Stimulation Of Collagen Gene Expression Research Articles

A20 suppresses canonical Smad-dependent fibroblast activation: novel function for an endogenous inflammatory modulator.

BackgroundThe ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)-mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc). Because recent studies implicate endogenous ligand-driven TLR signaling in SSc pathogenesis, we sought to investigate the regulation, role and mechanism of action of A20 in skin fibroblasts.MethodA20 expression and the effects of forced A20 expression or siRNA-mediated A20 knockdown on fibrotic responses induced by transforming growth factor-ß (TGF-ß) were evaluated was evaluated in explanted human skin fibroblasts. Additionally, A20 regulation by TGF-ß, and by adiponectin, a pleiotropic adipokine with anti-fibrotic activity, was evaluated.ResultsIn normal fibroblasts, TGF-ß induced sustained downregulation of A20, and abrogated its TLR4-dependent induction. Forced expression of A20 aborted the stimulation of collagen gene expression and myofibroblast transformation induced by TGF-ß, and disrupted canonical Smad signaling and Smad-dependent transcriptional responses. Conversely, siRNA-mediated knockdown of A20 enhanced the amplitude of fibrotic responses elicited by TGF-ß. Adiponectin, previously shown to block TLR-dependent fibrotic responses, elicited rapid and sustained increase in A20 accumulation in fibroblasts.ConclusionThese results identify the ubiquitin-editing enzyme A20 as a novel endogenous mechanism for negative regulation of fibrotic response intensity. Systemic sclerosis-associated genetic variants of A20 that cause impaired A20 expression or function, combined with direct suppression of A20 by TGF-ß within the fibrotic milieu, might play a significant functional role in persistence of fibrotic responses, while pharmacological augmentation of A20 inhibitory pathway activity might represent a novel therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1118-7) contains supplementary material, which is available to authorized users.

Peroxisome proliferator‐activated receptor‐γ abrogates Smad‐dependent collagen stimulation by targeting the p300 transcriptional coactivator

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.

Transforming Growth Factor-β (TGF-β ) and its Role in the Pathogenesis of Systemic Sclerosis: A Novel Target for Therapy?

One of the growth factors that appear to play a crucial role in the pathogenesis of systemic sclerosis is TGF-beta. The three functionally and structurally similar human isoforms of TGF-beta play important roles in embryonic development, in the regulation of tissue repair following injury and in immune responses. Systemic sclerosis fibroblasts express increased levels of TGF-beta receptors on their surface which in turn results in increased signalimg of TGF-beta induced collagen gene expression. Some of the patents and intricate pathways that mediate the stimulation of collagen gene expression by TGF-beta have recently been described and a potential inhibition of these pathways may lead to novel therapeutic targets for systemic sclerosis.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

Selective inhibition of activin receptor–like kinase 5 signaling blocks profibrotic transforming growth factor β responses in skin fibroblasts

