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Originally published In Press as doi:10.1074/jbc.M204405200 on July 30, 2002

J. Biol. Chem., Vol. 277, Issue 43, 41220-41229, October 25, 2002
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The Expression of novH in Adrenocortical Cells Is Down-regulated by TGFbeta 1 through c-Jun in a Smad-independent Manner*

Jérôme LafontDagger , Maryvonne LaurentDagger , Hélène ThiboutDagger , François Lallemand§, Yves Le BoucDagger , Azeddine Atfi§, and Cécile MartinerieDagger

From Dagger  INSERM U515 and § INSERM U482, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France

Received for publication, May 6, 2002, and in revised form, July 17, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The human NOV secreted glycoprotein (NOVH) is abundant in the fetal and adult adrenal cortex. The amount of NOVH increases in benign adrenocortical tumors and decreases in malignant adrenocortical tumors, suggesting that NOVH plays a role in tumorigenesis in the adrenal cortex. Transforming growth factor beta 1 (TGFbeta 1), fibroblast growth factor 2 (FGF2), and insulin growth factors (IGFs) play crucial roles in the physiology of the adrenal cortex. We investigated the effects of these factors on the expression of novH in the NCI H295R adrenocortical cell line. The amounts of NOVH protein and novH transcripts were down-regulated by TGFbeta 1 and up-regulated by FGF2, whereas IGFs had no effect. Furthermore, the TGFbeta 1-dependent inhibition of novH promoter activity was completely abrogated following site-directed mutation of two activating protein (AP-1) sequences (positions -473 and -447), whereas the stimulatory effect of FGF2 was not affected. Co-transfection with dominant negative forms of c-Jun and MEKK1 also abrogated novH-targeted regulation by TGFbeta 1, whereas the overproduction of Smad proteins or dominant negative forms of Smad had no effect. Taken together, these results suggest that c-Jun and MEKK1 signaling but not Smad signaling are involved in the TGFbeta 1-dependent decrease in NOVH in NCI H295R cells. In conclusion, our data provide evidence that novH is a new target of TGFbeta 1; unlike other members of the CCN (cyr61, ctgf, nov) family, however, its expression is repressed rather than induced.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The nov1 gene is a member of the CCN (cyr61 (1), ctgf (2), nov (3, 4)) family (5), which also includes elm1/wisp1 (6, 7), r-cop1/wisp2 (6, 8, 9), and wisp3 (6). It encodes a cysteine-rich secreted multimodular glycoprotein (10), which shares strong structural similarities with the other CCN proteins (5, 11). These proteins are involved in the regulation of cell proliferation, chemotaxis, angiogenesis, adhesion, and the formation of the extracellular matrix. In vivo, they appear to be involved in normal processes such as implantation, placentation, embryogenesis, differentiation, and development as well as pathological situations including wound healing, fibrotic disorders, and tumors (for a review see Ref. 11).

Relatively little is known about the functions of NOV. However, several reports have suggested that it up- or down-regulates cell proliferation, depending on the cell type, (3, 12, 13). There are also several lines of evidence indicating that NOV is involved in cell adhesion. Indeed, the multidomain structure of NOV and the other CCN proteins suggests that they bind to components of the extracellular matrix, including heparin-like oligomers (5). The finding that fibulin 1C, an extracellular matrix-associated protein (14, 15), interacts with the human NOV protein (NOVH) (16) suggests that NOVH has a role in signaling pathways involving the extracellular matrix. It was recently shown that recombinant NOV can promote the adhesion of vascular smooth muscle cells in vitro and that changes in nov expression occur following injury to the arterial walls (13).

In normal tissues, the expression of nov is tightly regulated during the development of the central nervous system (17, 18) and skeletal and visceral muscles (18) and during chondrogenesis.2 novH is highly expressed in the adrenal cortex during embryogenesis, and in adults novH is more strongly expressed in the adrenal cortex than in other endocrine tissues (19). Thus, novH may play an autocrine/paracrine role in the development and/or differentiation of these tissues.

Interestingly, the expression of novH is altered in several human tumors, including Wilms' tumors (4, 10) and adrenocortical tumors (19). In adrenocortical tumors, which have a very poor prognosis (20, 21), quantitative and qualitative changes in novH expression are correlated with the acquisition of the tumoral phenotype by adrenocortical tissue (19). Significant differences have been detected in the concentrations of NOVH and novH mRNA in benign and malignant tumors. Furthermore, the NOVH protein profiles are different in the two types of tumor, suggesting that novH plays a role in the early stages of tumorigenesis. The enhanced expression of novH in benign tumors may contribute to the benign phenotype by increasing cell adhesion, whereas the lower expression of novH in malignant tumors could be involved in cell invasiveness (19). Alternatively, the down-regulation of novH in malignant adrenocortical tumors suggests that novH could act as a tumor suppressor. This hypothesis is supported by the inverse correlation between tumorigenicity and novH expression in glioma cells (22) and by the fact that the ectopic expression of novH in glioma cells reduces their tumorigenicity in xenografts (23).3

Therefore, we decided to investigate the molecular mechanisms responsible for the alterations in novH expression in tumoral adrenocortical cells. For this purpose, in the present study we used the human NCI H295R cell line, which is derived from a human adrenocortical carcinoma that produces steroids (24, 25). A number of growth factors and cytokines such as epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha ), tumor necrosis growth factor (TNFalpha ), interleukins (26-28), insulin-like growth factors (IGFs) (for a review see Refs. 26 and 29), fibroblast growth factor 2 (FGF2) (26), and transforming growth factor beta 1 (TGFbeta 1) (for a review see Refs. 26 and 29) regulate adrenal growth and functions in normal and fetal adrenal glands. The concentration of IGF-II, which also plays a role in adrenocortical tumorigenesis (24, 30, 31), is inversely correlated to novH expression in several malignant adrenocortical tumors, suggesting that IGF-II regulates the expression of novH or vice versa (19). Moreover, FGF2 and TGFbeta 1 can induce the production of other members of the CCN family such as CTGF and CYR61 in fibroblasts and in some epithelial cells (32-36).

