Materials and methods

Reagents

Fetal bovine serum (FBS) was purchased from Gibco BRL (Rockville, MD), mithramycin and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) were obtained from Sigma (St Louis, MO), and okadaic acid was purchased from Upstate Biotechnology (Bedford, MA).

Cell culture

Human dermal fibroblasts derived from a 2-mo-old child (GM05756A) were obtained from Coriell Cell Repositories (Camden, NJ) and propagated in Dulbecco's minimal Eagle's medium (DMEM) supplemented with 10% FBS. Human adult dermal fibroblasts were obtained from healthy volunteers, following institutional approval and informed consent. Primary explant cultures were established in 25 cm² culture flasks in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 50 g amphotericin per ml. Fibroblast cultures independently isolated from different individuals were maintained as monolayers at 37°C in 90% air, 10% CO₂, and studied between the third and sixth subpassages. Cell viability was determined by trypan blue stain (Gibco BRL).

Plasmid constructions

The deletion and substitution mutant plasmids have been previously described (^{Ihn et al, 1996,1997}). Substitution mutations were generated using Quick Change site-directed mutagenesis kit (Stratagene) and confirmed by sequencing. Plasmids used in transient transfection assays were twice purified on CsCl gradients. At least two different plasmid preparations were used for each experiment.

Transient transfections and chloramphenicol acetyltransferase (CAT) assays

Human fibroblasts were grown to 90% confluence in 100 mm dishes in DMEM with 10% FBS. Monolayers were washed, and cells were transfected by the calcium phosphate technique (^{Ihn et al, 1996}) with 20 g of various deletion or mutant promoter–CAT constructs. pSV--galactosidase control vector (Promega, Madison, WI) was cotransfected to normalize for transfection efficiency. After incubation overnight, the medium was replaced with DMEM containing 0.1% bovine serum albumin or with DMEM containing 10% FBS, and incubation was continued for 48 h. Cells were harvested in 0.25 M Tris-HCl (pH 8) and fractured by freeze thawing. Extracts, normalized for protein content as measured by the Bio-Rad reagent, were incubated with butyryl-CoA [¹⁴C]chloramphenicol for 90 min at 37°C. Butyrated chloramphenicol was extracted using an organic solvent (2:1 mixture of tetramethylpentadecane and xylene) and quantitated by scintillation counting. Each experiment was performed in duplicate. In order to normalize for small variations in transfection efficiencies, the data were standardized with -galactosidase activity using a -Galactosidase Enzyme Assay System (Promega). The Mann–Whitney U test was used to determine statistical significance.

Preparation of nuclear extract

Nuclear extracts were prepared as described previously (Andrews et al, 1991;^{Ihn et al, 1996}). Briefly, confluent cells from five 150 mm dishes were washed with phosphate buffer saline (PBS) and scraped into 1 ml of cold buffer A (10 mM HEPES-KOH pH 7.9 at 4°C, 1.5 mM MgCl₂, 10 mM KCl, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate). The cells were allowed to swell on ice for 10 min and then vortexed for 10 s. After centrifugation for 3 min, the supernatant was discarded. The pellet was resuspended in 80 l of cold buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM ethylenediamine tetraacetic acid) and incubated on ice for 20 min for high salt extraction. Cellular debris was removed by centrifugation for 2 min at 4°C and the supernatant fraction was stored at -80°C until use. The protein concentration of the extract was determined using the Bio-Rad reagent.

DNA mobility shift assays

DNA mobility shift assays were performed as described (^{Ihn et al, 1997}). Radioactive probes (132-mer) were generated by polymerase chain reaction using [-³²P]adenosine triphosphate end-labeled primers or direct end-labeling of oligonucleotide probes. For DNA mobility shift assay, the binding reaction was carried out for 30 min in 20 l of binding buffer containing 10 000 cpm of labeled probe, 2 g of poly(dI-dC)poly(dI-dC), and nuclear extracts containing 5 g of protein. Where indicated, specific synthetic oligonucleotides or antibodies were included in the reaction mixture. Separation of free radiolabeled DNA from DNA–protein complexes was carried out on a 5% nondenaturing polyacrylamide gel in a 0.5 Tris borate electrophoresis buffer at 200 V at 4°C. Autoradiography was performed by overnight exposure to Kodak XOMAT film with intensifying screens at -80°C. The densities of bands were measured using a phosphoimager scanner.