Members of the transforming growth factor beta (TGFbeta) cytokine superfamily play critical roles in both homeostasis and disease. In light of their profibrotic effects, these molecules are implicated in the pathogenesis of fibrosis. In fibroblasts, TGFbeta signals through the activin receptor-like kinase 5 (ALK-5) type I TGFbeta and triggers Smad and MAP kinase signaling pathways. Because targeting of TGFbeta signaling represents a potential approach to the treatment of systemic sclerosis (SSc) and other fibrotic disorders, we investigated the modulation of intracellular TGFbeta signal transduction by SB431542, the first small-molecule inhibitor of ALK-5 to be described. Ligand-induced activation of the Smad signaling pathway in human dermal fibroblasts was examined by Western blot analysis and confocal immunocytochemistry. Modulation of profibrotic gene expression was investigated using Northern blot analysis, transient transfection assays, and confocal microscopy. Induction of TGFbeta production was evaluated by enzyme-linked immunosorbent assay. SB431542 abrogated TGFbeta-induced phosphorylation and nuclear importation of endogenous Smad2/3 and Smad4, and inhibited Smad3- and Smad2-dependent gene transcription. Treatment with SB431542 prevented TGFbeta-induced stimulation of collagen, fibronectin, plasminogen activator inhibitor 1, and connective tissue growth factor gene expression, TGFbeta autoinduction, and myofibroblast transdifferentiation, and it could reverse stimulation even when added to the cultures after TGFbeta. In contrast, STAT-6-mediated stimulation of collagen gene expression induced by interleukin-13 was not prevented by SB431542, indicating the specificity of blockade for ALK-5-dependent signaling. Furthermore, in contrast to its effects on receptor-activated Smad activation, SB431542 failed to prevent TGFbeta-induced activation of MAP kinases. The results indicate that SB431542 is a potent inhibitor of intracellular TGFbeta signaling in normal fibroblasts through selective interference with ALK-5-mediated Smad activation and Smad-dependent transcriptional responses. Therefore, SB431542 is useful as a novel experimental tool for gaining a detailed understanding of normal and aberrant TGFbeta signaling in SSc. Furthermore, as an anti-TGFbeta agent, SB431542 may represent a potential new approach to the treatment of fibrosis.

Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.

In fibroblasts, transforming growth factor beta (TGF beta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF beta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF beta/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) in normal skin fibroblasts and its effect on TGF beta-induced cellular responses. The expression and activity of PPAR gamma in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPAR gamma activation by naturally occurring and synthetic ligands on collagen synthesis and alpha-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR gamma expression constructs. The PPAR gamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPAR gamma resulted in modest suppression of basal collagen gene expression, it abrogated TGF beta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPAR gamma in fibroblasts, and was blocked by a selective antagonist of PPAR gamma signaling or by transfection of fibroblasts with dominant-negative PPAR gamma constructs. Furthermore, PPAR gamma ligands abrogated TGF beta-induced expression of alpha-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGF beta was suppressed by PPAR gamma despite the absence of a consensus PPAR gamma-response element in the targeted promoters. Ligand-induced activation of fibroblast PPAR gamma had no effect on protein expression of cellular Smad3 or Smad7. By abrogating of TGF beta-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPAR gamma may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPAR gamma activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.

Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-beta involves functional cooperation with p300/CBP transcriptional coactivators.

Transforming growth factor-beta (TGF-beta) stimulation of Type I collagen gene (COL1A2) transcription involves the Smad signal transduction pathway, but the mechanisms of Smad-mediated transcriptional activation are not fully understood. We now demonstrate that the ubiquitous transcriptional coactivators p300 and CREB-binding protein (CBP) enhanced basal as well as TGF-beta- or Smad3-induced COL1A2 promoter activity, and stimulated the expression of endogenous Type I collagen. The adenoviral E1A oncoprotein abrogated stimulation of COL1A2 activity in transfected fibroblasts, and reduced the basal level of collagen gene expression. This effect was due to specific interaction of E1A with cellular p300/CBP because (a) a mutant form of E1A defective in p300 binding failed to abrogate stimulation, and (b) forced expression of p300/CBP restored the ability of TGF-beta to stimulate COL1A2 promoter activity in the presence of E1A. The effect of p300 on COL1A2 transcription appeared to be due, in part, to its intrinsic acetyltransferase activity, as stimulation induced by a histone acetyltransferase-deficient mutant p300 was substantially reduced. Transactivation of COL1A2 by p300 involved the Smad signaling pathway, as Smad4-deficient cells failed to respond to p300, and stimulation was rescued by overexpression of Smad4. Furthermore, minimal constructs containing only the Smad-binding CAGACA element of COL1A2 were transactivated by p300 in the presence of TGF-beta. These results indicate, for the first time, that the multifunctional p300/CBP coactivators play a major role in Smad-dependent TGF-beta stimulation of collagen gene expression in fibroblasts. Oncogene (2000) 19, 3546 - 3555

Glomerular expression of p27Kip1 in diabetic db/db mouse: Role of hyperglycemia

Glomerular expression of p27Kip1 in diabetic db/db mouse: Role of hyperglycemia

Antagonistic Effects of Interferon-γ and Interleukin-4 on Fibroblast Cultures.

