Sep 20, 1994 - SPARC and thrombospondin genes are repressed by the c-jun oncogene in rat embryo fibroblasts. Amel Mettouchi, Florence Cabon',.
The EMBO Journal vol.13 no.23 pp.5668-5678, 1994
SPARC and thrombospondin genes are repressed by the c-jun oncogene in rat embryo fibroblasts
Amel Mettouchi, Florence Cabon', Nicole Montreau, Philippe Vernier2, Gilles Mercier, Daniel Blangy, Herve Tricoire3, Philippe Vigier4 and Bernard Binetruy5 IRSC, CNRS, UPR272, Laboratoire Virus et Differenciation, BP 8, 94801 Villejuif, 'INSERM U134, H6pital Pitie-Salp&riere, 47 bld de l'Hopital, 75651 Paris, 2lnsitut A.Fessard, CNRS, 91198 Gif sur Yvette, 3Institut de Physique Nucleaire, Centre Universitaire, 91406 Orsay and 4Institut Curie, CNRS, URA1443, Bat. 110, Centre Universitaire, 91405 Orsay, France 5Corresponding author Communicated by M.Yaniv
The sequence-specific transcription factor c-Jun displays oncogenic potential in mammalian cells either in cooperation with activated Ras in primary embryonic fibroblasts or alone in established cell lines. Although pathways for signal transduction leading to activation of c-Jun proteins have been extensively studied, little is known about the events downstream of c-Jun stimulation. We isolated cellular genes that are targets of c-Jun by differential screening of a cDNA library from primary rat embryo fibroblasts. Two transcripts with sequences similar to known genes were repressed following transitory expression of a c-Junencoding vector. They correspond to the SPARC and thrombospondin 1 (TS1) genes, encoding extracellular matrix proteins. These genes are tightly regulated during embryogenesis and in adult tissues and are involved in the control of cell growth. c-Jun transitory repression of these two genes was demonstrated both in primary cells and in FR3T3, an established fibroblast cell line. The repression was also detected in FR3T3 derivatives stably transformed by c-Jun or Ras. Although c-Jun regulation of the TS1 gene was found at the promoter level, preliminary results strongly suggest that repression of SPARC and TS1 gene expression are mediated by a secreted factor. In contrast, expression of these genes was unaffected by transformation with oncogenes from DNA viruses. Our results identify new, specific, probably indirect c-Jun target genes and suggest previously unsuspected regulatory roles for SPARC and thrombospondin in the control of cell growth. Key words: c-jun and ras oncogenes/SPARC/thrombospondin/transcription factor targets/transformation
Introduction c-jun encodes the major component of the sequencespecific transcriptional activator protein API. This gene represents a paradigm in studies of signal transduction
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from the plasma membrane to the nucleus because its expression and the activity of c-Jun protein are induced by a variety of stimuli (Angel et al., 1987; review by Vogt and Bos, 1990). c-Jun appears to be the nuclear target and the meeting point of various different regulatory pathways and its transactivating properties may trigger some of the corresponding cellular responses. Recent data show that c-jun is directly involved in oncogenic transformation: it is able to cooperate with an activated ras gene to transform primary rat embryo fibroblasts (REF) and, alone, can transform Ratla cells (Schutte et al.,1989). The transforming activity depends on c-Juninduced transactivation of AP1-regulated gene promotors (Alani et al.,1991). Oncogenic cooperation requires the Ras-dependent augmentation of c-Jun-induced transactivation via site-specific hyperphosphorylation of the c-Jun activation domain (Binetruy et al., 1991; Smeal et al., 1991). c-Jun is directly involved in another biological phenomenon: it is able to inhibit myogenic differentiation by interfering with the transcription factor myoD (Su et al., 1991; Bengal et al., 1992; Li et al., 1992). Very little is known about the downstream molecular mechanisms of cross-talk and interplay between transcription factors and signalling proteins. In particular, few genes have been identified as direct targets of c-Jun regulation (see review by Karin, 1992). In an attempt to identify new c-Jun cellular target genes we differentially screened a cDNA library of rat embryo fibroblast mRNA. After transitory overexpression of c-jun we identified several differentially regulated clones. They included three clones whose expression, after c-jun transfection, was lower than in control conditions. The clones corresponded to two genes encoding extracellular matrix proteins: SPARC (secreted protein acidic and rich in cysteine) and thrombospondin. Interactions between extracellular matrix proteins and cells have been shown