RNA preparation and northern blot analysis

Fibroblasts were grown to confluence in DMEM supplemented with 10% FBS and then incubated for 24 h in serum free medium (DMEM plus 0.1% bovine serum albumin) before addition of serum. Total RNA was extracted and analyzed by northern blotting as described previously (^{Ihn et al, 1997}). Filters were sequentially hybridized with radioactive probes for 2(I) procollagen, Sp1, Sp3 and glyceraldehyde-3-phosphate dehydrogenase. The filters were scanned with a Phosphoimager (Molecular Dynamics, Sunnyvale, CA).

Western blot analysis

Nuclear extract (15 g) was electrophoresed in 7.5% sodium dodecyl sulfate-polyacrylamide gel and transferred on to nitrocellulose filters and probed with anti-Sp1 or anti-Sp3 antibody (Santa Cruz) as described previously (^{Ihn and Trojanowska, 1997}). Then the filters were incubated for 30 min with horseradish peroxidase-conjugated goat antirabbit IgG. Sp1 and Sp3 were detected using an enhanced chemilunescent system (Amersham) according to the manufacturer's recommendations. The densities of bands were measured using densitometer.

Immunoprecipitation

Fibroblasts were washed three times with PBS and scraped into 500 l of lysis buffer (50 mM Tris-HCl pH 8.0 at 4°C, 150 mM NaCl, 2 mM ethylenediamine tetraacetic acid, 50 mM NaF, 1% Triton X-100, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 g each of aprotinin, leupeptin, and pepstein per ml). The cells were incubated at 4°C for 30 min with rotation. After centrifugation for 15 min, the supernatant was transferred to a fresh microcentrifuge tube and the extract was precleared with 20 l of protein A-sepharose for 1 h with rotation. The beads were pelleted, the supernatant was transferred to a new tube, and 20 1 of protein A-sepharose beads conjugated to Sp1-specific antibody was added. Immunoprecipitation was performed overnight at 4°C with rotation, after which the immunoprecipitated proteins were washed four times with lysis buffer. At the last wash, the beads were transferred to a new tube, resuspended in 30 l of sample buffer, and boiled for 5 min. The samples were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel and transferred on to nitrocellulose filters and probed with anti-Sp1 (Santa Cruz) or antiphosphoserine specific antibody (Zymed Laboratories). Sp1 and serine-phosphorylated Sp1 were detected in the same filters using an enhanced chemilunescent system as described above.

Results

Serum upregulates transcriptional activity of the human 2(I) collagen gene expression

Previous studies have shown that serum regulates collagen synthesis at the transcriptional level (^{Narayanan and Page, 1977,1987}). To investigate the effect of serum on the transcriptional activity of the human 2(I) collagen gene, we tested the 3.5 kb fragment of the human 2(I) collagen promoter linked to the CAT reporter gene in transient transfection assays. Serum induced a concentration-dependent increase in promoter activity of the 2(I) collagen gene, with maximum stimulation observed at 10% Figure 1. Serum stimulation resulted in an up to 6-fold induction of promoter activity. The potency of serum was greater than that of TGF- under similar culture conditions Figure 1.

Figure 1.

Serum induces concentration-dependent increase in promoter activity of the human 2(I) collagen gene. The plasmid carrying the -3500 kb fragment of the human collagen promoter sequence cloned upstream from the CAT reporter gene was transiently transfected into human fibroblasts as described under Materials and Methods. On the day after transfection, some dishes were incubated with various concentrations of serum or 3 ng TGF- per ml without serum for 24 h, whereas control dishes received medium only. Comparisons of promoter activity in response to serum were made between unstimulated and serum-stimulated promoter activities. Asterisks indicate statistically significant results (p <0.01).

