with Smads during TGF-␤ induction of PTHrP. USC have been treated with TGF-␤ (3 ng/ml) for different time points, the RNA was harvested, and changes in PTHrP expression levels were measured by real-time PCR. The relative expression of the gene is normalized to the relative expression of GAPDH. The data are presented as foldincrease with respect to control samples (collected at initial time t ⫽ 0). *Statistical significance at p ⱕ 0.05 for TGF-␤-treated samples compared with untreated control samples. For the next set of experiments, USC were incubated for 2 days with viral supernatant of RCASBP(A) followed by another 2 days incubation with RCASBP(B). The RCASBP viruses used were either empty or carrying ATF-2 or Smad3 cdnas. Subsequently, USC were treated with TGF-␤ (1 or 3 ng/ml) for 4 days, the RNA was harvested, and changes in PTHrP expression levels were measured by real-time PCR. The relative expression of each gene is normalized to the relative expression of GAPDH. The data are presented as fold-increase with respect to control samples (infected with empty RCAS and unstimulated). *Statistical significance at p ⱕ 0.05.

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Fig. 6. TGF-␤ does not induce activation of CREB. USC were transfected with 1 ␮g CRE-Luc luciferase reporter (A) or Gal4-CREB system (B) and stimulated with TGF-␤ (3 ng/ml) or PTHrP (10⫺7M) for 24 h and the cells were then assayed for luciferase activity. The graph shows relative luciferase units (RLU).

and RCASBP(B), had a 2-fold increase in PTHrP expression. Infection with viruses expressing either wild-type ATF-2 or Smad3 alone induced PTHrP by 2.1-fold and 1.9-fold, respectively. Simultaneous expression of ATF-2 and Smad3 was cooperative and resulted in a 2.9-fold increase in PTHrP gene expression (Fig. 5B). Thus, ATF-2 and Smad3 cooperatively induce PTHrP in chondrocytes. TGF-␤ effects are not associated with activation of CREB Since PTHrP inhibits chondrocyte maturation through activation of cAMP/PKA/CREB signaling, we initially examined TGF-␤ effects that are also dependent upon activation of this transcription factor. The effects of TGF-␤ and PTHrP were investigated on two separate CREB-responsive promoters, a cis-acting CRE-Luc and a trans-acting Gal4CREB reporter. While PTHrP stimulated the CRE-Luc reporter (40-fold) and the Gal4-CREB reporter (22.3-fold), TGF-␤ had no effect (Fig. 6A and B). Thus, TGF-␤ inhibitory effects on maturation are not dependent upon CREB signaling.

7A and B). Similarly, expression of dominant negative CREB enhanced colX expression in both TGF-␤ and in BMP-2-treated cultures. A-CREB blocked the inhibitory effect on colX in TGF-␤-treated cultures (Fig. 7A) and enhanced the stimulatory effect of BMP-2 on colX expression (Fig. 7B). These experiments suggest that CREB regulates differentiation by targeting important differentiationrelated genes independent of the individual signaling effects of TGF-␤ and BMP. Since prior studies have also suggested activation of ATF-2 by BMP signaling, we studied the effect of gain and loss of ATF-2 function on BMP effects in chondrocytes. Infection with dominant negative ATF-2 (A-ATF-2) increased the basal colX level while overexpression of wildtype ATF-2 diminished basal colX expression consistent with our prior results. However, neither dominant negative nor wild-type ATF-2 altered BMP-2-mediated effects on colX expression (Fig. 7C). The findings suggest that BMPs and TGF-␤ regulate colX through different transcriptional regulators and that ATF-2 is specific for TGF-␤ signaling. Discussion

CREB signaling alters TGF-␤ and BMP effects on colX, while ATF-2 is specific for TGF-␤ Additional experiments examined whether CREB modulates TGF-␤ and BMP effects on colX expression. Infection of the cultures with a dominant negative CREB (ACREB) results in an increase in baseline colX expression, suggesting a role for basal CREB activity in these cells (Fig.