Thus, we examined whether novH expression is affected by IGFs, FGF2, and TGFbeta 1 in NCI H295R cells. We showed that the expression of novH is not modulated by IGFs but is up-regulated by FGF2 and down-regulated by TGFbeta 1. These regulations occur at the transcriptional level. Further studies indicated that two AP-1 consensus binding sites (-473 and -447) within the novH promoter play a crucial role in TGFbeta 1 regulation but not in the stimulatory effect of FGF2. Finally, we provide evidence that c-Jun and MEKK1, but not Smad, can mediate the TGFbeta 1-dependent decrease of novH expression.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids

Reporter Constructs and Expression Vectors-- The novH promoter constructs p625NH-Luc and p492NH-Luc were derived from p625NH-CAT and p492NH-CAT, respectively (37). Following digestion with HindIII and BglII, the novH promoter fragments were subcloned into the promoterless p2KM[BT] luciferase reporter vector (38). p2540NH-Luc was obtained by replacing the 493-bp HindIII-Bsu36I fragment from p625NH-Luc with the 1.8-kb HindIII-Bsu36I fragment derived from the novH pBH7 subclone (4). The (CAGA)9-MLP-Luc reporter was a gift from Dr. J. M. Gauthier. Myc-Smad2, Smad3, Smad4, FLAG-Smad7, pCMV-TAM67, c-Jun-Ala, and MEKK1(K432A) have been described previously (39-42). The constitutively activate MKK7 and the dominant negative Smad4 mutant were gifts from Dr. E. Nishida and Dr. R. Derynck, respectively. The reporter construct containing four copies of the AP-1 enhancer (pAP-1-Luc) was purchased from Clontech.

Site-directed Mutagenesis-- Either one or both of the AP-1 consensus sites present at positions -473 (GGTGACAAACT) and -447 (CATGACTAAC) (Fig. 7A) of p625NH-Luc were changed to TGctgAA using a two-step PCR strategy. Both strands of all the constructs were fully sequenced (Genome Express, Grenoble, France) to confirm the mutations before use.

Cells

Cell Culture-- NCI H295R cells (ATCC) were maintained in Dulbecco's modified Eagle's/F12 medium supplemented with 2% Ultroser G (Invitrogen), 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite (Sigma), 200 units/ml penicillin, 200 µg/ml streptomycin, and 2.5 mM L-glutamine. Primary cultures of mouse astrocytes were obtained as follows. Brains from 3-4-day-old mice were dissected and crushed in minimum Eagle's medium. Cells were recovered by filtration through sterile filters (70-µm pores), resuspended in minimum Eagle's medium containing 25 mM Hepes, nonessential amino acids, and 10% fetal calf serum and plated out. Fibroblasts were allowed to adhere for 4 h, and any non-adhering astrocytes were replated on complete medium.

To eliminate the influence of serum, NCI H295R cells were transferred into Dulbecco's modified Eagle's/F12 medium supplemented with transferrin (5 µg/ml), selenium (5 ng/ml), 200 units/ml penicillin, 200 µg/ml streptomycin, and 2.5 mM L-glutamine. All experiments were performed with cell lines obtained from passages 2-8 following thawing or with primary cultures from passage 2.

Cytokine Treatment of Cell Cultures-- Adrenocortical cell lines (NCI H295R) plated out at a density of 5 × 106 cells per 100-mm dish were incubated in a serum-free medium for 24 h and then treated with TGFbeta 1 (Sigma) or FGF2 (R&D Systems) or left untreated as indicated in the text. In some experiments, cycloheximide (10 µg/ml, Sigma) and actinomycin D (5 µg/ml, Sigma) were added to the medium 1 h before the addition of the growth factor.

RNA Extraction and Northern Blotting-- Total RNA was isolated from cultured cells by use of the acid-guanidium-thiocyanate-phenol-chloroform extraction kit according to the manufacturer's instructions (Tri-Reagent, Sigma). Total RNA samples (10 µg) were loaded onto a 1% agarose-2.2 mol/liter formaldehyde gel, subjected to electrophoresis, and transferred onto nylon membranes. The membranes were hybridized as previously described with the 1.9-kb EcoRI novH probe or the 2.3-kb EcoRI-XhoI mouse nov (novM) probes (4, 10, 43) labeled by random hexamer priming (Amersham Biosciences) in the presence of [32P]dCTP. The signal for novH or novM was normalized according to the intensity of the gapdh signal (Clontech).

Luciferase Reporter Assays-- NCI H295R cells plated in 6-well plates (5 × 105 cells per well) were transfected using LipofectAMINE Plus (Invitrogen) as described in the user's manual. For reporter assays, the reporter constructs (0.5 µg) were co-transfected with 0.1 µg of pCMV-beta -galactosidase as an internal transfection control (Clontech). For assays in which the role of the transacting proteins was to be tested, 1.5 µg of the empty pcDNA3 (Invitrogen) vector or pcDNA3 encoding the protein of interest was used. In dose response experiments, the total amount of the expression vectors was kept constant by use of the empty vector. In each assay, cell cultures were serum starved prior to treatment with TGFbeta 1 (4 ng/ml) or FGF2 (10 ng/ml). Luciferase and beta -galactosidase activities were assayed by use of kits from Promega and PerkinElmer Life Sciences (Galacto-Star system), respectively. The data are presented as means ± S.E. of representative experiments performed in triplicate on at least two separate occasions.