Full figure and legend (14K)

Functional mapping of the serum response element in the 2(I) collagen promoter

To analyze the transcriptional regulation of the collagen gene by serum, we tested a series of 5'-deletions of the human 2(I) collagen promoter linked to the chloramphenicol acetyltransferase reporter gene in transient transfection assays Figure 2. Deletions to bp -353 did not significantly alter the level of inducibility, but further deletion to bp -264 abolished by half the serum stimulation. Subsequent deletion to bp -148 had no additional effect on serum stimulation, but deletion up to bp -108 abolished serum stimulation completely. Deletion of the GC-rich element (a Sp1/Sp3 binding site) between bp -353 and -264 significantly decreased not only basal promoter activity (^{Tamaki et al, 1995}) but also serum stimulation. Deletion to bp -148 increased promoter activity about 2.8-fold compared with the deletion to -186. These results further confirmed the location of the previously mapped repressor site between bp -164 and -159 (^{Ihn et al, 1996}). Subsequent deletion to bp -108 caused a decrease in basal promoter activity as well as serum induction. These effects most likely result from the removal of the previously identified positive constitutive cis-element containing a TCCTCC motif (another Sp1/Sp3 binding site) located between bp -128 and -123 in this promoter (^{Ihn et al, 1997}). As deleting between bp -353 and -264, or between bp -148 and -108 promoter fragment abolished by half the serum response, we tested whether these cis-elements also decrease the serum stimulation of collagen promoter activity using substitution mutants in the GC-rich element and/or the TCCTCC motif. As shown in Figure 3, collagen promoter constructs carrying substitution mutations in the TCCTCC motif or the three GC boxes abolished half the serum response. Double mutations in the TCCTCC motif and the three GC boxes were unresponsive to serum stimulation. Furthermore, none of the substitution mutations in other response elements previously identified in this promoter affected serum stimulation Figure 3.

Figure 2.

Functional mapping of the serum response elements in the a2(I) collagen promoter. Plasmids containing various lengths of the 2(I) collagen promoter sequence cloned upstream from the CAT reporter gene were transiently transfected into human fibroblasts. On the day after transfection, some dishes were incubated with 10% serum in DMEM for 24 h, whereas control dishes received medium only. Relative CAT activity indicates the human 2(I) collagen activity relative to untreated cells (serum free), whose value was set at 100. The diagrams on the left show the deletion end-points. The numbers on the right show the basal and serum-stimulated promoter activities of each deletion construct relative to -353 promoter, whose value was set at 100. The mean SE values for separate experiments are also shown. The number of experiments used to calculate the mean is shown in parentheses. Comparisons of promoter activities in response to serum were made between unstimulated and serum-stimulated promoters separately for each deletion construct. Asterisks indicate statistically significant results (p <0.001).

Full figure and legend (10K)

Figure 3.

The GC boxes and the TCCTCC motif mediate serum stimulation of the human 2(I) collagen promoter. Plasmids carrying substitution mutations (^{Ihn et al, 1996}) were used in transient transfections of cells stimulated with 10% serum/DMEM. Sequences with the mutated nucleotides are shown on the left. The stimulation ratio for serum is shown on the right. The mean SE for separate experiments are shown. The number of experiments used to calculate the mean is shown in parentheses. Comparisons were made between untreated and serum treated cells. Asterisks indicate statistically significant results (p <0.001).

Full figure and legend (15K)

As previously reported, the GC-rich element contains three GC boxes that contribute the basal transcriptional activity of the collagen promoter (^{Tamaki et al, 1995;Ihn et al, 1996;Ihn and Trojanowska, 1997}). We have tested the effects of the mutations in the individual GC boxes on serum stimulation. Mutating the individual GC boxes did not have an appreciative effect on serum stimulation Figure 4. A significant decrease in the promoter activity was observed with a double mutant of the second and third GC boxes, and almost half of the serum stimulation was abolished by mutating all three GC boxes Figure 4.

Figure 4.

Effect of serum on the GC-box region. The 2(I) collagen promoter construct carrying substitution mutations in the GC boxes located between -310 and -270 were transfected into human fibroblasts with or without serum stimulation. Mutated GC boxes are in black. Asterisks indicate statistically significant results (p <0.001).

Full figure and legend (13K)

Serum stimulation does not affect nuclear protein binding to the GC-rich element or the TCCTCC motif in the collagen promoter

To determine whether DNA–protein interactions in these regions are regulated by serum, we performed DNA mobility shift assays using the promoter fragment from bp -313 to -183 or bp -135 to -116 and nuclear extracts from human fibroblasts treated with serum up to 24 h. As shown in Figure 5(a, b), no changes in the DNA–protein complexes were observed in either of these regions. Furthermore, no changes in the DNA–protein complexes were observed even after the treatment with serum for 12 h or 24 h (data not shown).

Figure 5.