While TGF-␤ is established as a critical regulator of chondrocyte differentiation, the signaling pathways through which this factor acts are not completely understood. Previously we have shown that Smad2 and Smad3 play an important role as mediators of TGF-␤ effects on chondrocyte differentiation [21]. However, TGF-␤ activates parallel signaling pathways in other cells, including the MAP kinase

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Fig. 7. CREB signaling alters both BMP and TGF-␤ effects on colX while ATF-2 is specific to TGF-␤ signaling. USC were incubated for 2 days with viral supernatant of RCASBP(A) either empty or carrying dominant negative CREB (A-CREB) and treated with TGF-␤ (3 ng/ml) (A) or BMP-2 (50 ng/ml) (B) for the next 4 days. The RNA was harvested and changes in colX were measured by Northern blot of mRNA. The ethidium bromidestained 18s rRNA was used as a loading control (A and B). USC were incubated for 2 days with viral supernatant of RCASBP(A) either empty or carrying ATF-2 cdna or dominant negative and treated with BMP-2 (50 ng/ml) for the next 4 days. The RNA was harvested and changes in colX were measured by Northern blot of mRNA. The ethidium bromide-stained 18s rRNA was used as a loading control (C).

signaling pathways JNK, ERK, and p38 kinases, along with the downstream transcription factors ATF-2 and CREB [23]. Our study demonstrates that, in chondrocytes, ATF-2, but not CREB, is activated by TGF-␤ signaling and that it cooperates with Smad3 to mediate TGF-␤ effects on chondrocyte differentiation. Our findings establish that ATF-2 is rapidly phosphorylated by TGF-␤ through activation of p38 kinase. Pharmacological inhibition of p38 kinase resulted in decreased phosphorylation of ATF-2 in response to TGF-␤ as well as repression of TGF-␤-regulated gene transcription. Gain-and loss-of-function experiments using molecular regulators further support a role for ATF-2 as a mediator of TGF-␤ effects. Overexpression of wild-type ATF-2 alone, or in combination with Smad3, enhanced activation of the TGF␤-responsive reporter p3TP-Lux, while overexpression of dominant negative ATF-2 inhibited TGF-␤ or Smad3/ Smad4 stimulation of the reporter. Furthermore, similar observations were made on gene expression. While overexpression of either wild-type ATF-2 or Smad3 inhibit colX, in combination the effect is greater and mimics the maximal inhibitory effect of TGF-␤. Similarly, overexpression of either dominant negative ATF-2 or Smad3 alone partially