Immunoblotting-- For the detection of endogenous NOV, cultured NCI H295R cells were lysed in radioimmune precipitation assay buffer (50 mM Tris (pH 7.4), 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 mM EDTA, 20 mg/ml aprotinin, 20 mg/ml leupeptin). For the detection of secreted NOV, proteins from conditioned medium corresponding to 2 × 105 cells (unless otherwise indicated) were collected after incubation overnight at 4 °C with heparin-Sepharose as described previously (10).

Protein samples from the lysates (10 µg) or from the conditioned medium were subjected to electrophoresis in 12% reducing SDS-polyacrylamide gels before being transferred to polyvinylidene difluoride membranes (Hybond P, Amersham Biosciences) for immunological detection. The membrane was incubated with the K19M anti-NOVH (1:500 dilution) polyclonal antibody (10) for 1 h at 37 °C. Immunoreactive proteins were detected by ECL (Amersham Biosciences) according to the manufacturer's instructions. For the detection of proteins encoded by transfected expression vectors, protein samples (40 µg) derived from the same cell lysates used for luciferase were subjected to immunoblotting. The anti-Myc (9E10; Santa Cruz Biotechnology), monoclonal antibody was used to detect c-Myc-tagged Smad2, 3, and 4. Immunoreactive proteins were visualized by ECL.

Protein Kinase Assay-- JNK activity was determined as described previously (42) with minor modifications. Briefly, cells were lysed in a buffer containing 25 mM Hepes (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.5 mM dithiothreitol, 0.5% deoxycholate, 20 mM beta -glycerophosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 10% glycerol. Lysates were clarified by centrifugation, and endogenous kinases were immunoprecipitated using anti-JNK (C-17; Santa Cruz Biotechnology) antibody. Immune complexes were collected by binding to protein A-Sepharose and washed three times in lysis buffer and then twice with kinase assay buffer (25 mM Hepes, 20 mM beta -glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, and 100 µM sodium vanadate). JNK kinase was assayed for 20 min at 30 °C in 30 µl of a kinase reaction mixture containing 2 µg of GST-Jun (1-79; Biomol), 20 µM unlabeled ATP, and 5 µCi of [gamma -32P]ATP. The reaction was stopped by adding electrophoresis sample buffer, and proteins were separated on 12% SDS-polyacrylamide gels and visualized by autoradiography.

Densitometry-- Western blots were scanned with a GS700 imaging densitometer and processed with the Molecular Analyst data system (Bio-Rad). Northern blots were analyzed with a Storm PhosphorImager (Amersham Biosciences).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of novH in NCI H295R Is Down-regulated by TGFbeta 1 and Up-regulated by FGF2-- NCI H295R cells are considered to be a good cellular model for adrenocortical tumors (24). To determine whether the expression of novH is influenced by environmental conditions, we used Western blotting to examine the amount of NOVH present in a conditioned medium when these cells were plated out at different densities. In serum-free medium, the amount of NOVH detected, corresponding to the same number of NCI H295R cells (105) tested, increased with cell density (Fig. 1A). The concentration of secreted NOVH also increased in the presence of serum (Fig. 1B). Thus, the production of NOVH in NCI H295R cells may be regulated by cell-cell contact and by growth factors present in serum.


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  		Fig. 1.   The expression of novH increases with cell density and in the presence of serum in NCI H295R cells. A, Western blot analysis showing the amount of NOVH produced in 48 h by 4.5 × 105 (lane 1), 1.75 × 106 (lane 2), 4.5 × 106 (lane 3), and 107 (lane 4) NCI H295R cells in serum-free conditions. B, Western blot analysis showing the amount of NOVH produced by 107 NCI H295R cells in 48 h in the presence of serum (lane 5) and in serum-free conditions (lane 6). A fraction of each medium corresponding to 105 cells was incubated with heparin-Sepharose overnight at 4 °C before SDS-PAGE and immunodetection with K19M antibody at a 1:500 dilution (19). SF9/82-conditioned medium (20 µl) containing NOVH baculoviral recombinant protein (19) was used as a control (lane 7). The amount of NOVH increased by 1.25 ± 0.08-, 2.1 ± 0.57-, and 5.3 ± 0.11-fold in the presence of serum. Arrows indicate the two (48 and 31-32 kDa) NOVH isoforms produced in conditioned medium, and asterisks indicate the two (44 and 27 kDa) baculoviral recombinant isoforms of the NOVH protein.

We next tried to identify growth factors that affect the production of NOVH in these cells. We focused on TGFbeta 1, FGF2, and IGFs because they play important roles in adrenocortical development and physiology (see Ref. 26 for a review). As shown in Fig. 2, the amount of NOVH did not change following treatment with IGF-II or IGF-I; however, the amount of NOVH (in cell lysates or in medium) was decreased (~ 80%) by TGFbeta 1 (4 ng/ml) and increased (~3-fold) by FGF2 (10 ng/ml). The amount of NOVH increased only slightly in the presence of both factors, suggesting that TGFbeta 1 inhibits both basal and FGF2-induced expression of NOVH.


