Biomedical subjects

M Trojanowska

Publications and source records attributed to M Trojanowska.

At least 19 recordsLinked to original sources

PubMed2006

Endovascular repair of abdominal aortic aneurysms in patients with severe angulation of the proximal neck using a flexible stent-graft: European Multicenter Experience.

AIM: The aim of this paper was to report the results of a multicenter study on endovascular repair of abdominal aortic aneurysms (AAA) in patients with important angulation of proximal neck using a flexible stent-graft (Aorfix). METHODS: Endovascular repair of AAA using a flexible stent-graft was performed at 16 centers in 29 patients with angulation of proximal neck greater than 45 degrees. Twenty-three patients (79%) had angulation greater than 60 degrees and were therefore contraindicated for repair with other contemporary devices. RESULTS: Technical success was achieved in all but one case (96%). There was one postoperative death due to multiorgan failure following revision of groin wound for hemorrhage. No patients were converted to open repair. One patient had persisting proximal endoleak despite placement of proximal extension. One patient in whom wireform fractures had been detected died from ruptured aneurysm at nearly 4 year follow-up. CONCLUSION: Endovascular repair using a flexible stent-graft is feasible in patients with highly angulated necks. This stent-graft allows the possibility of a to offer repair for patients un-suitable for the currently available commercial grafts. Mid-term results are acceptable and need to be confirmed by longer follow-up and larger series.

Aged↗

PubMed2001

The inhibitory effects of camptothecin, a topoisomerase I inhibitor, on collagen synthesis in fibroblasts from patients with systemic sclerosis.

The main manifestation of systemic sclerosis (SSc) is the overproduction of extracellular matrix, predominantly type I collagen. This study was undertaken to evaluate the effects of noncytotoxic doses of the topoisomerase I inhibitor camptothecin (CPT) on collagen production in the activated dermal fibroblasts from patients with SSc and healthy donors. The fibroblasts were cultured in the presence or absence of CPT. Production of collagenous proteins by fibroblasts was determined in cell and matrix layers by ELISA and in conditioned media by [(3)H]proline incorporation, gel electrophoresis, and autoradiography. Expression of alpha2(I) collagen (COL1A2) mRNA was measured by northern blot, and the activity of COL1A2 promoter was determined by a chloramphenicol acetyltransferase assay. CPT (10(-7) M) decreased the deposition of type I collagen by 68%, of type III by 38%, and of type VI by 21% in SSc fibroblasts and to a lesser degree in healthy controls. Similarly, CPT (10(-8) M to 10(-6) M) significantly inhibited secretion of newly synthesized collagenous proteins into conditioned media by 50%. CPT (10(-8) M to 10(-6) M) caused a significant dose-dependent inhibition of COL1A2 mRNA levels and COL1A2 promoter activity, both by as much as 60%. The inhibitory effect of CPT on collagen production by fibroblasts from patients with SSc suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.

Adult↗

PubMed2001

Fli-1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent pathway.

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of this study was to investigate the role of Ets factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen alpha2(I) (COL1A2) mRNA was inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In vitro binding assays demonstrate that both Fli-1 and Ets-1 form DNA-protein complexes with sequences present in COL1A2 promoter. Furthermore, Fli-1 binding to the COL1A2 is enhanced via Sp1-dependent interaction. Studies using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (via protein-protein interaction) mechanisms and that Sp1 is an important mediator of the Fli-1 function. Furthermore, experiments using the Gal4 system in the context of different cell types as well as experiments with the COL1A2 promoter in different cell lines demonstrate that the direction and magnitude of the effect of Fli-1 is promoter- and cell context-specific. We propose that Fli-1 inhibits COL1A2 promoter activity by competition with Ets-1. In addition, we postulate that another factor (co-repressor) may be required for maximal inhibition after recruitment to the Fli-1-Sp1 complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins ultimately control ECM production in fibroblasts.

Base Sequence↗

PubMed2001

Spl phosphorylation induced by serum stimulates the human alpha2(I) collagen gene expression.