Serum treatment does not change DNA-protein binding in the human collagen promoter. Representative results of DNA mobility shift assays. Nuclear extracts were prepared from human fibroblasts that had been either incubated in serum free medium (lane 2) or stimulated with 10% FBS for the indicated time (lanes 3–5) up to 24 h and used in binding reactions with 5'-end-labeled collagen promoter fragment bp -313 to bp -183 (A) or bp -135 to -116 (B) probe as described under Materials and Methods. Lane 1 contains no nuclear extract. (C) Gel shift analyses were performed with 5'-end-labeled collagen promoter fragment bp -313 to bp -183 with or without anti-Sp1 or anti-Sp3 antibodies. Lane 1 contains no nuclear extract. (D) Gel shift analyses were performed with 5'-end-labeled collagen promoter fragment bp -135 to -116 in the absence (lane 3) or the presence of wild-type competitor (lane 1), anti-Sp1 antibodies (lane 2), anti-Sp3 antibodies (lane 4), or IgG (lane 5).

Full figure and legend (101K)

To further determine the nature of the nuclear proteins interacting these regions, we performed gel shift analyses with competitor oligonucleotides or antibodies against Sp1 or Sp3. As shown in Figure 5(c), collagen promoter fragment from -313 to -183 binds Sp1 and Sp3. Anti-Sp1 antibody supershifted the upper complex (lane 3) and anti-Sp3 antibody specifically interferes formation of the middle complex (lane 4), as demonstrated previously (^{Ihn and Trojanowska, 1997}). Simultaneous addition of the anti-Sp1 and anti-Sp3 antibodies abolished formation of these complexes (lane 5). Addition of IgG did not affect formation of the DNA–protein complex (lane 6). TCCTCC motif located between bp -128 and -123 also binds Sp1 and Sp3 Figure 5d. Five DNA–protein complexes were detected when gel shift assays were performed with TCCTCC motif and nuclear extracts. The same complex formation pattern has been shown before and three upper complexes can be competed off by unlabeled specific competitor oligonucleotide (Figure 5d, lane 1), but not by random sequences (^{Ihn et al, 1997}). The forth complex may represent nonspecific binding of the proteins to this promoter region, because this complex was competed off by random sequences (^{Ihn et al, 1997}) and appeared variably (^{Ihn et al, 1996}). Addition of the anti-Sp1 antibody caused the upper complex to be supershifted (Figure 5d, lane 2). Addition of the anti-Sp3 antibody prevented formation of the second and the third complexes specifically (lane 4), as demonstrated previously (^{Ihn and Trojanowska, 1997;Ihn et al, 1997}). These results suggest that Sp1 and Sp3 interact with these regions.

Furthermore, serum stimulation did not affect protein binding to other regions of the collagen promoter: a longer collagen promoter segment from bp -313 to -109 was used in DNase I protection and DNA mobility shift assays, and there was no difference in binding patterns between serum-stimulated and control extracts (data not shown).

Serum stimulation does not affect mRNA or protein synthesis of the transcription factor Sp1 or Sp3

We reported that both the GC-rich element and the TCCTCC motif constitute binding sites for the transcription factors Sp1 and Sp3 (^{Ihn et al, 1997;Ihn and Trojanowska, 1997}). Therefore, we investigated the effect of serum on mRNA or protein synthesis of Sp1 and Sp3. As shown in Figure 6(a), serum does not affect mRNA levels of Sp1 or Sp3 (1.1 0.2-fold, 0.9 0.3-fold, respectively). As shown in Figure 6(b), immunoblot analysis revealed that serum does not affect protein levels of Sp1 or Sp3 (1.2 0.3-fold, 1.1 0.2-fold, respectively).

Figure 6.

(A) Northern blot analysis of Sp1, Sp3, and 2(I) collagen gene expression in human dermal fibroblasts stimulated with serum. Comparisons of Sp1, Sp3, and 2(I) collagen mRNA expression in response to serum were made between treated and untreated cells. Fibroblasts were treated with serum for 24 h. Representative results of four experiments are shown. (B) Immunablot detection of Sp1 and Sp3 in human dermal fibroblasts stimulated with serum. Nuclear proteins were electrophoresed through an 8% SDS polyacrylamide gel, transferred to nitrocellulose membrane and probed with anti-Sp1 or anti-Sp3 antibody. Representative results of four experiments are shown. Lane 1, nuclear extract from dermal fibroblasts untreated with serum; lane 2, nuclear extract from dermal fibroblasts treated with serum for 24 h.