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  		Fig. 2.   Down-regulation of novH expression by TGFbeta 1 and up-regulation of novH expression by FGF2 in NCI H295R. Western blot analysis showing the amount of NOVH in NCI H295R or secreted into the medium following 24 h in serum-free medium with IGF-II (50 ng/ml), IGF-I (50 ng/ml), TGFbeta 1 (4 ng/ml), FGF2 (10 ng/ml), or FGF2 (10 ng/ml) plus TGFbeta 1 (4 ng/ml). C, untreated control. Arrows indicate the two isoforms of NOVH.

The amount of novH mRNA was also affected by TGFbeta 1 and FGF2 in a dose-dependent manner (Fig. 3A). Time-course experiments showed (Fig. 3B) that novH down-regulation by TGFbeta 1 (4 ng/ml) was maximal after 24 h (72 ± 20%, n = 8), whereas stimulation by FGF2 (10 ng/ml) reached a maximum after 6 h, and this level was maintained for at least 24 h (2.5 ± 0.4-fold, n = 8).


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  		Fig. 3.   Dose-response and time-course expression of novH in NCI H295R cells treated with TGFbeta 1 and FGF2. A, Northern blot analysis of novH in NCI H295R treated with increasing concentrations of TGFbeta 1 or FGF2 for 24 h or not treated. B, Northern blot analysis of novH in NCI H295R cells treated with TGFbeta 1 (4 ng/ml) or FGF2 (10 ng/ml) for increasing periods of time. Blots were successively hybridized with novH and gapdh probes and subjected to autoradiography. Densitometric analyses of the normalized novH/gapdh concentrations are presented in the lower panels. AU, arbitrary units.

Next, we examined whether protein synthesis was required for the regulation of novH by TGFbeta 1 or FGF2 in NCI H295R. Pretreatment with the translation inhibitor cycloheximide 1 h before the addition of TGFbeta 1 or FGF2 did not block the effects of TGFbeta 1 but completely abolished the effects of FGF2 (Fig. 4A). Thus, TGFbeta 1 directly regulates the expression of novH, and FGF2 requires de novo protein synthesis, which could be rapidly induced.

To gain further insight into the molecular mechanisms by which TGFbeta 1 and FGF2 regulate the amount of novH mRNA, we investigated the effects of these factors on the steady-state levels of transcripts. From Fig. 4B it can be seen that when transcription was blocked with actinomycin D for 9 h, the basal level of novH mRNA decreased (~3-fold), indicating that the half-life of novH transcripts is less than 9 h in these cells. Under these conditions, TGFbeta 1 and FGF2 did not significantly modulate the inhibitory effect of actinomycin D. This is consistent with the hypothesis that these factors regulate the expression of novH at the transcriptional level.


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  		Fig. 4.   Regulation of the expression of novH by TGFbeta 1 and FGF2 following treatment with cycloheximide (CHX) or actinomycin D (AD). Northern blot analysis of novH in NCI H295R (10 µg of total RNA). A, cells were pretreated for 1 h with CHX (10 µg/ml) before the addition of TGFbeta 1 and FGF2 for 24 h. B, cells were pretreated for 1 h with AD (5 µg/ml) before the addition of TGFbeta 1 and FGF2 for 9 h. As the expression of gapdh was affected by AD in these conditions, the amount of novH mRNA was normalized relative to the 18 S ribosomal RNA. Densitometric analyses of the normalized novH RNA concentrations are presented in the lower panels. Two independent experiments gave the same results. C, control; AU, arbitrary units.

TGFbeta 1 and FGF2 Regulate novH Promoter Activity-- To better understand the mechanism by which TGFbeta 1 and FGF2 regulate the transcriptional activity of novH, we analyzed their effects on the novH promoter fused to the luciferase reporter gene in transient transfections in NCI H295R cells. We assessed the regulation of three different promoter constructs, p2540NH-Luc (-2540 to +87), p625NH-Luc (-625 to +87), and p492NH-Luc (-492 to +87) by TGFbeta 1 and FGF2 (Fig. 5A). Treatment of all three constructs with TGFbeta 1 resulted in ~50% inhibition, and treatment with FGF2 resulted in ~150% stimulation (Fig. 5, B and C). Thus, the promoter region between -2540 and -492 is not involved in the regulation of novH expression by these two factors. Furthermore, the stimulatory effect of FGF2 on p625NH-Luc promoter activity was reduced following the addition of TGFbeta 1 (Fig. 5D), which is consistent with our results for the endogenous NOVH. Therefore, we carefully examined the promoter region beyond position -492 to try to identify any specific cis-acting elements that could be involved in the regulation of novH by TGFbeta 1 and FGF2.


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  		Fig. 5.   Effects of TGFbeta 1 and FGF2 on novH promoter activity. A, schematic structures of novH-Luc reporter constructs. The potential consensus sequences of the transcription binding sites are indicated (37). B and C, NCI H295R cells were transfected with p2540NH-Luc, p625NH-Luc, or p492NH-Luc (0.5 µg). Cells were or were not treated with TGFbeta 1 or FGF2 24 h prior to lysis and subjected to a luciferase assay. The results are expressed as the mean ± S.E. of a representative experiment performed in triplicate. D, NCI H295R cells were transfected with p625NH-Luc. 4 h later the cells were or were not treated with FGF2. After 20 h they were treated with TGFbeta 1 in the presence or absence of FGF2 (10 ng/ml). Cells were subjected to the luciferase assay 24 h later. The increase in p625NH-Luc promoter activity (~370%) due to FGF2 in these experiments compared with control was probably due to longer treatment times of cells with the growth factor. The results are expressed as the mean of a representative experiment, performed in triplicate, ± S.E. AU, arbitrary units.