to be important for normal cell growth and development. The most abundant of these proteins have a structural role and are major adhesive macromolecules. In contrast, proteins present in lower amounts, including SPARC and thrombospondin, are thought to play a modulatory role in cell-cell interactions. The synthesis of these proteins is regulated and specific to particular tissues or embryonic stages and is stimulated by growth factors (for reviews see Sage and Bomstein, 1991; Adams and Lawler, 1993; Lane and Sage, 1994). Although their precise functions are still largely unknown, several studies suggest their involvement in regulation of cell growth. SPARC displays different effects on DNA synthesis in endothelial cells and fibroblasts depending on the cell line, the SPARC peptide domain studied and the concentration used (Funk and Sage, 1993). At low concentration a particular SPARC-derived peptide inhibits DNA synthesis by bovine endothelial cells and increases
New c-Jun targets: SPARC and thrombospondin
DNA synthesis by human and bovine fibroblasts. However, fibroblast growth is inhibited by high .doses of the same SPARC peptide. Thrombospondin has been characterized as an inhibitor of angiogenesis in vivo and in vitro and it might have a major adhesive effect (Sage and Bornstein, 1991). Overexpression of exogenous thrombospondin confers to NIH 3T3 cells various properties characteristic of transformed cells, but not tumourigenicity (Castle et al., 1993). Because of the involvement of SPARC and thrombospondin in cell division and cell adhesion control, understanding the regulation of their gene expression might give new insights into their functions. In this paper we show that transfection with the c-jun oncogene induces a substantial repression of the expression of these two genes. Furthermore, mRNA and protein synthesis from these genes were also inhibited in cell lines stably transformed by c-jun or ras in comparison with that in normal fibroblasts. These results identify new c-Jun targets and suggest previously unsuspected regulatory roles for these extracellular matrix proteins in the control of cell growth.
Results Differential screening after transitory overexpression of c-Jun identifies SPARC and thrombospondin as new cellular targets To identify target genes of c-Jun, a subtracted cDNA library constructed using mRNA isolated from REF cells was differentially screened. A preliminary kinetic experiment was performed: mRNA from mock- and c-juntransfected cells were purified at each of a series of times and analysed by Northern blot. Transin, a known endogeneous c-Jun target gene (Matrisian et al., 1985), was maximally induced in c-jun-transfected cells after 24 h (data not shown). Thus, to study the early downstream events involved in c-jun transforming potential, both library and cDNA probes were synthesized with mRNAs isolated 24 h after transfection. Better to detect transcripts modulated by c-Jun, a subtracted cDNA library was constructed from c-jun + ras-transfected cells. cDNA subtraction was performed with poly(A)+ mRNA extracted from Fisher rat embryos and hydroxyapatite chromatography to ensure the best possible representation of the library. Under these experimental conditions, the cloned cDNAs were 10-fold enriched in rare transcripts as compared with the initial cDNA population (see Materials and methods) and the number of clones to be screened was reduced in the same proportion. We then sought to identify targets of c-Jun by differential screening of the subtracted library with radiolabelled probes synthesized from either mock-transfected cells or cells transfected with a c-jun expression vector. These probes were also subtracted with poly(A)+ mRNA extracted from Fisher rat embryos and each hybridized to replicas of the library. The two resulting hybridization signals were compared and 39 clones (out of 4000) giving different signals thereby identified genes potentially regulated by c-jun: 2/3 of them gave a stronger signal with the cDNA probe from c-jun-transfected cells (called 'activated') and 1/3 a stronger signal with the control probe (called 'repressed') (data not shown). The presence of repressed clones in the library is likely to be due in part to their expression in non-transfected cells and in part to the homogenization of transcript abundance
Table I. Characteristics of clones differentially repressed in c-jun transfections Clone
Insert size Similar mouse gene sequencesa
(bp) 19 37 15
70 90 124
Coding region of SPARC (460-530) 3' Non-coding region of SPARC (1880-1970) 3' Non-coding region of TSI (1870-1990)
aThe region of similarity with mRNA sequences of known genes is indicated and in brackets the sequence position is given.
