Regulation Of Collagen Gene Expression Research Articles

Human chondrocyte culture models for studying cyclooxygenase expression and prostaglandin regulation of collagen gene expression

ObjectiveSince articular chondrocytes and synovial fibroblasts are particularly responsive to interleukin-1 (IL-1) with respect to stimulation of prostaglandin E2(PGE2) biosynthesis, we have used them as models to examine feedback modulatory effects of PGE2, which blocks or attenuates the direct effects of IL-1β on cell-specific collagen gene expression.MethodsImmortalized human chondrocytes were developed for studying responses to cytokines and prostaglandins. Regulatory sequences of the type II collagen gene (COL2A1) in reporter gene constructs were analyzed in transient transfection experiments. Endogenous expression of COL2A1 mRNA, as well as aggrecan, biglycan, and decorin mRNAs, and IL-1-inducible cyclooxygenase (COX-2), phospholipase A2 (PLA2), and inducible nitric oxide synthetase (iNOS) mRNAs were analyzed by RT-PCR.ResultsPrevious work has shown that IL-1β inhibits, while prostaglandins stimulate COL2A1 expression. In different immortalized chondrocyte cell lines, the ability to respond to IL-1β with increased levels of COX-2, PLA2, and iNOS mRNAs depends upon expression of the differentiated chrondrocyte phenotype.ConclusionsOur studies suggest that some IL-1-induced responses in chondrocytes may require differentiation-specific transcription factors that could serve as therapeutic targets for arthritis.

アンチオキシダントと遺伝子発現制御

アンチオキシダントと遺伝子発現制御

活性持続型ビタミンＣ（Ａｓｃ２‐Ｐ）による遺伝子発現の制御

活性持続型ビタミンＣ（Ａｓｃ２‐Ｐ）による遺伝子発現の制御

Dexamethasone Abrogates the Fibrogenic Effect of Transforming Growth Factor-β in Rat Granuloma and Granulation Tissue Fibroblasts

Administration of TGF-beta, a fibrogenic inflammatory growth factor, promotes fibrosis and scarring. Dexamethasone, an anti-inflammatory steroid, inhibits wound healing and reduces fibrosis. The current studies were initiated to determine whether the co-administration of dexamethasone was able to abrogate the fibrogenic effect of TGF-beta. Polyvinyl alcohol sponges were implanted subcutaneously on the abdominal area of rats and directly injected with vehicle, dexamethasone, TGF-beta, or dexamethasone plus TGF-beta. Dexamethasone was able to block the fibrogenic effect of TGF-beta. Collagen and noncollagen protein synthesis was measured as a function of TGF-beta or dexamethasone concentrations in fibroblasts isolated from granulation tissue. Addition of dexamethasone to cultures treated simultaneously with TGF-beta blocked the fibrogenic response of TGF-beta. To study the molecular regulation of collagen gene expression by TGF-beta or dexamethasone, fibroblasts derived from granulation tissue were stably transfected with the ColCat 3.6 plasmid, which contains the rat pro alpha1(I) collagen promoter linked to the chloramphenicol acetyltransferase (CAT) gene. Dexamethasone decreased CAT activity whereas TGF-beta increased the activity of this reporter gene. The increase in CAT activity observed with TGF-beta treatment was significantly decreased when dexamethasone was added to the cultures, although CAT activity did not return to control level. Since collagen synthesis in fibroblasts treated simultaneously with dexamethasone and TGF-beta1 was found to be the same as that of untreated samples, the data indicate that there is a dexamethasone-mediated posttranscriptional regulation of pro alpha1(I) collagen mRNA. These studies demonstrate that at the in vivo level, the cellular level, and the molecular level, dexamethasone is able to block the fibrogenic effect of TGF-beta.

Suppression of Stellate Cell Type I Collagen Gene Expression Involves AP-2 Transmodulation of Nuclear Factor-1-dependent Gene Transcription

The regulation of collagen gene expression was studied in culture-activated rat hepatic stellate cells, the fibrogenic effector cell involved in hepatic fibrogenesis. Treatment of cells with a 5-lipoxygenase-specific inhibitor caused a reduction in alphaI(I) collagen mRNA transcript abundance, which suggested that leukotriene production was involved in maintaining the activated cell's high level of collagen mRNA production. The underlying mechanism involved a decrease in collagen gene transcription. Suppression of gene transcription was localized to an nuclear factor-1 (NF-1) binding domain in the proximal promoter and an AP-2 binding domain adjacent to it. Gel retardation assays demonstrated that an increase in AP-2 binding adjacent to the NF-1 site was likely to be the transmodulator responsible for the suppression of the NF-1-dependent gene expression. The data suggest that post-translational alterations in AP-2 activity are responsible for this unappreciated mechanism of regulating the collagen gene.

Collagen Types I and III Localization by In Situ Hybridization and Immunohistochemistry in the Partially Obstructed Young Rabbit Bladder

Purpose The objective of the study was to investigate the alteration of collagen expression patterns at the transcription and translation levels during partial outlet obstruction and to compare changes in collagen types I and III gene expression, and protein deposition during early and subacute phases of obstruction. Materials and Methods Following 1, 2, 7, 14 and 21 days of partial bladder obstruction in 2-week-old rabbits bladder tissues were evaluated for collagen types I and III messenger ribonucleic acid localization by in situ hybridization and for protein localization by immunohistochemical study. Results There is up regulation of collagen types I and III gene expression after obstruction. Up regulation of collagen gene expression immediately after obstruction is within the lamina propria and the message moves into the muscular layer as obstruction progresses. Protein deposition for both collagen types is analogous to that of gene expression. Conclusions Up regulation of collagen types I and III gene expression, and increased protein deposition after partial obstruction demonstrate that the alteration in collagen synthesis is at least partially transcription regulated. This alteration starts from superficial layers of the bladder wall and becomes deeper as the pathological process progresses.

Collagen types I and III localization by in situ hybridization and immunohistochemistry in the partially obstructed young rabbit bladder.

The objective of the study was to investigate the alteration of collagen expression patterns at the transcription and translation levels during partial outlet obstruction and to compare changes in collagen types I and III gene expression, and protein deposition during early and subacute phases of obstruction. Following 1, 2, 7, 14 and 21 days of partial bladder obstruction in 2-week-old rabbits bladder tissues were evaluated for collagen types I and III messenger ribonucleic acid localization by in situ hybridization and for protein localization by immunohistochemical study. There is up regulation of collagen types I and III gene expression after obstruction. Up regulation of collagen gene expression immediately after obstruction is within the lamina propria and the message moves into the muscular layer as obstruction progresses. Protein deposition for both collagen types is analogous to that of gene expression. Up regulation of collagen types I and III gene expression, and increased protein deposition after partial obstruction demonstrate that the alteration in collagen synthesis is at least partially transcription regulated. This alteration starts from superficial layers of the bladder wall and becomes deeper as the pathological process progresses.

Serotonin regulation of gene expression in uterine extracellular matrix: reciprocal effects on collagens and collagenase

The regulation of collagen gene expression by serotonin was investigated in rat uterine smooth muscle cells. Serotonin treatment of myometrial cells caused decreases of up to 10-fold in levels of type I collagen mRNA. Decreases in secreted type 1 collagen protein paralleled decreases in collagen mRNA. The effective half-life of collagen mRNA in serotonin-treated cells was approximately 1.7 days. Selective 5-HT 2 receptor agonists mimicked the effects of serotonin, while the effects of serotonin were blocked by 5-HT 2 antagonists. Nuclear run-on analysis showed that serotonin-dependent decreases in collagen mRNA are accompanied by decreased transcription. Progesterone analogs, which inhibit the serotonin-dependent activation of the gene for interstitial collagenase, had no effect on the ability of serotonin to decrease collagen mRNA. Conversely, the cell-permeable cAMP analog, 8-bromo-cAMP, mimicked the effects of serotonin on type I collagen mRNA and protein. Serotonin also decreased levels of the mRNAs for type III collagen and fibronectin, but had no effect on the mRNAs for type IV collagen. These results indicate that serotonin, previously shown to upregulate the interstitial collagenase gene, downregulates the gene for type I collagen and other extracellular matrix proteins, possibly by a novel mechanism of action downstream of 5-HT 2 receptor binding.

Systemic sclerosis: current pathogenetic concepts and future prospects for targeted therapy

Although there is no cure for systemic sclerosis, the disease is certainly treatable. And the more that is understood of its pathogenesis, the better the prospects for treatment. Excessive collagen deposition in the skin and viscera is responsible for the morbidity and mortality. The immune system plays a critical role in setting up the fibrotic process, but impaired regulation of collagen gene expression probably underlies the persistence of fibrosis. The development of effective antifibrotic drugs, in particular agents that inhibit transcription factors involved in the activation of type I collagen genes, is likely to be the way forward for treatment of the later stages of diffuse systemic sclerosis, with immunomodulatory therapy for the earlier stages.

Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A.

Expression of type I collagen genes is highly regulated and becomes abnormal in various pathological conditions, from excessive collagen production in fibrotic diseases to their downregulation in transformed cells. Some inflammatory cytokines and other ligands, capable of eliciting intracellular phosphorylation, can profoundly alter collagen gene expression. We investigated the role of serine/threonine protein phosphatases (PP) in the regulation of collagen gene expression. Biosynthesis of the endogenous type I procollagen, and expression of Pro alpha 1(I) promoter-luciferase (Luc) constructs transfected in NIH3T3 fibroblasts, were evaluated in response to PP2A and PP1 inhibitor okadaic acid (OA) and exogenously expressed PP catalytic subunits. OA suppressed type I collagen gene expression as judged by reduced rates of protein synthesis, steady state levels of Pro alpha 1(I) collagen mRNA and expression of Luc driven by Pro alpha 1 (I) collagen promoter in OA-treated cells. Co-transfection of Pro alpha 1(I)-Luc with expression vectors containing PP2A, but not PP1, stimulated collagen promoter activity. These results strongly suggest that OA acts via PP2A-mediated dephosphorylation of an unidentified transcription factor(s) or cofactor(s) needed to activate Pro alpha 1(I) collagen promoter.

Increased α1(I) Procollagen Gene Expression in Tight Skin (TSK) Mice Myocardial Fibroblasts Is Due to a Reduced Interaction of a Negative Regulatory Sequence with AP-1 Transcription Factor

The TSK mouse, a model of fibrosis, displays exaggerated connective tissue accumulation in skin and visceral organs including the heart. To study the mechanisms of myocardial fibrosis in TSK mice, we established several strains of TSK mice myocardial fibroblasts in culture and examined the regulation of collagen gene expression in these cells. These strains displayed increased collagen gene expression in comparison with myocardial fibroblasts established from normal mice. On an average, the TSK myocardial fibroblast cultures showed a 4-fold increase in collagen synthesis and 4.4- and 3.6-fold increases, respectively, in alpha 1(I) and alpha 1(III) collagen mRNA steady state levels. The increased alpha 1(I) and alpha 1(III) collagen mRNA levels were mainly due to increased transcription rates (3.4- and 3.8-fold higher, respectively) of the respective genes. Furthermore, we showed that the up-regulation of alpha 1(I) procollagen gene transcription in TSK mice myocardial fibroblasts was due to the lack of the strong inhibitory influence of a regulatory sequence contained in the promoter region encompassing nucleotides -675 to -804. Nuclear extracts from TSK mice myocardial fibroblasts showed lower DNA binding activity to oligonucleotides spanning the mapped regulatory sequence as well as to a consensus AP-1 sequence, but not to a consensus SP-1 sequence, and supershift experiments with an AP-1 antibody confirmed the interaction of these oligonucleotides with AP-1 protein. These observations indicate that a strong negative regulatory sequence contained within -0.675 to -0.804 kilobase of the alpha 1(I) procollagen promoter binds AP-1 transcription factor and mediates inhibition of gene transcription in normal murine myocardial fibroblasts. The TSK mice myocardial fibroblasts lack this inhibitory control, due to lower available amounts and/or decreased binding activity to this inhibitory sequence, and hence display increased alpha 1(I) procollagen gene expression.

Differential regulation of collagen gene expression in granulation tissue and nonrepair connective tissues in vitamin C‐deficient guinea pigs

Poor wound healing during vitamin C deficiency is thought to be due to decreased hydroxylation of proline residues in collagen. In non-repair connective tissues of guinea pigs, however, procollagen gene expression is not decreased until weight loss occurs during the third and fourth weeks of scurvy (phase II) with only a moderate decrease in proline hydroxylation. Decreased procollagen gene expression is related to the induction of insulin-like growth factor binding proteins 1 and 2 that inhibit insulin-like growth factor-I action. We examined wound healing and granulation tissue formation during phase I of vitamin C deficiency. Synthetic sponges were implanted on day 7 of vitamin C deficiency and analyzed at 6 and 10 days after surgery, when there was no weight loss or induction of insulin-like growth factor binding proteins. Healing of incisions was almost complete at 10 days after surgery in normal controls but not in scorbutic animals. The area around the incision and implant exhibited excessive angiogenesis and hemorrhaging of vessels in the scorbutic animals at 6 and 10 days after surgery. At 10 days after surgery, collagen synthesis in the implants of scorbutic guinea pigs was 36% lower than control values, with a normal extent of proline hydroxylation. Concentrations of messenger RNAs for types I and III procollagens were slightly increased by scurvy at 6 days after surgery but were decreased by 26% and 40%, respectively, at 10 days. Fibronectin mRNA levels were unaffected by scurvy at both time points. Our results suggest that poor wound healing in phase I of scurvy may be related to defective interstitial procollagen gene expression and defective blood vessel formation, but it does not involve inhibition of proline hydroxylation or induction of insulin-like growth factor binding proteins. mRNA for insulin-like growth factor-II, transforming growth factor-beta(1), and transforming growth factor-beta(2) were significantly expressed in implants, but their patterns of expression did not correlate with changes in procollagen gene expression.

Characterization of spatial distribution and aspects of regulation of collagen gene expression in keloids

Characterization of spatial distribution and aspects of regulation of collagen gene expression in keloids

Regulation of Collagen Gene Expression in Keloids and Hypertrophic Scars

The synthesis of type I and III collagens in cultured skin fibroblasts from normal skin, normal scar, hypertrophic scar, and keloids was examined. The ratio of type I/III collagen was significantly elevated in keloids compared to that in the other groups. When mRNA steady-state levels coding for α1(I) procollagen were determined, it was apparent that this increase in the type I/III collagen ratio in keloids was paralleled by a specific increase in α1(I) procollagen mRNA. This specific increase in α1(I) procollagen mRNA in keloids was the result of increased gene expression because the transcription rate of the α1(I) procollagen gene was significantly elevated in keloids, as determined by nuclear runoff transcription. The rate of transcription of the α(I) procollagen gene was also elevated in hypertrophic scars, although no concomitant increase in α1(I) procollagen mRNA levels or alteration in the type I/III collagen ratio was observed. These data indicate that the rate of gene transcription of α1(I) procollagen is increased in both hypertrophic scars and keloids, but only keloids exhibit increased steady-state levels of α1(I) procollagen mRNA and concurrent increases in type I collagen. These results suggest that at least two distinct mechanisms, one pretranscriptional and one post-transcriptional, regulate type I collagen synthesis. It is possible, therefore, that in keloids, neither mechanism functions efficiently to down-regulate type I collagen. In hypertrophic scars, however, the post-transcriptional mechanisms are able to decrease elevated levels of mRNA coding for α1 (I) procollagen that result from increased transcriptional of the α1(I) procollagen gene.

Expression and accumulation of interstitial collagen in the neonatal rat heart

Significant physiological changes occur in the heart following birth including increased arterial blood pressure and heart rate. Concurrently, biochemical and structural alterations are evident in the neonatal heart in response to these dynamic physiological properties. Prominent among these is the elaborate development of the cardiac extracellular matrix, composed primarily of interstitial collagen. The collagenous fibers, together with other matrix components, form an elastic, stress-tolerant network which functions in the dissipation of force throughout the heart wall. The present studies have used biochemical and molecular techniques to show the temporal and spatial patterns of interstitial collagen accumulation and expression during late fetal and neonatal development of the rat heart. The use of biochemical and particularly molecular methodologies allows the analysis of the expression of matrix components at a resolution previously not attained by structural studies alone. These data show relative increases in interstitial collagen immediately following birth as well as spatial differences in collagen mRNAs within the heart. The data presented provide further evidence for a role of mechanical stimulation in the regulation of collagen gene expression during this period of heart development.

Feedback regulation of collagen gene expression: a Trojan horse approach.