We also investigated the ability of TGFbeta 1 and FGF2 to modulate the expression of nov in cells from other species. Using primary cultures of mouse astrocytes, we observed that FGF2 had no effect, whereas TGFbeta 1 also decreased the amount of nov RNA in these mouse cells (Fig. 6). Therefore, we compared the sequences of the human (novH) and murine (novM) promoter sequences (Fig. 7A). We found two consensus sequences corresponding to AP-1 binding sites in the novH promoter region (at positions -473 and -447). Interestingly, these sequences were also present in the novM promoter region.


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  		Fig. 6.   Regulation of the expression of novM by TGFbeta 1 and FGF2 in mouse astrocytes. Northern blot analysis of novM in primary cultures of mouse astrocytes (15 µg total RNA) treated with TGFbeta 1(4 ng/ml), FGF2 (10 ng/ml), or not treated (C, control) for 24 or 48 h. Blots were successively hybridized with novM and GAPDH probes and processed for autoradiography.


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  		Fig. 7.   Regulation of the novH promoter by TGFbeta 1 through AP-1 sites. A, alignment of the ~700-bp region encompassing the human and mouse nov promoter. The top strand represents the human (H) nov promoter and the bottom strand represents the mouse (M) nov promoter (deposited in the GenBankTM/EMBL data bank under the accession number AJ431713). Nucleotides of the human nov promoter are numbered relative to the transcription start site (37). Because the transcription start site of novM has not yet been determined, nucleotides are numbered relative to the first coding ATG. Several putative cis-regulatory elements for known transcription factors conserved in the two promoters are indicated in boldface. B, schematic diagram of the AP-1 mutants used in the transient transfection assays. The substitutions at positions -473 and -447 of AP-1 are indicated. C and D, NCI H295R cells were transiently transfected with p625NH-Luc or AP-1-mutated constructs and the internal CMV-beta -galactosidase control. Cells were or were not treated with FGF2 (C) or TGF beta  1 (D) 24 h prior to lysis and subjected to the luciferase assay. The mean luciferase activity ± S.E. of a representative experiment performed in triplicate is presented. AU, arbitrary units.

The AP-1 family of transcription factors is implicated in various regulatory activities of TGFbeta 1 (44-47) and FGF2 (48, 49). To determine the role of AP-1 in the regulation of nov expression by TGFbeta 1 and FGF2, we used site-directed mutagenesis to alter the AP-1 sites (Fig. 7B). Each of the point mutations resulted in a substantial decrease in basal promoter activity (~4-5-fold) when these constructs were used to transfect NCI H295R. No further effect was observed when both AP-1 sites were mutated simultaneously (Fig. 7, B and C).

As shown in Fig. 7C, none of the AP-1 mutations prevented FGF2 from stimulating novH promoter activity (~100%), indicating that this process does not involve the binding of AP-1 to these sites. However, each of these mutations completely abrogated the effects of TGFbeta 1 on novH promoter activity in these cells (Fig. 7D) even following stimulation by FGF2 (data not shown). These data show that these AP-1 sites mediate the inhibitory effects of TGFbeta 1 and also suggest that the mechanism by which TGFbeta 1 inhibits FGF2 stimulation of novH expression is not a direct competition between transcription factors for binding on AP-1 sites.

C-Jun and MEKK1 Are Involved in the Effects of TGFbeta 1 on novH Expression-- Next, we were interested in determining which signaling pathways mediate the inhibitory effect of TGFbeta 1 on novH expression. TGFbeta 1 signaling is mediated by two types of serine-threonine kinase receptors (50, 51). The highly conserved Smad proteins act as downstream signal transducers (51, 52). Smad2 and Smad3 are restricted to the TGFbeta /activin pathway. After phosphorylation by TGFbeta 1-activated type I receptors, pathway-restricted Smads form heteromeric complexes with Smad4 and then translocate to the nucleus where they control the expression of a number of genes (53). TGFbeta also initiates other pathways such as the SAPK/JNK pathway (42). This intracellular signal leads to the phosphorylation of c-Jun by JNK, which increases its transcriptional potential (54-56). c-Jun is a member of the AP-1 family of transcription factors, which can bind to and activate transcription from AP-1 or 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element sites (57). Several lines of evidence have indicated that c-Jun is a downstream target of TGFbeta signaling (42). However, only a few examples of a down-regulation of gene regulation by TGFbeta 1 involving AP-1 and c-Jun have been reported (41, 58).

To investigate whether the down-regulation of novH expression by TGFbeta 1 involves c-Jun, we examined whether a dominant negative form of c-Jun (TAM67) lacking the region between amino acids 3 and 122 and encompassing the transactivation domain and the SAPK/JNK binding site could abrogate the effect of TGFbeta 1 on the p625NH-Luc reporter construct (42). As shown in Fig. 8A, transient transfection with increasing amounts of TAM67 significantly blocked the effects of TGFbeta 1 on p625NH-Luc promoter activity. Similar results were also obtained (Fig. 8A) with another dominant negative form of c-Jun in which the JNK phosphorylation sites (Ser-63 and Ser-73) were replaced by alanine (41). These data therefore strongly suggest that c-Jun through the JNK pathway plays a crucial role in the down-regulation of novH expression by TGFbeta 1.