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blocked the inhibitory effect of TGF-␤ on colX expression, while coexpression of the two dominant negatives completely blocked TGF-␤ effects. A similar pattern of cooperation between these two transcription factors was demonstrated on regulation of PTHrP, a TGF-␤-responsive gene in cartilage. Overall, our data suggest that ATF-2 and Smad3 act together to regulate TGF-␤-mediated inhibition of colX and induction of PTHrP. These effects are specific for TGF-␤, since overexpression of either wild-type or dominant negative ATF-2 did not influence BMP regulation of colX. ATF-2 is a member of the CREB/ATF family of transcription factors. These transcription factors translocate to the nucleus and constitutively bind to DNA at CRE-like DNA sequences. These factors are activated following their phosphorylation by upstream kinases at specific sites [44,45]. ATF-2 is activated by phosphorylation at Thr69 and Thr71 and to a lesser extent at Ser90 [45]. Phosphorylation is mediated by ERKs, JNK, and p38 kinases [25,27] but is insensitive to cAMP/PKA activation (despite being a low-affinity substrate for PKA in vitro) [46,47]. The current experiments show that ATF-2 phosphorylation occurs following exposure of chondrocytes to TGF-␤ and is dependent upon p38 kinase. ATF-2 binds to a CRE-like element to mediate the effects of TGF-␤ on the promoters of several genes, including fibronectin [33], cyclinD1 [34], and cyclinA [35]. Analysis of these promoters reveals that ATF-2 binds to the CRE-like site as either a homodimer or a heterodimer with either c-Jun or CREB. Heterodimerization alters ATF-2 binding specificity and affinity for various binding sites. For example, heterodimerization of ATF-2 with c-Jun results in a higher affinity for AP-1 sites than for its usual CRE-like binding sites [44]. This paradigm was supported in chondrocytes using the P3TP-Lux reporter, which has three AP-1 sites adjacent to the Smad3 binding site. Coexpression of ATF-2 and c-Jun in chondrocytes markedly enhanced transactivation of the p3TP-Lux reporter and cooperatively enhanced Smad3 effects on the promoter in both the absence and presence of TGF-␤. Retroviral infection was used to coexpress various combinations of transcription factors in chondrocytes with high efficiency. Typically, infection of a cell by a replicationcompetent retrovirus prevents subsequent superinfection by another virus of the same subgroup by interfering with subsequent receptor binding [43]. Vectors utilizing distinct receptors do not block each other, thus allowing the possibility of the delivery of two genes simultaneously. Preliminary experiments were performed using a retrovirus carrying human placental alkaline phosphatase, whose resistance to heat allows differentiation from the endogenous gene, in order to establish the experimental conditions necessary to establish dual infection with high efficiency [41]. Optimal infection occurred following incubation with RCASBP(A) for 48 h followed by incubation with RCASBP(B). Using this method, we showed that ATF-2 and Smad3 cooperate to

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inhibit colX and stimulate PTHrP expression and mimic TGF-␤ effects in chondrocytes. Furthermore, dominant negative ATF-2 and Smad3 both blocked TGF-␤ inhibitory effects on chondrocyte maturation, with enhanced effects present when both dominant negatives were coexpressed. Thus, both gain and loss of function experiments establish a role for a cooperative effect of ATF-2 and Smad signaling in transduction of TGF-␤ effects in chondrocytes. Previously, we have shown that PTHrP activates CREB signaling and that dominant negative CREB inhibits PTHrP effects on chondrocyte differentiation and proliferation [48]. Although TGF-␤ did not activate CREB transcription, dominant negative CREB enhanced the basal rate of chondrocyte differentiation and also blocked the inhibitory effect of TGF-␤ on colX. Similarly, dominant negative CREB enhanced the effect of BMP-2 as a stimulator of chondrocyte maturation. These findings suggest that CREB signaling is independent of either TGF-␤ or BMP signaling pathways and is a potent independent suppressor of chondrocyte maturation. Genetic models support the important role for ATF-2 in skeletal development. Homozygous mice expressing a mutant form of ATF-2 are runted and have altered growth plate morphology similar to that observed in human hypochondrodysplasia [49]. Beier et al. [34,35] proposed that a reduced chondrocyte proliferation accompanied by a decrease in cyclin A and cyclin D levels was responsible for the observed dwarfism. ATF-2 gene deletion results in immediate postnatal death due to meconium aspiration, but the skeletal phenotype has not been described [50]. Our findings demonstrate that ATF-2 is an important target of TGF-␤ signaling in chondrocytes and is involved in regulating the rate of chondrocyte maturation. Future experiments will further elucidate the mechanisms by which ATF-2 modulates skeletal growth during development.

Acknowledgments The authors thank Dr. J.Massague for the p3TP-Lux reporter construct, Dr. R.Derynck for the Smads 3 and 4 dominant negatives and cdnas, Dr. Charles Vinson for the dominant negatives A-CREB and A-ATF2, Dr. Stephen H. Hughes for the RCASBP(A,B) vectors, and the Genetics Institute for the recombinant BMP-2. The authors also appreciate the technical assistance of April Frankenberg. This work has been supported by a National Health Award AR38945 (to R.O.).

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