Serum has been known to stimulate collagen production by dermal fibroblasts. As part of an ongoing study of the molecular mechanisms of collagen production, we have investigated transcriptional regulation of the human alpha2(I) collagen gene by serum in human dermal fibroblasts. Serum responsive elements were mapped by deletion analysis between bp -353 and -264, and between -148 and -108 in the alpha2(I) collagen promoter. Further functional analysis of the alpha2(I) collagen promoter containing various substitution mutations revealed that serum stimulation of this promoter is mediated equally by a GC-rich region located between bp -303 and -271 and by the TCCTCC motif located between bp -123 and -128, both of which constitute binding sites for transcription factor Spl and Sp3. No differences were observed in electrophoretic mobility shift assays between unstimulated and serum stimulated fibroblasts. The Spl inhibitor mithramycin blocked stimulation of the alpha2(I) collagen promoter activity by serum. Furthermore, immunoprecipitation analysis showed that serum stimulation increased Spl phosphorylation. In conclusion, this study characterized response elements that mediate serum stimulation of the human alpha2(I) collagen promoter and suggests that serum stimulation was mediated via Sp1/Sp3 binding sites in this promoter.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2001

Differential regulation of transforming growth factor-beta receptors type I and II by platelet-derived growth factor in human dermal fibroblasts.

BACKGROUND: Elevated expression of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta have been observed in a number of fibrotic diseases, including systemic sclerosis (SSc). This suggests a possible interaction between these factors in establishing a profibrotic programme in dermal fibroblasts. OBJECTIVES: To examine the effects of PDGF isoforms on the expression of TGF-beta receptors in human dermal fibroblasts. METHODS: Steady-state mRNA levels of TGF-beta receptor I and II (TbetaR-I and TbetaR-II) were analysed by northern blot. TbetaR-I protein levels were analysed by immunoprecipitation of 35S metabolically labelled cells. TbetaR-II protein levels were analysed by western blot. RESULTS: Steady-state mRNA levels of TbetaR-I and TbetaR-II were induced in response to PDGF isoforms. PDGF-AA and PDGF-AB stimulated both receptors with similar potency, whereas PDGF-BB was less potent. The MEK1 (mitogen-activated protein kinase [MAPK] or extracellular signal regulated kinase) inhibitor, PD98059, abrogated the stimulatory effect of PDGF-AB. In contrast to mRNA levels, only TbetaR-II protein levels were elevated in response to PDGF. CONCLUSIONS: These data suggest that PDGF receptor alpha and MAPK mediate stimulation of TGF-beta receptors by PDGF. Furthermore, TGF-beta receptor protein levels are discordantly regulated by PDGF.

Activin Receptors, Type I↗

PubMed2000

Ets factors and regulation of the extracellular matrix.

Ets factors are critical mediators of extracellular matrix (ECM) remodelling. As the spectrum of Ets-regulated target genes widens, so does their role in various pathological and physiological processes. Regulation of matrix degrading proteases by Ets factors in tumor invasion and metastasis is well established. Emerging evidence suggests that they may also play a role in the pathology of autoimmune diseases. Newly characterized Ets target genes such as tenascin-C and collagen type I suggest their role in diseases characterized by aberrant collagen deposition (fibrosis). Ets function is also critical in bone and cartilage development. There is increasing knowledge of the complex regulatory mechanisms involved in transcription of Ets target genes. Ets factors may function as activators or as repressors via association with specific cofactors depending on the promoter context. Signaling pathways can modulate the activation status of Ets factors and their transcriptional partners. Precise understanding of the role of Ets factors in the complex cellular network governing the expression of ECM proteins and the enzymes that degrade them will be a focus of future studies.

3T3 Cells↗

PubMed2000

Interaction of smad3 with a proximal smad-binding element of the human alpha2(I) procollagen gene promoter required for transcriptional activation by TGF-beta.