Full figure and legend (29K)

Mithramycin blocks serum stimulation of the human 2(I) collagen gene expression

Mithramycin is known as a specific inhibitor of Sp1 binding (^{Blume et al, 1991}). First, the effect of mithramycin on the binding of Sp1 and Sp3 to the 2(I) collagen promoter, gel shift analyses were performed using nuclear extracts with or without addition of mithramycin. As shown in Figure 7(a), mithramycin abolished the binding of Sp1 to the collagen promoter, but not the binding of Sp3. Therefore, this reagent was used to determine the role of Sp1 in serum stimulation of 2(I) collagen promoter activity. Consistent with the reported role of Sp1 in maintaining constitutive expression of the human 2(I) collagen gene (^{Ihn and Trojanowska, 1997}), mithramycin treatment decreased basal promoter activity of the human 2(I) collagen gene up to 40% Figure 7b. Serum stimulation of this promoter activity was abolished in a concentration-dependent manner. Furthermore, serum stimulation of the human 2(I) collagen mRNA expression was also abolished by mithramycin in a concentration-dependent manner Figure 7c. These results suggest that Sp1 is involved in serum stimulation of the human 2(I) collagen expression.

Figure 7.

Mithramycin blocks serum stimulation of the human 2(I) collagen gene expression.(A) Representative results of DNA mobility shift assays. Nuclear extracts were prepared from human fibroblasts that had been either incubated without mithramycin (dimethyl sulfoxide only, lane 1) or with mithramycin dissolved in dimethyl sulfoxide (lane 2) and used in binding reactions with 5'-end-labeled collagen promoter fragment bp -313 to bp -183. (B) The plasmid carrying the -353 deletion of the human 2(I) collagen promoter was used in transient transfections of cells stimulated with or without 10% serum/DMEM. After transfection, some dishes were incubated with various amounts of mithramycin, whereas control dishes received dimethyl sulfoxide only. Cell viability was determined by trypan blue stain. Comparisons were made between untreated and mithramycin-treated cells. Asterisks indicate statistically significant results (p <0.001). (C) Northern blot analyses of the 2(I) collagen gene in the absence or presence of various amounts of mithramycin. The histograms summarize the data of four independent experiments. Asterisks indicate statistically significant results (p <0.01).

Full figure and legend (28K)

H7, a serine/threonine kinase inhibitor, abolishes serum stimulation of the human 2(I) collagen promoter activity

Recent studies showed that serum increases Sp1 phosphorylation due to serine phosphorylation (^{Alroy et al, 1999;Black et al, 1999}). H7 is known as a specific serine/threonine kinase inhibitor. This reagent was used to determine that serine phosphorylation is involved in serum response. As shown in Figure 8(a), H7 treatment abolished serum response in a concentration-dependent manner; however, okadaic acid, a specific serine/threonine phosphatase inhibitor, did not affect serum response Figure 8b.

Figure 8.

H7, a serine/threonine kinase inhibitor, abolishes serum stimulation of the human 2(I) collagen promoter activity. The plasmid carrying the -353 deletion of the human 2(I) collagen promoter was used in transient transfections of cells stimulated with or without 10% serum/DMEM. After transfection, some dishes were incubated with various amounts of H7 (A) or okadaic acid, a specific serine/threonine phosphatase inhibitor (B), whereas control dishes received DMSO only. Cell viability was determined by trypan blue stain. Comparisons were made between untreated and H7 or okadaic acid treated cells. Asterisks indicate statistically significant results (p <0.001).

Full figure and legend (9K)

Serum stimulation increased Sp1 phosphorylation in dermal fibroblasts

Sp1 phosphorylation was assessed in human dermal fibroblasts following serum stimulation using immunoprecipitation. Serine phosphorylation was determined using anti-phosphoserine specific antibody. As shown in Figure 9, serum stimulation of fibroblasts led to an increased Sp1 phosphorylation (2.7 0.5-fold, n = 4), whereas Sp1 levels were not affected. Furthermore, H7 abolished increased Sp1 phosphorylation by serum (Figure 9, lane 3). Serum stimulation of fibroblasts led to an increased Sp1 serine phosphorylation in a concentration-dependent manner, which was maximum at 10% (data not shown).