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  		Fig. 8.   Effect of the JNK pathway and of MEKK1 on the TGFbeta 1-mediated down-regulation of novH transcription. A, effect of dominant negative forms of c-Jun (TAM67 and c-Jun-Ala). NCI H295R cells were co-transfected with p625NH-Luc (0.4 µg) and increasing concentrations of TAM67 (0.2, 0.4, 0.8, or 1.2 µg) or c-Jun-Ala (1.2 µg). The total amount of transfecting DNA was kept constant (1.6 µg) by adding an empty pCDNA3 vector. Transfected cells were treated with TGFbeta 1 (4 ng/ml) or not treated 24 h prior to the luciferase assay. Results are presented as the ratio of the luciferase activity of TGFbeta 1-treated cultures to the luciferase activity of untreated control cultures for each concentration of dominant negative constructs. Mean values ± S.E. are presented. B, effect of TGFbeta 1 on JNK activity. NCI H295R cells were exposed to TGFbeta 1 (10 ng/ml) for the indicated times. Cell lysates were immunoprecipitated with anti-JNK (Santa Cruz Biotechnology), and immunoprecipitates were subjected to an in vitro kinase assay using GST-Jun (1-79) as a substrate. The phosphorylated proteins were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Immunoblotting of a whole cell extract using anti-JNK showed that similar amounts of JNK proteins were present in each sample. Results are representative of at least three experiments. C, effect of a dominant negative form of MEKK1 (K432A). NCI H295R cells were co-transfected with p625NH-Luc (0.4 µg) and increasing concentrations of a dominant form of MEKK1 (K432A) (0.2, 0.4, 0.8, or 1.2 µg) or pAP-1-Luc (0.4 µg) with MEKK1 (K432A) (1.2 µg). The total amount of transfecting DNA was kept constant (1.6 µg) by adding an empty pCDNA3 vector. Transfected cells were treated as in A. Results are presented as in A. AU, arbitrary units.

We carried out further experiments to determine whether the activation of JNK contributes to the down-regulation of novH expression by TGFbeta 1. NCI H295R cells were treated for various periods of time with TGFbeta 1, and endogenous JNK activity was examined by an immune complex kinase assay using GST-Jun (1-79) as a substrate. Under our experimental conditions, the basal phosphorylation level of GST-Jun observed was relatively high (Fig 8B), and it remained elevated without any significant increase for all the time periods studied (up to 24 h). A weak but not reproducible increase was detected at 6 and 24 h in this representative experiment. We also checked whether JNK, was not transiently activated within the first 15 min as has been reported in some cells (59). Immunoblotting analysis of total cell lysates from NCI H295R with the anti-JNK antibody demonstrated that approximately equivalent amounts of the JNK, protein were present (Fig 8B). Thus, although c-Jun must be phosphorylated by JNK if novH is to be down-regulated by TGFbeta 1, because TGFbeta 1 did not significantly activate JNK, our results suggest that the basal level of JNK activity detected in these cells is sufficient for this inhibition to occur. Consistent with this, the production of increasing concentrations of a constitutively active MKK7 protein, a specific activator of JNK (60), neither decreased the basal level nor increased the TGFbeta 1-induced down-regulation of the novH promoter activity, which was still inhibited by ~50% (data not shown).

MEKK1 is an upstream activator of the JNK pathways that is also able to mediate the effects of TGFbeta 1 activation on AP-1-responsive promoters (59). Transient transfection of NCI H295R cells with a dominant negative interfering MEKK1 mutant (K432A) significantly blocked the TGFbeta 1-induced down-regulation of both p625NH-Luc and AP1-Luc promoter activities (Fig. 8C). These results suggest that additional components besides those activated by the JNK pathway are involved in the TGFbeta 1-mediated inhibition of these two reporter constructs.

The Smad Pathway Is Not Required for the Down-regulation of novH Expression by TGFbeta 1-- No Smad-binding elements (CAGA) have been found in the p625NH-Luc promoter sequence (61, 62); however, the Smad and JNK pathways may converge at the transcriptional levels (58). In particular, c-Jun physically interacts with Smad2, Smad3, and Smad4 (63), resulting in a synergy of activation on AP-1 site-mediated transcription (63, 64). In contrast, c-Jun was shown to repress a TGFbeta 1-inducible promoter containing the Smad3/4 binding element CAGA (58, 61). MEKK1 was also shown to modulate Smad2-mediated transcriptional activation selectively (65).

To determine whether the Smad pathway was functional in NCI H295R cells, we used the CAGA reporter containing nine copies of the Smad-binding site derived from the PAI-1 promoter (61). Treatment of the transfected NCI H295R cells with TGFbeta 1 led to a ~100-fold increase in the CAGA reporter activity (Fig. 9A), indicating that TGFbeta 1 can induce the Smad pathway in these cells.


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  		Fig. 9.   Effect of Smad proteins and of dominant forms of Smad on the TGFbeta 1-mediated down-regulation of novH promoter activity. A, NCI H295R cells were transfected with (CAGA)9-MLP-Luc reporter vector. Cells were or were not treated with TGFbeta 1 (4 ng/ml) 24 h prior to lysis and subjected to the luciferase assay. B, NCI H295R cells were co-transfected with p625NH-Luc (0.4 µg) and increasing concentrations of Smad2, Smad3, or Smad4 (0.2, 0.4, 0.8, and 1.2 µg). Total amount of transfecting DNA was kept constant (1.6 µg) by adding the empty pCDNA3 vector. Cells were treated with TGFbeta 1 (4 ng/ml) or not treated 24 h prior to the luciferase assay. The mean luciferase activity ± S.E. of a representative experiment performed in triplicate is presented. Protein samples (20 µg) derived from the same Smad cell lysates used for the luciferase assay were subjected to Western blot analysis. c-Myc-tagged Smad2, Smad3, and Smad4 proteins were detected by use of a monoclonal anti-c-Myc antibody (bottom). C and D, NCI H295R cells were co-transfected with p625NH-Luc (0.4 µg) and increasing concentrations of either a dominant negative form of Smad4 or Smad7 (0.4, 0.8, and 1.2 µg). As a control, NCI H295R cells were also co-transfected with (CAGA)9-MLP-Luc reporter vector and DN Smad4 or Smad7 (1.2 µg). The total amount of transfecting DNA was kept constant (1.6 µg) by adding the empty pCDNA3 vector. Cells were treated as in A and B. Results are presented as the ratio of the luciferase activity of TGFbeta 1-treated cultures to the luciferase activity of untreated control cultures for each concentration of DN Smad4 or Smad7 constructs. Mean values ± S.E. are presented.