The mechanisms involved in feedback regulation of type I procollagen synthesis by the N-terminal propeptide of the pro alpha 1(I) chain, termed Col 1, are poorly understood. We have constructed a metallothionein-human collagen chimeric minigene (pMTCol) that codes for a Col 1 fusion protein but lacks a signal peptide sequence and, therefore, would be expected to direct the synthesis of the fusion protein to the cytosol. Baby hamster kidney cells and fetal calf ligament cells, transfected with pMTCol, transcribed the gene and synthesized an intracellular antigen that was identified as the fusion protein with a monospecific antibody. Transfected fetal calf ligament fibroblasts showed significantly reduced levels of endogenously produced type I collagen, as determined by imaging and digital quantitation of immunofluorescence by confocal microscopy; synthesis of fibronectin, thrombospondin, and SPARC (secreted protein, acidic and rich in cysteine) was unchanged or increased in these cells. This recombinant approach offers the potential for a systematic analysis of feedback regulation of collagen synthesis.

Regulation of Collagen Gene Expression by Transformed Human Fibroblasts: Decreased Type I and Type III Collagen RNA Transcription

The regulation of collagen gene expression in normal diploid human fetal fibroblasts (KMS-6 cells), and fibroblasts immortally transformed by treatment of KMS-6 with Co-60 gamma rays (KMST-6 cells) was compared to that of ones tumorigenically transformed by treatment of KMST-6 cells with Harvey murine sarcoma virus (KMST-6-Ras cells). Synthesized collagenous protein decreased to approximately 30% of that of normal fetal fibroblasts in both transformed cell lines, and the relative rate of collagen synthesis to total protein synthesis decreased about sixfold in KMST-6 cells and twelvefold in KMST-6-Ras cells. The m-RNA levels of type I collagen in both of these cell lines decreased to approximately 20% of that of the control fibroblasts, whereas type III collagen m-RNA levels decreased to only 9% of that of the control. The copy number of the collagen gene in both transformed cell lines was unaltered. The transcriptional rates of collagen alpha 1(I) and collagen alpha 1(III) in both cell lines decreased to 20% and 7% respectively of that of control. These data indicate that collagen synthesis was reduced at the transcriptional level in these transformed human fibroblasts.

Regulation of collagen gene expression by transformed human fibroblasts

Regulation of collagen gene expression by transformed human fibroblasts

Synergistic Regulation of Collagen Gene Expression in Human Chondrocytes by Tumor Necrosis Factor‐α and Interleukin‐1βa

Annals of the New York Academy of SciencesVolume 580, Issue 1 p. 536-539 Synergistic Regulation of Collagen Gene Expression in Human Chondrocytes by Tumor Necrosis Factor-α and Interleukin-1βa MARY B. GOLDRING, MARY B. GOLDRING Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorJAMES BIRKHEAD, JAMES BIRKHEAD Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorLINDA J. SANDELL, LINDA J. SANDELL Departments of Orthopaedics and Biochemistry University of Washington Seattle, Washington 98195Search for more papers by this authorSTEPHEN M. KRANE, STEPHEN M. KRANE Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this author MARY B. GOLDRING, MARY B. GOLDRING Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorJAMES BIRKHEAD, JAMES BIRKHEAD Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this authorLINDA J. SANDELL, LINDA J. SANDELL Departments of Orthopaedics and Biochemistry University of Washington Seattle, Washington 98195Search for more papers by this authorSTEPHEN M. KRANE, STEPHEN M. KRANE Department of Medicine Harvard Medical School, Boston, Massachusetts 02114 The Medical Services (Arthritis Unit) Massachusetts General Hospital Boston, Massachusetts 02114Search for more papers by this author First published: February 1990 https://doi.org/10.1111/j.1749-6632.1990.tb17983.xCitations: 16 a This work was supported by Grants AR-34390 and AR-03564 from the National Institutes of Health. This is Publication Number 1051 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume580, Issue1Structure, Molecular Biology, and Pathology of CollagenFebruary 1990Pages 536-539 RelatedInformation

Increased Collagen Synthesis Accompanying Elevated m-RNA Levels in Cultured Werner's Syndrome Fibroblasts

Although Werner's syndrome (WS) is a premature aging disease and its fibroblasts typically grow poorly in culture, WS may cause abnormalities in connective tissue metabolism that are seldom seen in normal aging, such as scleroderma-like skin. In a preliminary report, we described increased collagen synthesis in fibroblasts derived from two WS patients. The present study was undertaken to determine the degree of the regulation of collagen gene expression in dermal fibroblasts from two other patients. Overproduction of collagenase sensitive protein was observed in WS fibroblasts. Collagen m-RNA levels, that were determined by hybridization of RNA blots with specific cDNA were about 2 times greater than those in the control cells. These results suggest that control of collagen synthesis in WS fibroblasts is altered at the transcriptional level.