Transcription of the alpha2(I) collagen gene (COL1A2) in fibroblasts is potently induced by transforming growth factor-beta (TGF-beta). Smad family proteins function as intracellular signal transducers for TGF-beta that convey information from the cell membrane to the nucleus. In the present study, we establish the functional requirement for endogenous Smad3 and Smad4 in TGF-beta-stimulated COL1A2 transcription in human skin fibroblasts in vitro. Furthermore, using transfections with a series of 5' deletions of the human COL1A2 promoter, we identify a proximal region between -353 and -148 bp, which is required for full stimulation of transcription by a constitutively active TGF-beta type I receptor. This region of the COL1A2 promoter contains a CAGA motif also found in the promoter of the plasminogen activator inhibitor-1. Substitutions disrupting this sequence decreased the binding of nuclear extracts or recombinant Smad3 to the CAGACA oligonucleotide, and markedly reduced the transcriptional response to TGF-beta or overexpressed Smad3 in transient transfection assays. The insertion of tandem repeats of CAGACA conferred TGF-beta stimulation to a heterologous minimal promoter-reporter construct. Inhibition of endogenous Smad expression in fibroblasts by antisense oligonucleotides or cDNA against Smad3 or Smad4, and transfection of COL1A2 promoter constructs into Smad4-deficient breast adenocarcinoma cells, indicated the critical role of Smads for the full TGF-beta response. The importance of Smad binding to the CAGACA box of COL1A2 was further established by transcriptional decoy oligonucleotide competition. Taken together, the results identify a functional Smad-binding element of the COL1A2 promoter harboring a CAGACA consensus sequence that is both necessary and sufficient for stimulation by TGF-beta, and demonstrate that interaction of this Smad-binding element with endogenous Smads is required for the full TGF-beta response in fibroblasts.

Adenocarcinoma↗

PubMed2000

Oncostatin M: a cytokine upregulated in human cirrhosis, increases collagen production by human hepatic stellate cells.

BACKGROUND/AIMS: Hepatic stellate cells are predominantly responsible for the increased extracellular matrix seen in cirrhosis. The cytokine oncostatin M has been implicated in fibrogenesis in vitro in other cell types and in vivo in other tissues, although its effect on hepatic stellate cells or in cirrhosis is unknown. METHODS: To examine the effect of oncostatin M on collagen production by human hepatic stellate cells in culture, collagen protein was measured and collagen alpha2(1) mRNA was quantified by Northern analysis. Tissue inhibitor of metalloproteinase-1 (an inhibitor of collagen degradation) mRNA was measured in response to oncostation M stimulation. To explore the potential biological significance of this work to human liver disease, oncostatin M messenger RNA in normal and cirrhotic human liver was measured. RESULTS: Oncostatin M induced in a 2-fold increase in collagen secretion. The potency of induction of collagen protein secretion was equal to that observed after transforming growth factor beta stimulation. An increase in endogenous collagen alpha2(1) mRNA could not be detected. This suggested a post-transcriptional mechanism for the increase in collagen protein. In response to oncostatin M stimulation, there was a 2-fold increase in the tissue inhibitor or metalloproteinase-1 mRNA. Oncostatin M mRNA was detected in 6/6 cirrhotic livers and 1/7 normal livers after 28 PCR cycles. CONCLUSION: These results suggest that oncostatin M expression is upregulated in cirrhosis where it may have a role as a profibrogenic cytokine in hepatic stellate cells.

Cells, Cultured↗

PubMed1999

Ets transcription factors cooperate with Sp1 to activate the human tenascin-C promoter.

Tenascin-C (TN-C), an extracellular matrix glycoprotein is expressed during embryonic development, but is present only at low levels in normal adult tissues. TN-C is re-expressed during wound healing, fibrotic diseases and in cancer. To better understand the mechanisms that control TN-C gene expression, we examined the regulation of the human TN-C promoter in human fibroblasts. We demonstrate that a short segment of the TN-C promoter between bp -133 and -27 contains three evolutionarily conserved Ets binding sites (EBS). These three EBSs bind in vitro expressed Fli1 protein and mediate transactivation of the TN-C gene by Fli1. Furthermore, two proximal EBSs contribute significantly to basal activity of the TN-C promoter. GABP, which is present in human fibroblast nuclear extracts, interacts with the two proximal EBSs. In addition, several Sp1 and Sp3 binding sites have been located in close proximity to the EBSs within this promoter region. The studies performed in Drosophila cells demonstrate that either Fli1 or GABPalpha+beta1 functionally interact with Sp1 resulting in a synergistic stimulation of the TN-C promoter activity. In conclusion, this study shows for the first time that the TN-C gene is regulated by Ets proteins, which together with Sp1 act as potent activators of TN-C expression.