We therefore co-transfected NCI H295R cells with p625NH-Luc and increasing concentrations of either the Smad2, Smad3, or Smad4 expression vector. As presented in Fig. 9B, we observed that the overexpression of Smad2, Smad3, or Smad4 in the absence of TGFbeta 1 did not significantly affect the basal novH promoter activity. This is consistent with previous studies of CAGA-mediated transcription (61, 66). More importantly, TGFbeta 1 still down-regulated novH promoter activity as efficiently as it does in the absence of co-transfected Smad proteins. In all of these experiments, the expression of the c-Myc tagged-Smad proteins was checked by immunoblotting using an anti-c-Myc monoclonal antibody (Fig. 9B). A similar conclusion could be drawn when Smad2 or Smad3 were transfected together with Smad4 (data not shown). We also investigated the effects on this process of the overexpression of a dominant negative interfering form of Smad4 (DN Smad4) and Smad7, a natural inhibitor of the Smad pathway (67, 68). The expression of both DN Smad4 and Smad7 in NCI H295R significantly decreased the TGFbeta 1-induced CAGA promoter activity but did not affect the TGFbeta 1-dependent down-regulation of novH promoter activity (Figs. 9, C and D). Thus, these results suggest that Smad signaling does not participate in the TGFbeta 1-dependent down-regulation of novH expression.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because IGF-I, IGF-II, FGF2, and TGFbeta 1 are involved in the physiological functions of adrenocortical cells, (26, 29), we analyzed their effects on the expression of novH in the NCI H295R cell line, which is derived from a human adrenocortical carcinoma (25). This cell line allowed us to show for the first time that novH expression is up-regulated by FGF2 and down-regulated by TGFbeta 1, whereas IGF-I and II have no influence. These data suggested that FGF2 and TGFbeta 1, which are involved in the development of various tumors (69, 70) and are also produced by adrenocortical cells (71, 72), could be considered as potential candidates involved in the modulation of novH expression in adrenocortical tumors (19). Our results also show that TGFbeta 1 reduces the up-regulation of novH expression induced by FGF2, suggesting that the basal level of novH expression results from a balance between the actions of these two growth factors. This balance may vary during tumorigenesis, and FGF2 detected in adrenocortical tumors (72) may play a major role in the overexpression of novH during the earlier stages of tumorigenesis. We cannot exclude the possibility that other factors also influence the levels of novH expression during adrenocortical tumorigenesis.

FGF2 has been shown to up-regulate the expression of novH in NCI H295R cells; however, we showed that down-regulation of the expression of novH by TGFbeta 1 is not restricted to tumoral adrenocortical cells, because it was also observed in primary astrocytes and could be detected in human as well as murine cells. The regulation of novH by TGFbeta 1 might, however, present some specificity, because it has not been reported in human prostatic cells (36).

novH is the first member of the CCN family that has been shown to be down-regulated by TGFbeta 1. The other members of this family such as ctgf and cyr61 are induced by TGFbeta 1 in different cell systems (33, 36). It is noteworthy that nov is also regulated oppositely from ctgf and cyr61 in chicken embryo fibroblasts. In these cells, ctgf and cyr61 behave as immediate-early genes induced by serum and oncogenes (73, 74), whereas the expression of nov is down-regulated by these factors and associated with quiescence (75). Primary cultures of mouse astrocytes are another cell type in which the expression of nov and ctgf is inversely regulated by TGFbeta 1.4 These observations suggest that NOV and CTGF and CYR61 have antagonistic functions in certain cell systems. However, the expression of nov and ctgf has been reported to be down-regulated by Wilms' tumor suppressor gene 1 (WT1) in renal cells (37, 76), indicating that these two molecules may also cooperate in some cells.

Under our experimental conditions, the expression of ctgf and cyr61 was barely detectable in NCI H295R cells, and the expression of ctgf was only slightly stimulated by TGFbeta 1.4 In fibroblasts, CTGF can function as a downstream mediator of TGFbeta 1 activity. For example, it can stimulate cell proliferation and extracellular matrix protein synthesis (77, 78). In vivo, CTGF plays a role in TGFbeta 1-mediated formation of granulation tissue and cooperates with TGFbeta 1 to induce persistent fibrosis (79). Whether NOVH mediates a function of TGFbeta 1 in adrenocortical cells remains to be determined. However, no correlation could be found between the levels of novH expression in NCI H295R cells treated with TGFbeta 1 or FGF2 and their proliferation state as assessed by [3H]thymidine incorporation.4 TGFbeta 1 has been reported to be a strong inhibitor of steroidogenesis in adrenocortical cells (26); therefore, novH may antagonize the effect of TGFbeta 1 in this function. This hypothesis is currently under investigation. TGFbeta 1 can potentiate tumorigenesis by down-regulating the genes involved in cell-cell adhesion and by up-regulating the expression of genes involved in cell-extracellular matrix association, ultimately improving the migration and invasiveness of the cell (69). These properties are more consistent with NOVH having a role as an adhesive protein (13) that is able to regulate the expression of genes involved in extracellular matrix remodeling.3