A major characteristic of scleroderma (SSc) fibroblasts is an increased biosynthesis of extracellular matrix macromolecules that could be linked to impaired regulation by cytokines. We investigated the effects of two cytokines from T lymphocytes, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma), on normal and scleroderma fibroblast cultures. In both types of fibroblasts, IL-4 strongly stimulated collagen synthesis, whereas IFN-gamma was a potent inhibitor. The effects of these cytokines were localized at the pre-translational level, and both mRNA steady-state level and protein synthesis were equally affected. SSc fibroblasts responded to IL-4 and IFN-gamma as well as normal fibroblasts. When fibroblasts were incubated with combinations of both cytokines, IFN-gamma completely suppressed the stimulation of collagen gene expression induced by IL-4. Northern blot and western blot analyses demonstrated that IFN-gamma induced a rapid and strong decrease in the expression of the IL-4 receptor-alpha by fibroblasts. This effect might explain the antagonistic effects of IFN-gamma on the IL-4-dependent enhancement of collagen synthesis. Thus, our data suggest that the alteration of collagen production in scleroderma fibroblasts does not depend on an altered sensitivity of these cells to stimulatory or inhibitory cytokines but is more likely the consequence of an imbalance in the local production of autocrine or paracrine regulatory factors.

Involvement of oxidation in LDL-induced collagen gene regulation in mesangial cells

Oxidized low-density lipoprotein (Ox-LDL) is present in the lesions of focal segmental glomerulosclerosis (FSGS), but the role of Ox-LDL in the disease process is not clear. Recent studies have shown that LDL stimulates the type IV collagen mRNA expression in cultured mesangial cells. Thus, we examined whether oxidative stress is responsible for the stimulation of LDL-induced collagen gene expression in cultured human mesangial cells (HMCs). When quiescent HMCs were exposed to serum-free media containing LDL for 48 hours, peroxidation of LDL was induced as shown by the increased production of thiobarbituric acid-reactive substances (TBARS). LDL stimulated the alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA expression in a dose-dependent manner. At a concentration of 200 micrograms/ml, LDL enhanced the levels of alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA by 3.7-, 3.8- and 3.2-fold, respectively, over the levels in the control cells. These transcripts were further increased 5.4-, 6.7-, and 5.9-fold, respectively, by the addition of 500 micrograms/ml of LDL. Cu(2+)-catalyzed Ox-LDL at a concentration of 200 micrograms/ml also stimulated the alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA expression 4.4-, 5.9-, and 2.8-fold, respectively, compared with the control cells. The addition of monoclonal antibody (mAb) OL-10, which recognizes the malondialdehyde (MDA)-modified peptide epitope, or vitamin E (50 microM) to cultured HMC exposed to LDL markedly inhibited the stimulation of collagen gene expression. When HMCs were incubated with MDA (200 microM), alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA levels increased by two- to threefold compared to control cells. Immunohistochemical staining utilizing mAb OL-10 demonstrated the presence of MDA-modified proteins in the cytoplasm of HMC exposed to either LDL or MDA. These results suggest that peroxidative products of LDL stimulate collagen gene expression possibly via modification of proteins, which are responsible for the expression of collagen genes in cultured HMCs. Given that, lipid peroxidation of LDL may be implicated in the development of glomerulosclerosis by facilitating excessive mesangial matrix generation.