resulting from the subtraction procedure. Complete or partial sequences of the inserts and computer analysis showed that most of them (both activated and repressed) were previously unknown sequences. These clones are currently being characterized. We describe herein an analysis of cDNA inserts from three clones, all of which were repressed by c-Jun (Table I). They present substantial (>80%) sequence similarities with two known mouse genes encoding: (i) thrombospondin (clone 15); (ii) SPARC (clones 19 and 37). The small size of the inserts probably results from the thermal degradation effect of the subtraction procedure used during construction of the library. The sequence differences to known sequences presumably correspond to species differences between rat and mouse. mRNA from control or c-jun- or ras-transfected REF cells were probed with radiolabelled cDNA inserts by Northern blotting. The probe from insert 15 gave a single hybridization signal at 6.3 kb (Figure 1A). Thrombospondins are encoded by a gene family and two of the genes have been characterized: TS 1 and TS2, their transcripts are all -6 kb long in the mouse (Frazier, 1991). Clone 15 presumably corresponds to TS 1, as it displayed no sequence similarity with TS2. Clones 19 and 37 only hybridized with the SPARC sequence and the 2.1 kb mRNA identified (Figure IA) corresponds to the size previously described for SPARC mRNA (Lane and Sage, 1994). Northern blots from the same fibroblast mRNAs probed with full-length TS1 and SPARC cloned cDNAs (gifts of Drs Nischt and Bornstein) hybridized with the same 6.3 and 2.1 kb bands respectively (data not shown). The identity of these genes was further confirmed at the protein level: anti-SPARC and anti-TS 1 antibodies detected the corresponding proteins at their expected sizes (see Figure 7). Thus our cDNA clones correspond to the SPARC and TS 1 genes. Northern blot analysis confirmed that SPARC and TS1 gene expression were inhibited by overexpression of exogeneous c-Jun (Figure 1A and C). Specific signals were quantified (see Materials and methods) and corrected for the amount of RNA loaded using signals from GAPDH mRNA. RSV-c-Jun transfection induced repression of TS1 to 42% and SPARC to 53% of the expression level in mock-transfected cells. Overexpression of an activated ras in REF cells reproducibly repressed the SPARC gene (60% of the control level), but not TS1 (122%). Since the repression levels were relatively high in these transiently transfected cells, we checked whether it was compatible with the transfection efficiency. The most sensitive test we found to measure the proportion of transfected cells was immunofluorescence assays using an anti-p-galactosidase serum after transfection with an RSV-P-Gal construct (Figure lB and C). The mean number of positive cells in
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Fig. 1. Levels of SPARC and TS 1 mRNA after transitory transfections of REF cells with c-jun or ras expression vectors. (A) REF cells were either mock-transfected (C lanes) or transfected with c-jun (J) or ras (R) and incubated for 24 h prior to RNA isolation. Twenty micrograms of total RNA were loaded per lane and blotted, filters were probed with insert 15 (TS1), insert 19 (SPARC) and a GAPDH cDNA as the control. Labelling intensities, corrected for the corresponding GAPDH signal, were normalized to I for signals from the control lane and are given under each lane. Arrows indicate the apparent size (in kb) of the bands. (B) Measurement of transfection efficiency. REF cells were either mock-transfected (left) or transfected with RSV-,B-Gal (right) and fixed 2 days later. Positive cells were revealed by immunofluorescence using anti-,3-galactosidase antibodies. (C) Histogram showing mean labelling intensities of three Northern blots similar to A (ratios of SPARC or TS1 signals to GAPDH signal are given and normalized to 100% for mock-transfected cells) and three immunofluorescence experiments as in B.