Because the expression of novH is directly regulated by TGFbeta 1 and different elements of the TGFbeta 1 signaling pathway can also be altered in cancer (69), we analyzed the signaling pathway involved in the TGFbeta 1-mediated down-regulation of novH. Comparison of the human nov promoter region, which is targeted by TGFbeta 1, with the corresponding mouse sequences revealed a high degree of sequence homology (69%). These conserved regions included several consensus sequences involved in the binding of transcription factors (such as USF, NFkappa B, NFY, and AP-1), suggesting that novH and novM could be subjected to common regulations. Our data provide an example of one of those, as the expression of novH and novM can be down-regulated by TGFbeta 1. We further demonstrated that down-regulation of novH expression is mediated by AP-1 sites, which are found in the same region of the two promoters. Our results suggest that the TGFbeta 1 signaling pathway targets AP-1 sites in the novH promoter to inhibit the expression of novH. However, we consistently observed that the mutation of AP-1 sites also results in a decrease in the basal activity of the novH promoter. The molecular mechanisms involved in maintaining the expression of novH in unstimulated NCI H295R cells are currently unknown. The identification of environmental cues that regulate the expression of novH will help to clarify this point.

Whereas both ctgf and novH can be regulated by TGFbeta 1, quite different promoter sequences are involved in this regulation, because the Smad pathway is responsible for the up-regulation of ctgf expression by TGFbeta 1 in fibroblasts (80). The induction of gene expression by TGFbeta 1 involving Smad or AP-1 binding sequences and c-Jun has been well documented (81, 82), but there are only a few reports of the down-regulation of gene expression by TGFbeta 1 involving c-Jun (41, 58). For example, TGFbeta 1 down-regulates the expression of the gene that encodes the metalloproteinase MMP12 (83) through AP-1 sites, but this inhibitory effect is dependent on signaling through Smad3. Our results showed that the mechanism by which novH is negatively regulated by TGFbeta 1 in NCI H295R cells is different. We demonstrated that although the Smad pathway in these cells was induced by TGFbeta 1, which is in agreement with a previous report (84), this pathway is not involved in the TGFbeta 1-mediated inhibition of novH expression. Our data concerning the novH promoter mutated in the AP-1 sites and the dominant forms of c-Jun mutated in the JNK binding domain or in JNK-specific phosphorylation indicate that the SAPK/JNK pathway is required in this regulation. Two other studies (59, 85) showed that the activation of JNK independently of Smads leads to the regulation of fibronectin and insulin-like growth factor binding protein 5 (IGFBP5) by TGFbeta 1. In contrast, our data suggest that the down-regulation of novH expression by TGFbeta 1 requires a basal level of JNK activity to phosphorylate c-Jun and an additional TGFbeta 1-dependent mechanism. We provide evidence that MEKK1 could play a crucial role in this regulation in a manner that does not involve the activation of JNK. It has recently been shown that MEKK1 is able to directly activate, independently of JNK, other proteins such as p300/cAMP-response element-binding protein-binding protein (86). It has also been reported that by reinforcing the association between c-Jun and TGIF, TGFbeta 1 leads to the repression of AP1-mediated transcriptional activity (41). It is therefore tempting to speculate that when NCI H295R are treated with TGFbeta 1, a factor specifically regulated by MEKK1 could participate in an interaction between c-Jun and TGIF, resulting in the down-regulation of novH expression. However, the molecular mechanism by which MEKK1 could participate in the TGFbeta 1-negative regulation of novH expression awaits further investigation. The study of novH regulation in NCI H295R cells, in which the Smad pathway is functional, may therefore represent a good model for a better understanding of the molecular mechanisms involved in the TGFbeta 1-mediated inhibition of gene expression.

In summary, the data presented here demonstrate that novH is a new target for TGFbeta 1. Further studies aimed at determining which of the functions of TGFbeta 1 are mediated by novH might be useful to the development of therapeutic agents for the treatment of diseases involving also other members of the CCN family such as fibrosis or cancer.

    ACKNOWLEDGEMENT

We thank Dr. C. Dubois for helpful discussions and critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ431713.

To whom correspondence should be addressed: INSERM U515, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France. Tel.: 33-1-4928-4664 or 4631; Fax: 33-1-4343-1065; Email: martiner@st-antoine.inserm.fr.

Published, JBC Papers in Press, July 30, 2002, DOI 10.1074/jbc.M204405200

2 M. Laurent, unpublished results.

3 M. Laurent, C. Martinerie, H. Thibout, M. P. Hoffman, F. Verrechia, Y. Le Bouc, A. Mauviel, and H. Kleinman, submitted for publication.

4 C. Martinerie and M. Laurent, unpublished results.

    ABBREVIATIONS

The abbreviations used are: nov (NOV), nephroblastoma overexpressed gene (protein); novH (NOVH), human nov gene (protein); novM, mouse nov gene; CCN, cyr61, ctgf, nov family; TGFbeta 1, transforming growth factor beta 1; IGF, insulin-like growth factor; FGF2, fibroblast growth factor 2; AP-1, activator protein 1; MLP, major late promoter; CAT, chloramphenicol acetyl transferase; MEKK1, mitogen-activated/extra cellular response kinase kinase 1; MKK7, mitogen-activated kinase kinase 7; JNK, c-Jun NH2-terminal protein kinase; SAPK, stress activated protein kinase; GST, glutathione S-transferase; gapdh, glyceraldheyde-3-phosphate dehydrogenase (gene); DN Smad4, dominant negative Smad4 protein.

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