Figure 9.

Serum stimulation increased Sp1 phosphorylation in dermal fibroblasts. Sp1 phosphorylation was assessed in human dermal fibroblasts following serum stimulation in the absence or presence of H7 using immunoprecipitation. Serine phosphorylation was determined using antiphosphoserine specific antibody. Sp1 levels were determined using anti-Sp1 antibody. IP, immunoprecipitation; Sp1, anti-Sp1 antibody; phosphoserine, antiphosphoserine antibody.

Full figure and legend (16K)

Discussion

Several cytokines have been reported to be important modulators of collagen synthesis in human dermal fibroblasts. TGF- (^{Rossi et al, 1988}), OSM (^{Ihn et al, 1997}), interleukin-4 (^{Postlethwaite et al, 1992}), and serum (^{Narayanan et al, 1997}) upregulate collagen production, whereas interferon- (^{Kahari et al, 1990}), TNF- (^{Kahari et al, 1990}), and interleukin-10 (^{Reitamo et al, 1994}) downregulate it. Previous studies have characterized response elements of these cytokines in the human 2(I) collagen promoter. TGF- and TNF- have been shown to share the same response element of this promoter, which was mapped between bp -378 and -255 containing three Sp1 binding sites and CCAAT/enhancer-binding proteins binding sites (^{Inagaki et al, 1994,1995;Greenwel et al, 1997,2000}). In a different study, however, TGF- and TNF- response element was mapped to a bp -265 to -241 region that contains an AP-1 binding site and an nuclear factor-KB binding site (^{Chung et al, 1996;Kouba et al, 1999}). The reason for the discrepancy between these studies is presently not clear. We characterized an OSM response element in the human 2(I) collagen promoter that is the Sp1/Sp3 binding site located between bp -128 and -123 (^{Ihn et al, 1997}). The response element mediating modulation of 2(I) collagen promoter activity by other cytokines has not been reported.

In this study, we have characterized serum response elements in the human 2(I) collagen promoter, which overlap an OSM response element and a TGF-/TNF- response element. We have also shown that the serum response elements map to the same promoter region as the previously characterized constitutive response element (^{Ihn et al, 1996}). Previous studies showed that these response elements are binding sites for Sp1/Sp3 (^{Ihn et al, 1997;Ihn and Trojanowska, 1997}). Our previous study has also shown that constitutive activity of the human 2(I) collagen promoter is regulated equivalently by the three positive cis-acting elements at bp -300 (GC-boxes), -125 (TCCTCC motif), and -80 (CBF binding site) (^{Ihn et al, 1996}). Furthermore, our previous data suggested that a repressor that binds to a promoter region located between the GC boxes and the TCCTCC motif interferes with the activation of the promoter via these two positive cis-elements, but activation by the third positive response element that binds CBF seemed to be unaffected by this repressor (^{Ihn et al, 1996}). This study showed that serum utilises two of the three positive response elements of the human 2(I) collagen promoter. These results suggest that the human collagen promoter contains multiple Sp1/Sp3 binding sites that are also response elements of several cytokines and that these multiple Sp1/Sp3 binding sites may be necessary to ensure high levels of expression of this abundant protein.

Serum is a complex mixture of biomolecules that exert various physiologic effects on dermal fibroblasts. Its major components include various plasma proteins, peptides, lipids, carbohydrates as well as growth factors and cytokines, including TGF-. The first serum response element of the collagen promoter contains three GC-rich motifs and overlap with a previously characterized TGF- response element (^{Inagaki et al, 1994}); however, TGF-, which is mostly present in serum in its latent form (^{O'Connor-McCourt and Wakefield, 1987}), does not seem to mediate serum response, because a pan-specific TGF- antibody (R&D Systems) did not inhibit serum stimulation (data not shown). Moreover, TGF- treatment has been shown to increase binding of nuclear factors to the -315–284 region (^{Inagaki et al, 1994}), but in our study no increase was observed after serum treatment to the same region Figure 5a.