three independant experiments was 36%, with higher values in some areas of the plates (Figure IB). This high percentage demonstrated that primary REF cells are good recipient cells for transitory experiments. Nevertheless, this value is not compatible with the repression (by 50% for SPARC and 60% for TS 1 and sometimes higher, as in Figure 1A) exerted by c-Jun on SPARC and TS 1 gene expression, suggesting a complex mechanism involving, for example, a diffusable factor, released by transfected
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cells, capable of mediating repression in non-transfected cells. To confirm that the regulation evidenced in primary cells is a general phenomenon, we studied SPARC and TS 1 expression in FR3T3, an established cell line derived from REF cells (Seif and Cuzin, 1977). mRNA isolated from mock- and c-jun-transfected FR3T3 cells were analysed by Northern blotting (Figure 2A). A probe made with transin cDNA was used as a control for c-Jun-
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activated genes. The amount of transin mRNA increased 9-fold following c-jun transfection. The same mRNA blots were probed for TSI and SPARC transcripts. Consistent with the findings in REF cells, c-jun transfection reduced the amounts of TS 1 mRNA to 72% and SPARC mRNA to 82% of control levels (Figure 2A and B). ras overexpression in FR3T3 cells had the opposite effect of that in primary cells: repression of TS1, but not SPARC (Figure 2B). These contrasting results obtained with Ras could be due to the differences in the endogenous AP1 components available and/or in the Ras-regulated pathways in REF and FR3T3 cells. The measured transfection efficiency in FR3T3 (19%) was again not compatible with the repression observed after c-jun transfection (for example, 30% repression in the case of TS1). These results indicate that, after transitory transfection, the c-Jun-induced repression of these genes is not restricted to primary cells but is also reproduced in an established fibroblast cell line. They also further suggest the involvement of a diffusable factor mediating c-Jun repression. To test this hypothesis, FR3T3 cells were either mocktransfected or transfected overnight with the c-jun vector and the medium changed the following day. After 24 h the medium was recovered, diluted with one volume of fresh medium and given to non-transfected fresh FR3T3 cells. mRNA was isolated 8 h later and analysed by Northern blotting (Figure 3A). Specific signals were quantified. The conditioned medium from c-jun-transfected cells induced repression of SPARC and TS I gene expression (to 52 and 77%, respectively, of signals obtained with control cells) to levels similar to those observed in transfected cells themselves. No change in transin (data not shown) and GAPDH mRNA levels were detected.
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Serum and oncogene products have opposite effects on the regulation of SPARC and TS1 genes To understand further the regulation of SPARC and TS 1 by c-Jun we analysed the effects of different stimuli or oncogene products known to act upstream or in association with c-Jun. Serum stimulation leads to an increase in both c-jun and TS1 transcripts (Bornstein, 1992; Karin, 1992). The serum-responsiveness of SPARC is unknown, but SPARC gene expression is activated by TGF, stimulation (Wrana et al., 1991). We therefore tested the response of this gene to serum in our cellular system. After serum starvation, REF cells were stimulated by addition of 20% serum. RNA was extracted at various times and was analysed by Northern blotting (Figure 4A). The SPARC mRNA level was higher after a 1 h serum stimulation. Hence, SPARC and TS1 belong to the immediate-early class of genes stimulated by the serum. Furthermore, in REF cells, serum stimulation and c-jun overexpression appear to have opposite effects on the regulation of the SPARC and TSl genes. We investigated whether endogenous c-Jun has the same effect as transfected c-Jun on SPARC and TS 1 expression and tested the effects of a more specific c-Jun activator, the tumour promotor 1 2-O-tetradecanoylphorbol- 1 3-acetate (TPA). TPA directly activates c-Jun protein via a post-translational mechanism (Boyle et al., 1991). FR3T3 cells were treated with TPA for 2, 6 and 24 h and mRNA was analysed by Northern blotting (Figure 4B). Although detectable after 6 h of TPA treatment, SPARC and TS 1 repression were maximal only after 24 h. Transin induction is mediated by the c-Fos-c-Jun heterodimer. We therefore also checked the effects of c-Fos 5671
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Fig. 3. The effects of c-Jun are mediated by a secreted factor. (A) Conditioned medium from either mock- (C) or c-jun-transfected (J) FR3T3 cells was harvested and incubated with normal FR3T3 cells (see text). Northern blots were hybridized with the three probes together. (B) Co-cultures of FR3T3 and either FRcJ-4 or FREJRas cells. The indicated number of cells (upper panel) of each cell line were seeded together and RNA was isolated 44 h later. Northern blots were hybridized with the three probes together. Bands intensities (expressed as a percentage of FR3T3 alone values and corrected for GAPDH) of the respective corresponding signals are given for TSI and SPARC (lower panel). The 'cell num. corr.' quantifications take into account the doubling time differences between the cell lines (see Table II) and correspond to values corrected (X2 in the case of 5x 105 transformed cells and X3 for I X 106) for the dilution of signals due to the presence of transformed cells.
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