Adult↗

PubMed1999

Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad 3.

Transforming growth factor-beta (TGF-beta) stimulates the transcription of the alpha2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-beta. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-beta. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-beta response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-beta. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-beta-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-beta-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-beta treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-beta-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-beta signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-beta; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-beta.

Cells, Cultured↗

PubMed1998

Pathogenesis of fibrosis: type 1 collagen and the skin.

This review on the pathogenesis of fibrosis emphasizes the similarities between tissue repair, a tightly regulated salutary biological response, and fibrosis, an unregulated pathological process. It focuses on the transcriptional regulation of type I collagen, the role of cytokines in fibroblast activation, integrins as examples of cell-matrix signaling pathways, and the heterogeneity of fibroblast populations as factors contributing to fibrosis. Tissue remodeling and the role of matrix metalloproteinases and metalloproteinase inhibitors are mentioned briefly. The capacity of extracellular matrix to modulate cellular function is a recurring theme.

Cell Adhesion↗

PubMed1998

Increased expression of TGF-beta receptors by scleroderma fibroblasts: evidence for contribution of autocrine TGF-beta signaling to scleroderma phenotype.

Scleroderma fibroblasts exhibit numerous phenotypic differences when compared with healthy skin fibroblasts. Some of these differences, in particular overexpression of collagen type I and other extracellular matrix proteins, parallel the effect of transforming growth factor-beta (TGF-beta) on dermal fibroblasts, suggesting that the scleroderma fibroblast phenotype may result from activation of autocrine TGF-beta signaling. To test this hypothesis we examined the role of TGF-beta Type I and Type II receptors in regulating collagen type I transcription. We have shown that overexpression of either Type I or Type II receptors significantly (3-4-fold) increases alpha2 (I) collagen promoter activity in transient transfection assays in dermal fibroblasts. Addition of anti-TGF-beta antibody abolished, whereas addition of plasmin enhanced, the stimulatory effect of receptor overexpression on collagen promoter activity, suggesting that this effect depends on autocrine TGF-beta. Moreover, these cotransfection experiments indicated that expression levels of TGF-beta receptors is a limiting factor in the autocrine regulation of collagen type I transcription by TGF-beta. Comparison of the TGF-beta receptor Type I and Type II mRA expression levels in scleroderma and normal fibroblasts have indicated elevated expression (2-fold) of both receptor types in scleroderma cells, which correlated with increased binding of TGF-beta. Significantly, elevated TGF-beta receptor levels correlated with elevated alpha2 (I) collagen mRNA levels. These results suggest that the elevated production of collagen type I by scleroderma fibroblasts results from overexpression of TGF-beta receptors.

Collagen↗

PubMed1997

Oncostatin M stimulates transcription of the human alpha2(I) collagen gene via the Sp1/Sp3-binding site.

Oncostatin M (OSM), a member of the hematopoietic cytokine family, has been implicated in excessive bone growth and in the process of fibrosis. As part of an ongoing study of the molecular mechanisms of fibrosis, we have investigated the transcriptional regulation of the alpha2(I) collagen gene by OSM in human fibroblasts. An OSM response element was mapped by deletional analysis between base pairs (bp) -148 and -108 in the alpha2(I) collagen promoter. Further functional analysis of the alpha2(I) collagen promoter containing various substitution mutations revealed that both the basal activity and OSM stimulation of this promoter are mediated by a TCCTCC motif located between bp -128 and -123. Furthermore, three copies of the 12-bp synthetic alpha2(I) collagen promoter fragment containing the "TCC" motif conferred OSM inducibility to the otherwise unresponsive thymidine kinase promoter. Electrophoretic mobility shift assays demonstrated that the TCCTCC motif constitutes a novel binding site for the transcription factors Sp1 and Sp3. No differences have been observed in in vitro gel shift binding assays between unstimulated and OSM-stimulated fibroblasts. However, subtle conformational changes were detected in the region of the promoter surrounding TCC repeats after OSM stimulation using in vivo footprint analysis. In conclusion, this study characterized a dual-function response element that mediates the basal activity and OSM stimulation of the human alpha2(I) collagen promoter.

Binding Sites↗

PubMed1997

Sp3 is a transcriptional activator of the human alpha2(I) collagen gene.

Sp3 is an ubiquitously expressed transcription factor, closely related to Sp1 but, unlike Sp1, it often functions as a transcriptional repressor. In this study we investigated the role of Sp3 in regulating the transcription of the human alpha2(I) collagen gene. We show that Sp1 and Sp3 specifically bind to three of the previously characterized cis -elements in this promoter, including two positive cis-elements between -303 and -271 and -128 and -123, and a repressor site between -164 and -159, but do not bind to the fourth cis-element bound by CBF. Functional analyses of Sp3 and Sp1 in Drosophila cells indicate that each protein transactivates the human alpha2(I) collagen promoter with equal potency and, when tested together, have an additive effect on the promoter activity. Furthermore, in vitro transcription assays demonstrate that both Sp1 and Sp3 are capable of supporting transcription from the collagen promoter independently of each other. However, when activities of both Sp1 and Sp3 are blocked with specific antibodies, in vitro transcription from this promoter is almost completely abolished. The results of this study demonstrate that Sp3 is as potent an activator of the human alpha2(I) collagen promoter as is Sp1 and that a transcriptional activity of the human alpha2(I) promoter is dependent on both proteins.

Collagen↗

PubMed1997

Basic fibroblast growth factor inhibits basal and transforming growth factor-beta induced collagen alpha 2(I) gene expression in scleroderma and normal fibroblasts.

OBJECTIVE: Studies have shown that scleroderma (systemic sclerosis, SSc) and normal fibroblasts respond differently to basic fibroblast growth factor (bFGF), SSc fibroblasts being less responsive than normal fibroblasts in mitogenic assays in vitro, bFGF also stimulates the expression of platelet derived growth factor-alpha (PDGF-alpha) receptors in normal fibroblasts, but not in SSc fibroblasts. Conversely, transforming growth factor-beta (TGF-beta) stimulates PDGF-alpha receptor expression in SSc fibroblasts, but not in normal fibroblasts. Since bFGF has been shown to inhibit collagen gene expression in several cell types, we examined responses of SSc and normal fibroblasts to bFGF alone and in combination with TGF-beta with regard to collagen alpha 2(I) (COL1A2) expression. METHODS: Fibroblasts were obtained by skin biopsy from affected areas of patients with diffuse cutaneous SSc and from healthy donors and propagated in vitro. The effects of bFGF and TGF-beta on the COL1A2 mRNA expression levels in SSc and healthy fibroblasts were analyzed by Northern blot. The effects of bFGF on the COL1A2 promoter activities in both cell types were analyzed by transient transfection assays. The effects of bFGF and TGF-beta on collagen protein synthesis were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. RESULTS: While bFGF diminished COL1A2 mRNA in both SSc and normal cells, COL1A2 mRNA quantities in the SSc fibroblasts were not depressed to the levels expressed by normal controls. As anticipated, TGF-beta strongly induced COL1A2 mRNA levels in normal fibroblasts, and to a lesser degree in SSc fibroblasts. When cells were incubated with both TGF-beta and bFGF, the stimulatory effect of TGF-beta was completely suppressed in both cell types. bFGF decreased COL1A2 promoter activity in both cell types, suggesting that COL1A2 inhibition by bFGF occurs at least partially at the transcriptional level. The effects of bFGF and TGF-beta on the collagen protein synthesis correlated well with mRNA data, in that TGF-beta stimulated, while bFGF strongly inhibited, collagen synthesis. CONCLUSION: bFGF is a potent inhibitor of basal and TGF-beta stimulated collagen expression in human fibroblasts, and this effect is not different between SSc and healthy fibroblasts.

Collagen↗

PubMed1996

Transcriptional regulation of the human alpha2(I) collagen gene. Combined action of upstream stimulatory and inhibitory cis-acting elements.

This study identifies three regions of the human alpha2(I) collagen promoter involved in the binding of nuclear factors. These regions include sequences from -173 to -155 (footprint I), -133 to -119 (footprint II), and -101 to -72 (footprint III). A novel positive cis-element containing a TCCTCC motif was identified within footprint II. In addition, we demonstrated that a pyrimidine-rich region within footprint I is a binding site for a transcriptional repressor, and a CCAAT motif within footprint III is a binding site for a transcriptional activator. Comparative functional analysis of the cis-acting elements within the proximal 350 base pairs of this promoter, including previously characterized Sp1 binding sites at -300, indicates that constitutive activity of this promoter is regulated equivalently by the three positive cis-acting elements at -300, -125, and -80. Mutations in the repressor site at -160 increase constitutive activity by 4-6-fold. However, simultaneous mutations of the repressor site and the cis-regulatory element at either the -300 or -125 sites result in no increase in constitutive transcription activity suggesting interaction between the activators and repressor elements. In contrast, simultaneous mutation of the CCAAT motif and the repressor site results in about a 4-fold increase, suggesting that activation via the CCAAT motif may be independent of this repressor.

Animals↗

PubMed1996

IL-4 upregulates tenascin synthesis in scleroderma and healthy skin fibroblasts.

Tenascin (TN), a large extracellular matrix glycoprotein, is transiently expressed during embryonic development, but is absent from most normal adult tissues. TN is reexpressed, however, in healing wounds, in the stroma of some tumors, and in fibrotic diseases such as systemic sclerosis (SSc) and rheumatoid arthritis. To clarify the mechanisms regulating TN expression, we studied the effects of selected cytokines (PDGF, bFGF, TGF-beta, IL-1, IL-4, IL-6, IFN-gamma, and TNF-alpha) found in fibrotic tissue on TN expression by dermal fibroblasts. IL-4 strongly induced TN protein levels (up to 10-fold over the basal level), whereas PDGF and bFGF were less potent inducers of TN than IL-4. All other cytokines tested, including TGF-alpha1, did not stimulate TN synthesis. IL-4 also increased TN mRNA expression, and this effect was blocked by actinomycin D. Cycloheximide increased basal TN mRNA expression and induced TN mRNA in IL-4-treated fibroblasts, suggesting that repressor protein(s) may regulate transcription of the TN gene. Although no differences in constitutive TN expression or effects of cytokines on TN expression were observed between SSc and healthy fibroblasts, these data are consistent with the observations that high levels of both IL-4 and TN are present in the affected skin of patients with SSc. These results suggest that the high level of TN found in the affected tissue of patients with SSc results from the high level of IL-4 present.

Adult↗

PubMed1995

Characterization of a GC-rich region containing Sp1 binding site(s) as a constitutive responsive element of the alpha 2(I) collagen gene in human fibroblasts.

To analyze regulatory elements in the human alpha 2(I) collagen gene (COL1A2) promoter, a series of deletion mutants from -323 to -186 base pairs was tested in transient transfection assays in human fibroblasts. A strong positive responsive element was mapped to a GC-rich region located between base pairs -303 and -271. This region contains three binding sites (GC-boxes) resembling recognition sites for the transcription factor Sp1. Substitution mutations in the GC-boxes abolished binding to the GC-rich region in gel shift analyses and resulted in 90% reduction of promoter activity in transient transfection assays. We demonstrated that transcription factor Sp1 is essential for binding based on the following observations. 1) Sp1 consensus binding site alone competes by binding to the GC-rich region in the DNase I protection assay; 2) both Sp1 consensus binding site and Sp1 antibodies prevent the formation of a DNA-protein complex in the mobility shift assay; 3) anti-Sp1 antibodies recognize a component of the complex competed for by Sp1 consensus binding site.

Base Sequence↗