Costimulation of Fibroblast Collagen and Transforming Growth Factor β1 Gene Expression by Monocyte Chemoattractant Protein-1 via Specific Receptors

Recent studies indicate potential roles of monocyte chemotactic protein-1 (MCP-1) in recruitment of monocytes to sites of inflammation. However, their increased expression does not always correlate with monocyte influx, suggesting other possible biological activities for this member of the C-C chemokine family. In view of its potential role in regulating extracellular matrix expression in fibrotic disorders, the effects of MCP-1 on lung fibroblast collagen expression were evaluated. Isolated rat lung fibroblasts were treated with increasing doses of MCP-1 for variable periods of time and examined for effects on collagen synthesis and expression of procollagen alpha1(I) mRNA expression. The results show that MCP-1 was able to stimulate collagen expression in these cells in a dose-dependent manner but required over 24 h for significant elevation to occur. In view of this delayed time course, the possibility of mediation via endogenous transforming growth factor beta (TGFbeta) was tested by the ability of anti-TGFbeta antibody to inhibit this MCP-1 stimulation of collagen expression. Significant but incomplete inhibition by this antibody was observed. Pretreatment of the cells with antisense but not by sense or missense TGFbeta1 oligodeoxyribonucleotides caused essentially complete inhibition of this MCP-1 stimulatory effect. Furthermore, MCP-1 treatment was found to also stimulate TGFbeta secretion and mRNA expression, which was also abolished by pretreatment with antisense TGFbeta1 oligodeoxyribonucleotides. The kinetics of TGFbeta expression indicates that significant increase preceded that for collagen expression. Binding studies using 125I-labeled MCP-1 indicated the presence of specific and saturable binding sites with a dissociation constant consistent with the dose response curves for stimulation of fibroblast collagen synthesis and TGFbeta activity by MCP-1. These results taken together suggest that MCP-1 stimulates fibroblast collagen expression via specific receptors and endogenous up-regulation of TGFbeta expression. The latter then results in autocrine and/or juxtacrine stimulation of collagen gene expression.

Alcohol and iron: a radical combination?

HepatologyVolume 23, Issue 6 p. 1700-1703 Hepatology ElsewhereFree Access Alcohol and iron: A radical combination? K Houglum, K HouglumSearch for more papers by this author K Houglum, K HouglumSearch for more papers by this author First published: June 1996 https://doi.org/10.1002/hep.510230656Citations: 6AboutPDF 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 onFacebookTwitterLinkedInRedditWechat References 1 Houglum K, Filip M, Witztum JL, Chojkier M. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload. J Clin Invest 1990; 86: 1991– 1998. 2 Pierce RA, Glaug MR, Greco RS, Mackenzie JW, Boyd CD, Deak SB. Increased procollagen mRNA levels in CCl4-induced liver fibrosis in rats. J Biol Chem 1987; 262: 1652– 1658. 3 Chojkier M, Houglum K, Solis-Herruzo J, Brenner DA. Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation? J Biol Chem 1989; 264: 16957– 16962. 4 Parola M, Pinzani M, Casini A, Albano E, Poll G, Gentilini A, Gentilini P, et al. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells. Biochem Biophys Res Com-mun 1993; 194: 1044– 1050. 5 Lee KS, Buck M, Houglum K, Chojkier M. Activation of hepatic stellate cells by TGFα and collagen type I is mediated by oxida-tive stress through c-myb expression. J Clin Invest 1995; 96: 2461– 2468. 6 Casini A, Ceni E, Salzano R, Schuppan D, Milani S, Pellegrini G, Surrenti C. Regulation of undulin synthesis and gene expression in human fat-storing cells by acetaldehyde and transforming growth factor-β 1: comparison with fibronectin. Biochem Biophys Res Commun 1994; 199: 1019– 1026. 7 Tuma DJ, Thiele GM, Xu DS, Klassen LW, Sorrell MF. Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during chronic ethanol administration [Abstract]. Hepatology 1995; 22: 226A. 8 Irving MG, Halliday JW, Powell LW. Association between alcoholism and increased hepatic iron stores. Alcohol Clin Exp Res 1988; 12: 7– 13. 9 Houglum K, Bedossa P, Chojkier M. TGF β and collagen α(I) gene expression are increased in hepatic acinar zone 1 of rats with iron overload. Am J Physiol 1994; 267: G908– G913. 10 Pietrangelo A, Gualdi R, Giovanna C, Montosi G, Ventura E. Molecular and cellular aspects of iron-induced hepatic cirrhosis in rodents. J Clin Invest 1995; 95: 1824– 1831. 11 Nakatsukasa H, Evarts RP, Hsia C, Thorgeirsson SS. Transforming growth factor-β1 and type I procollagen transcripts during regeneration and early fibrosis of rat liver. Lab Invest 1990; 63: 171– 180. 12 Sadrzadeh SM, Nanji AA, Price PL. The oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one reduces hepatic-free iron, lipid peroxidation and fat accumulation in chronically ethanol-fed rats. J Pharmacol Exp Ther 1994; 269: 232– 236. 13 Kadiiska MB, MJ Burkitt, Q-H Ziang, Mason RP. Iron supplementation generates hydroxy radicals in vivo, and ESR spin-trapping investigation. J Clin Invest 1995; 96: 1824– 1831. Citing Literature Volume23, Issue6June 1996Pages 1700-1703 ReferencesRelatedInformation