The c-fos promoter has been studied extensively as a model of growth factor regulated promoter. Serum is known to induce the transcription of c-fos gene via the serum response element (SRE), which is centered 310 bp upstream of the start site of c-fos transcription (^{Treisman, 1985}). At the SRE, a ternary complex forms that contains serum response factor (SRF) (^{Norman et al, 1988}) and an Ets domain protein, ternary complex factor (TCF), which can only bind the SRE via interaction with SRF (^{Shaw et al, 1989}). The TCF are regulated by MAP kinase phosphorylation in response to extracellular signals (^{Gille et al, 1992}). Another transcription factor MEF2 is also known to mediate serum induction, especially of c-jun gene (^{Han and Prywes, 1995}); however, the mechanism of serum induction of c-jun gene transcription via MEF2 is presently not clear.

In this study, serum induction of the human 2(I) collagen gene was demonstrated to be regulated via Sp1/Sp3-binding sites. Serum response elements of the human 2(I) collagen promoter were mapped to Sp1/Sp3 binding sites and serum induction was blocked by mithramycin, an inhibitor of Sp1 binding. Sp1 is a ubiquitous transcription factor that regulates the constitutive activity of many genes. Furthermore, several recent reports suggest that Sp1 is involved in mediating responses to various environmental stimuli, including epidermal growth factor stimulation of gastrin gene (^{Merchant et al, 1995}), and cyclic adenosine monophosphate-dependent stimulation of the CYP11A gene (^{Venepally and Waterman, 1995}). Sp1 is also known to be involved in mediating the serum signal to other genes that lack SRE (^{Azizkhan et al, 1993;Xie and Herschman, 1996}). In these previous studies, serum stimulation did not affect nuclear protein binding to the response element. In this study, serum did not affect Sp1 or Sp3 binding to the GC-rich element or the TCCTCC motif in the human 2(I) collagen promoter. Expression levels of transcription factors can modulate the transcription of some genes (^{Kubo et al, 1995}). Serum stimulation, however, does not affect mRNA or protein synthesis of Sp1 or Sp3 in this study.

Recent studies showed that serum increases Sp1 phosphorylation due to serine phosphorylation (^{Alroy et al, 1999;Black et al, 1999}). In this study, serum increases Sp1 phosphorylation also in dermal fibroblasts. Sp1 can be phosphorylated in vitro by protein kinase CK2 (^{Armstrong et al, 1997}), DNA-dependent protein kinase (^{Jackson et al, 1990}), and protein kinase A (^{Rohlff et al, 1997}). Sp1 can be phosphorylated at multiple sites by these kinases, which are likely to be important in regulation of its activity in response to diverse signals.

^{Greenwel et al (1995)} reported that tyrosine dephosphorylation of an Sp1-containing complex induces the 2(I) collagen transcription. Moreover, they also showed that H7, a serine/threonine kinase inhibitor, abolished TGF-1 induction of the 2(I) collagen gene expression. In this study, H7 treatment abolished serum response Figure 8a, but okadaic acid did not affect serum response Figure 8b. These results, including ours, suggest that the signals that induce tyrosine dephosphorylation and/or serine phosphorylation of Sp1-containing complexes induce the 2(I) collagen transcript. Further studies are needed to understand the signaling pathways that regulate collagen genes.

Lack of increased levels or DNA binding of Sp1 indicates that serum stimulation is likely to involve alterations in the association of Sp1 with other proteins. Our previous study suggested that OSM affects the interaction of Sp1 with histones or other chromatin components (^{Ihn et al, 1997}). Such a mechanism has also recently been reported in TGF-/TNF- regulation of the mouse 2(I) collagen promoter, in which cytokine treatment affected the interaction of CTF/NF1 with histone H3 (^{Alevizopoulos et al, 1995;Alevizopoulos and Mermod, 1996}). At this point, further studies are needed to understand the transcriptional mechanism of serum induction of the human 2(I) collagen gene.

In conclusion, this study characterized the cis-regulatory elements in the 2(I) collagen promoter that mediate activation of this gene in response to serum, which are binding sites for Sp1. Both response elements (the GC-rich element and the TCCTCC motif) contribute equivalently to constitute expression of this gene (^{Ihn et al, 1996}) and each has been implicated in mediating TGF-/TNF- stimulation (^{Inagaki et al, 1994,1995}) or in mediating OSM stimulation (^{Ihn et al, 1997}). The results of this study further support a role for Sp1, the GC-rich element, and the TCCTCC motif not only in the regulation of basal transcription of the human 2(I) collagen gene, but also in the modulation of the expression of this gene in response to various environmental factors.

References

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