Mycophenolate mofetil (RS 61443): nothing new under the sun or an important break-through in the field of transplantation?

of glucose concentration on angiotensin 11-induced hypertrophy of proximal tubular cells in culture. Biochem Biophys Res Commun 1991; 176: 902-909 14. Flyvbjerg A, Bornfeldt KE, Marshall SM, Arnqvist HJ, Orskov H. Kidney IGF-I mRNA in initial renal hypertrophy in experimental diabetes in rats. Diabetologia 1990; 33: 334-338 15. Wolf G, Sharma K, Chen Y, Ericksen M, Ziyadeh FN. High glucose-induced proliferation in mesangial cells is reversed by autocrine TGF-0. Kidney Int 1992; 42: 647-656 16. Crowley ST, Brownlee M, Edelstein D et al. Effects of nonenzymatic glycosylation of mesangial matrix on proliferation of mesangial cells. Diabetes 1991; 40: 540-547 17. Nakamura T, Fukui M, Ebihara I et al. mRNA expression of growth factors in glomeruli from diabetic rats. Diabetes 1993; 42: 450-456 18. Ayo SH, Radnik RA, Glass WF et al. Increased extracellular matrix synthesis and mRNA in mesangial cells grown in highglucose medium. Am J Physiol 1991; 260: F185-F191 19. Ziyadeh FN, Sharma K, Ericksen M, Wolf G. Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-/?. J Clin Invest 1994; 93: 536-542 Nephrol Dial Transplant 1995: Editorial Comments

Overlapping Pathways Mediate the Opposing Actions of Tumor Necrosis Factor-α and Transforming Growth Factor-β on α2(I) Collagen Gene Transcription

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are multifunctional peptides intimately involved in the process of extracellular matrix remodeling. We recently showed that TGF-beta stimulates the human alpha 2(I) collagen gene by increasing the affinity of an Sp1-containing transcriptional complex bound to an upstream sequence termed the TbRE (Inagaki, Y., Truter, S. and Ramirez, F. (1994) J. Biol. Chem. 269, 14828-14834). Here, we report that the TbRE-bound complex also mediates the inhibitory signal of TNF-alpha. Nuclear proteins from cells treated with TNF-alpha bind to the TbRE sequence substantially more strongly than those from untreated cells. Additionally, TNF-alpha increases binding of a second protein complex that recognizes the negatively cis-acting element located immediately next to the TbRE. Thus, we postulate that TNF-alpha counteracts the TGF-beta-elicited stimulation of collagen gene expression through overlapping nuclear signaling pathways. One modifies the TGF-beta-targeted transcriptional complex, probably by reducing its stimulatory effect on collagen transcription. The other acts on the binding of the adjacent factor, presumably by increasing its effectiveness in repressing the activity of the collagen promoter. The convergence of the TGF-beta and TNF-alpha pathways on the same sequence of the alpha 2(I) collagen promoter is yet another example of combinatorial gene regulation achieved through composite response elements.

Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-beta.

Previous investigations have demonstrated that growing mesangial cells in high glucose concentration stimulates extracellular matrix synthesis and also increases the expression of TGF-beta. We tested whether the stimulation of extracellular matrix production is mediated by autocrine activation of TGF-beta, a known prosclerotic cytokine. Addition of neutralizing anti-TGF-beta antibody, but not normal rabbit IgG, significantly reduced the high glucose-stimulated incorporation of 3[H]proline. Denaturing SDS-PAGE revealed that mainly collagen types I and IV were stimulated by high (450 mg/dl) D-glucose. This high glucose-mediated increase in collagen synthesis was reduced by the anti-TGF-beta antibody. Treatment of mesangial cells grown in normal (100 mg/dl) D-glucose with 2 ng/ml recombinant TGF-beta 1 mimicked the effects of high glucose. Furthermore, the anti-TGF-beta antibody significantly reduced the increase in mRNA levels encoding alpha 2(I) and alpha 1(IV) collagens induced by high glucose. Thus, the high glucose-stimulated increase of collagen production in mesangial cells is mediated, at least in part, by autocrine TGF-beta activation. We postulate that the interception of the glomerular activity of TGF-beta may be an effective intervention in the management of diabetic nephropathy.

Effect of Topical Retinoic Acids on the Levels of Collagen mRNA During the Repair of UVB-Induced Dermal Damage in the Hairless Mouse and the Possible Role of TGF-β as a Mediator

Topically applied retinoic acids have been found to enhance the gene expression for collagen types I and III in the skin of UVB-irradiated hairless mice. Prior damage is required because the effect is not observed in the skin of age-matched, non-irradiated control animals. Immunochemical methods have shown an increase in TGF-beta 1 and, to a lesser extent, of TGF-beta 2 in the epidermis following retinoic acid treatment. There were no changes in mRNA levels for any of the isotypes of TGF-beta induced by retinoic acid treatment. This study suggests that TGF-beta may mediate the effect of retinoic acids on dermal repair through the stimulation of collagen gene expression.

Ascorbic acid stimulation of collagen biosynthesis independent of hydroxylation

Ascorbic acid stimulates collagen gene expression in cultured fibroblasts but mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Because another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts, we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mmol/L) induced lipid peroxidation and stimulated collagen alpha 1(I) gene transcription in cultured human fibroblasts. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 mumol/L) or methylene blue (10 mumol/L) prevented the stimulation of collagen gene expression. Addition of malondialdehyde (200 mumol/L), a product of lipid peroxidation, to cultured human fibroblasts also increased two- to threefold collagen production and procollagen alpha 1(I) mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes, and this step may be necessary for stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.

Stimulation of Collagen Gene Expression by Ascorbic Acid in Cultured Human Fibroblasts: A role for lipid peroxidation?

Ascorbic acid stimulates collagen gene expression in cultural fibroblasts (Lyons, B. L., and Schwartz, R. L. (1984) Nucleic Acids Res. 12, 2569-2579), but the mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Since another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts (Brenner, D. A., and Chojkier, M. (1987) J. Biol. Chem 262, 17690-17696), we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mM) induced lipid peroxidation in cultured human fibroblasts judging by the production of thiobarbituric acid-reactive substances and carbonyl groups, and by the presence of malondialdehyde- and 4-hydroxynonenal-protein adducts. Ascorbic acid stimulated (2-3-fold) the net production of collagen relative to total proteins, the levels of procollagen alpha 1 (I) mRNA and the transcription of this gene. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with alpha-tocopherol (50 microM) or methylene blue (10 microM) prevented the stimulation of collagen gene expression. The addition of malondialdehyde (200 microM), a product of lipid peroxidation, to cultured human fibroblasts also increased 2-3-fold collagen production and procollagen alpha 1 (I) mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes and this step may be necessary for the stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts.