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Background/aims: In the cirrhotic liver, gene expression of the multifunctional cytokine oncostatin M (OSM) is up-regulated, but its cellular origin is unknown. Therefore, we investigated the expression of OSM protein and its specific receptor subunits, OSMRbeta and LIFRbeta in normal and cirrhotic human liver using immunohistochemical and Western blot analysis., Results: OSM protein was expressed in Kupffer cells, variably in normal liver but consistently in cirrhosis. OSMRbeta was expressed at low level in hepatocytes of all normal livers examined, but in no cirrhotic sample. In contrast, LIFRbeta receptor was expressed weakly in normal livers, but much more intensely in cirrhosis, in reactive ductules, bile duct epithelial cells and perisinusoidal areas. Double immunostaining showed co-localization of LIFRbeta with cytokeratin 7, proliferating cell nuclear antigen (PCNA) and leukemia inhibitory factor (LIF), in bile duct epithelial cells, but not with alpha-smooth muscle actin, a myofibroblast marker., Conclusions: In human liver, OSM protein is expressed in Kupffer cells, variably in normals but universally in cirrhosis. The differential expression pattern of OSM and its receptors could allow for differential OSM signaling by alternative utilization of receptors to promote hepatocyte proliferation in acute injury and, with its homologue LIF, for the bile ductular reaction in cirrhosis.

The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.

Objective: Transforming growth factor beta (TGFbeta) induces profibrotic responses in normal fibroblasts, and plays a fundamental role in the pathogenesis of fibrosis in scleroderma (systemic sclerosis [SSc]). The intensity of cellular responses elicited by cytokines is modulated by transcriptional coactivators such as the histone acetylase p300. The objective of these studies was to delineate the physiologic role of p300 in Smad-dependent profibrotic responses elicited by TGFbeta., Methods: Ectopic p300 was transiently expressed in normal dermal fibroblasts. Cellular p300 levels were suppressed using p300-specific ribozymes. The regulation of gene expression was examined by transient transfection assays, Northern blotting, and immunoblot analysis. The expression of p300 in normal and scleroderma fibroblasts was evaluated by confocal microscopy and immunoblotting, and p300 levels in skin from mice with experimental scleroderma were assessed by immunohistochemistry., Results: In normal fibroblasts, TGFbeta induced an increase in the levels of p300. Forced expression of ectopic p300 in these cells dramatically enhanced the magnitude of TGFbeta responses, whereas selective depletion of p300 using ribozyme resulted in abrogation of TGFbeta-induced collagen synthesis and promoter activity. Furthermore, TGFbeta lost its ability to induce Smad-dependent transcription in p300-depleted fibroblasts. These responses could be fully rescued with ectopic p300. Abrogation of Smad-mediated TGFbeta signaling was not due to alterations in the levels or the ligand-dependent phosphorylation or intracellular trafficking of endogenous Smads. Immunohistochemical analysis demonstrated substantially increased p300 expression in lesional skin from mice with chronic graft-versus-host disease, an animal model of scleroderma. Furthermore, levels of p300 were 2-3-fold higher in cultured fibroblasts derived from SSc patients than in fibroblasts from matched normal controls., Conclusion: These results establish, for the first time, that the coactivator histone acetylase p300, itself a target of TGFbeta regulation, is an essential component of the cellular TGFbeta signal transduction pathways mediating stimulation of collagen synthesis in fibroblasts. Since the cellular abundance of p300 appears to govern the intensity of profibrotic responses elicited by TGFbeta, elevated p300 expression in lesional tissue may contribute to the progression of skin fibrosis in scleroderma.

Oncostatin M (OSM), a member of the interleukin-6 family of cytokines, is thought to be expressed mostly by activated T-lymphocytes and monocytes in adult animals. However, here we report specific constitutive tissue expression of OSM in the pancreas, kidney, testes, spleen, stomach, and brain, but not liver or lung, of three adult rodent species.

Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (approximately 2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK1 may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-beta. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-beta. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF-beta cooperated in TIMP-1 up-regulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-1 gene.

Purpose of Review: Systemic sclerosis is a complex disease manifesting itself by fibrosis of skin and other internal organs. Fibroblasts isolated from scleroderma lesions and cultured in vitro are characterized by increased synthesis of collagen and other extracellular matrix proteins, consistent with the disease phenotype. Cultured systemic sclerosis fibroblasts therefore serve as a principal experimental model for studying the molecular and cellular mechanisms involved in collagen overproduction in this disease. This review will discuss recent findings related to intracellular signal transduction pathways implicated in deregulated extracellular matrix deposition by systemic sclerosis fibroblasts., Recent Findings: Recent findings suggest that constitutively elevated synthesis of extracellular matrix by cultured systemic sclerosis fibroblasts is, at least in part, due to the aberrant activation of the autocrine transforming growth factor-beta signaling. Enhanced constitutive transforming growth factor-beta signaling may result from the elevated levels of transforming growth factor-beta receptor type I and/or inappropriate activation of Smad3. These alterations of the transforming growth factor-beta signaling in systemic sclerosis fibroblasts may facilitate increased collagen production in vivo even under conditions of low ligand availability. However, there exist many inconsistencies among published reports regarding the detailed mechanisms of this pathway in systemic sclerosis fibroblasts, and additional studies in this area are needed. Other signaling molecules implicated in fibrotic phenotype include several members of the protein kinase C family, mammalian target of rapamycin, mitogen-activated protein kinase, necdin, reactive oxygen species, and sphingolipids. These signaling pathways may work in conjunction with transforming growth factor-beta signaling to regulate the behavior of systemic sclerosis fibroblasts., Summary: Alterations in multiple signaling pathways contribute to elevated extracellular matrix synthesis by systemic sclerosis fibroblasts. Improved understanding of the key signaling molecules may provide a novel avenue for therapeutic interventions.

Lysophosphatidic acid (LPA) is a serum-derived pleiotropic mediator with a potential role in wound repair. Since extracellular matrix (ECM) deposition is a critical part of wound healing, this study was designed to examine whether LPA is involved in ECM regulation. Using human dermal fibroblasts, we demonstrate that LPA counteracts transforming growth factor-beta (TGF-beta) stimulation of type I collagen mRNA and protein. This factor elicits its inhibitory effects at the posttranscriptional level via destabilization of type I collagen mRNA. Furthermore, using the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, we show that the extracellular signal-regulated kinase (ERK) pathway is a negative regulator of the TGF-beta-induced stabilization of type I collagen mRNA, and that the activation of the ERK pathway by LPA mediates their inhibitory effects on collagen production. In conclusion, this study describes a novel function for LPA as an antagonist of TGF-beta induced ECM deposition. These findings may be relevant to physiologic wound repair and may be useful in designing therapeutic agents to prevent excessive scarring.

The mammalian target of rapamycin (mTOR) is a multifunctional protein involved in the regulation of cell growth, proliferation, and differentiation. The goal of this study was to determine the role of mTOR in type I collagen regulation. The pharmacological inhibitor of phosphatidylinositol (PI) 3-kinase, LY294002, significantly inhibited collagen type I protein and mRNA levels. The effects of LY294002 were more pronounced on the collagen alpha1(I) chain, which was inhibited at the transcriptional and mRNA stability levels versus collagen alpha2(I) chain, which was inhibited through a decrease in mRNA stability. In contrast, addition of the PI 3-kinase inhibitor, wortmannin, did not alter type I collagen steady-state mRNA levels. This observation and further experiments using an inactive LY294002 analogue suggested that collagen mRNA levels are inhibited independent of PI 3-kinase. Additional experiments have established that mTOR positively regulates collagen type I synthesis in human fibroblasts. These conclusions are based on results demonstrating that inhibition of mTOR activity using a specific inhibitor, rapamycin, reduced collagen mRNA levels. Furthermore, decreasing mTOR expression by about 50% by using small interfering RNA resulted in a significant decrease of collagen mRNA (75% COL1A1 decrease and 28% COL1A2 decrease) and protein levels. Thus, mTOR plays an essential role in regulating basal expression of collagen type I gene in dermal fibroblasts. Together, our data suggest that the classical PI 3-kinase pathway, which places mTOR downstream of PI 3-kinase, is not involved in mTOR-dependent regulation of type I collagen synthesis in dermal fibroblasts. Because collagen overproduction is a main feature of fibrosis, identification of mTOR as a critical mediator of its regulation may provide a suitable target for drug or gene therapy.

Objective: Aberrant transforming growth factor beta (TGFbeta) signaling has been implicated in the pathogenesis of scleroderma (systemic sclerosis [SSc]), but the contribution of specific components in this pathway to SSc fibroblast phenotype remains unclear. This study was undertaken to delineate the role of TGFbeta receptor type I (TGFbetaRI) and TGFbetaRII in collagen overexpression by SSc fibroblasts., Methods: Primary dermal fibroblasts from SSc patients and healthy adults were studied (n = 10 matched pairs). Adenoviral vectors were generated for TGFbetaRI (AdTGFbetaRI), TGFbetaRII (AdTGFbetaRII), and kinase-deficient TGFbetaRII (AdDeltakRII). TGFbetaRI basal protein levels were analyzed by (35)S-methionine labeling/immunoprecipitation and immunohistochemistry. Type I collagen and TGFbetaRII basal protein levels were analyzed by Western blot and newly secreted collagen by (3)H-proline incorporation assay., Results: Analysis of endogenous TGFbetaRI and TGFbetaRII protein levels revealed that SSc TGFbetaRI levels were increased 1.7-fold (P = 0.008; n = 7) compared with levels in healthy controls, while TGFbetaRII levels were decreased by 30% (P = 0.03; n = 7). This increased TGFbetaRI:TGFbetaRII ratio correlated with SSc collagen overexpression. To determine the consequences of altered TGFbetaRI:TGFbetaRII ratio on collagen expression, healthy fibroblasts were transduced with AdTGFbetaRI or AdTGFbetaRII. Forced expression of TGFbetaRI in the range corresponding to elevated SSc TGFbetaRI levels increased basal collagen expression in a dose-dependent manner, while similar TGFbetaRII overexpression had no effect, although transduction of fibroblasts at higher multiplicities of infection led to a marked reduction of basal collagen levels. Blockade of TGFbeta signaling via AdDeltakRII resulted in approximately 50% inhibition of basal collagen levels in healthy fibroblasts and in 5 of 9 SSc cell lines. A subset of SSc fibroblasts (4 of 9 cell lines) was resistant to this treatment. SSc fibroblasts with the highest levels of TGFbetaRI were the least responsive to collagen inhibition via DeltakRII., Conclusion: This study indicates that an increased TGFbetaRI:TGFbetaRII ratio may underlie aberrant TGFbeta signaling in SSc and contribute to elevated basal collagen production, which is insensitive to TGFbeta signaling blockade via DeltakRII.

The Ets family consists of a large number of evolutionarily conserved transcription factors, many of which have been implicated in tumor progression. Extensive studies on this family of proteins have focused so far mainly on the biochemical properties and cellular functions of individual factors. Since most of the Ets factors can bind to the core consensus DNA sequence GGAA/T in vitro, it has been a challenge to differentiate redundant from specific functions of various Ets proteins in vivo. Recent findings, however, suggest that such apparent redundancy may in fact be a central component of a network of differentially regulated specific Ets factors, resulting in distinct biological and pathological consequences. The programmed "Ets conversion" appears to play a critical role during tumor progression, especially in control of cellular changes during epithelial-mesenchymal transition and metastasis. Coordination of multiple Ets gene functions also mediates interactions between tumor and stromal cells. As such, these new insights may provide a novel view of the Ets gene family as well as a focal point for studying the complex biological control involved in tumor progression., (Copyright 2004 Wiley-Liss, Inc.)

In cultured human dermal fibroblasts, transforming growth factor (TGF)-beta induced the mRNA expression of tenascin-C (TN-C). The molecular mechanism(s) underlying this process is not presently understood. In this study, we performed serial 5' deletion and a transient transfection analysis to define a region in the TN-C promoter mediating the inducible responsiveness to TGF-beta. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. A DNA affinity precipitation assay revealed that Smad2/Smad3 bound to this site in a transient and specific manner. Overexpression of Smad3 or Smad4 activated the TN-C promoter activity and superinduced the TN-C promoter activity stimulated by TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the TN-C promoter activity in a synergistic manner. Mutation of the Smad-binding sites, the Ets-binding sites, or Sp1/3-binding sites in the TN-C promoter abrogated the TGF-beta/Smad-inducible promoter activity. Immunoprecipitation analysis revealed that Smad3, Sp1, and Ets1 form a transcriptionally active complex. Furthermore, the interaction between Smads and CBP/p300 in TGF-beta signaling was confirmed. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the TN-C promoter mediating responsiveness to TGF-beta involving Smad3/4, Sp1, Ets1, and CBP/p300.

Systemic sclerosis (SSc, scleroderma) is a complex disease manifesting itself by fibrosis of skin and other internal organs. Fibroblasts isolated from SSc lesions and cultured in vitro are characterized by the increased synthesis of collagen and other extracellular matrix (ECM) proteins, consistent with the disease phenotype. Cultured SSc fibroblasts therefore serve as a principal experimental model for studying the molecular and cellular mechanisms involved in collagen overproduction in this disease. This article provides an overview of transcription factors that are deregulated in SSc fibroblasts. The possible origin of SSc fibroblasts is also discussed.

Latent human cytomegalovirus (HCMV) infection has been implicated in diseases characterized by tissue remodeling. Because of recent evidence indicating the possibility of a partial HCMV reactivation, the purpose of this study was to examine the role of the HCMV immediate early (IE) genes in the regulation of extracellular matrix (ECM) related host genes. Adenoviral vector expressing IE1 was generated to allow efficient gene delivery into human fibroblasts. IE1 stimulated the prolonged expression of connective tissue growth factor (CTGF) and TIMP1. IE1-dependent stimulation of CTGF was partially mediated by TGF-beta. Moreover, whereas collagenous proteins and collagen type 1 mRNA were only transiently induced by IE1 in the majority of fibroblasts, in selected fibroblast strains IE1 induced persistent ECM upregulation for up to 120 hours. This study suggests that transient or limited HCMV reactivation may play a direct role in abnormal matrix remodeling in GVHD, scleroderma, atherosclerosis and other HCMV-linked diseases.

Levels of pulmonary and activation-regulated chemokine (PARC) mRNA and protein are increased in the lungs of patients with pulmonary fibrosis. The purpose of this study was to establish whether PARC could be directly involved in development of pulmonary fibrosis by stimulating collagen production in lung fibroblasts. Exposure to PARC increased production of collagen mRNA and protein by 3- to 4-fold in normal adult lung and dermal fibroblast cells. Collagen mRNA transiently increased after 3-6 h of activation with PARC, with an increase in collagen protein detected after 24 h of activation. At the same time, PARC had less pronounced effect on fibroblast proliferation, not exceeding 50% increase over control nonstimulated cells. PARC intracellular signaling led to activation of ERK1/2, but not p38, in fibroblasts; pharmacologic inhibition of ERK, but not p38, also blocked PARC's effect on collagen production. Inhibition experiments with pertussis toxin suggested that PARC receptor is G protein-coupled. Thus, PARC is a member of the CC chemokine family that acts directly as a profibrotic factor.

The molecular and cellular mechanisms that maintain proper collagen homeostasis in healthy human skin and are responsible for the dysregulated collagen synthesis in scleroderma remain primarily unknown. This study demonstrates that Fli1 is a physiological negative regulator of collagen gene expression in dermal fibroblasts in vitro and in human skin in vivo. This conclusion is supported by the analyses of mouse embryonic fibroblasts from Fli1(-/-), Fli1(+/-), and Fli1(+/+) mice. In cultured human and mouse fibroblasts Fli1 expression levels are inversely correlated with the collagen type I expression levels. These in vitro observations were validated in vivo. In healthy human skin Fli1 protein is expressed in fibroblasts and endothelial cells. Significantly, absence of Fli1 expression in individual fibroblasts correlates with elevated collagen synthesis. In contrast to healthy skin, Fli1 protein is consistently absent from fibroblasts and significantly reduced in endothelial cells in clinically involved scleroderma skin, which correlates with enhanced collagen synthesis in systemic sclerosis skin. This study supports the role of Fli1 as a suppressor of collagen transcription in human skin in vivo. Persistent down-regulation of Fli1 in scleroderma fibroblasts in vivo may directly contribute to uncontrolled matrix deposition in scleroderma skin.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.

Objective: To examine the mechanism of collagen induction by connective tissue growth factor (CTGF), a profibrotic cytokine overexpressed in the skin of patients with systemic sclerosis (SSc)., Methods: Dermal fibroblasts from 7 SSc patients and 7 matched healthy adult donors were stimulated with CTGF in the presence or absence of the culture-medium supplement, insulin-transferrin-selenium (ITS). Expression of collagen protein was analyzed by a (3)H-proline incorporation assay. To identify the signaling pathways mediating CTGF induction of collagen, pharmacologic inhibitors were used, including rottlerin, a protein kinase C delta (PKC delta) inhibitor., Results: Collagen levels in both SSc and normal fibroblasts were increased after treatment with transforming growth factor beta in serum-free medium, whereas no stimulation was observed following addition of CTGF. In the presence of ITS, CTGF (2.5 ng/ml) potently stimulated collagenous protein levels in SSc cell lines (n = 5); however, CTGF was not stimulatory in the majority of normal fibroblasts (n = 6). ITS alone induced collagen levels in normal fibroblasts to the levels observed in SSc skin fibroblasts, thereby diminishing the hallmark difference in basal collagen levels in these cell types. Insulin was the ITS component responsible for promoting the basal and CTGF stimulation of collagenous proteins. Rottlerin, the PKC delta inhibitor, down-regulated collagen synthesis in normal and SSc fibroblasts cultured in ITS, and inhibited the stimulatory effects of CTGF in cooperation with insulin or of insulin (500 ng/ml) alone., Conclusion: Increased responsiveness of SSc fibroblasts to CTGF-mediated collagen synthesis requires the costimulatory activation of insulin signaling pathways to induce matrix production. Blockade of this effect via rottlerin may suggest that PKC delta is a downstream signaling molecule necessary for CTGF stimulation of collagen synthesis.

Transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) are ubiquitously expressed in various forms of tissue fibrosis, including fibrotic diseases of the kidney. To clarify the common and divergent roles of these growth factors in the cells responsible for pathological extracellular matrix (ECM) deposition in renal fibrosis, the effects of TGF-beta and CTGF on ECM expression in primary human mesangial (HMCs) and human proximal tubule epithelial cells (HTECs) were studied. Both TGF-beta and CTGF significantly induced collagen protein expression with similar potency in HMCs. Additionally, alpha(2)(I)-collagen promoter activity and mRNA levels were similarly induced by TGF-beta and CTGF in HMCs. However, only TGF-beta stimulated collagenous protein synthesis in HTECs. HTEC expression of tenascin-C (TN-C) was increased by TGF-beta and CTGF, although TGF-beta was the more potent inducer. Thus both growth factors elicit similar profibrogenic effects on ECM production in HMCs, while promoting divergent effects in HTECs. CTGF induction of TN-C, a marker of epithelial-mesenchymal transdifferentiation (EMT), with no significant induction of collagenous protein synthesis in HTECs, may suggest a more predominant role for CTGF in EMT rather than induction of excessive collagen deposition by HTECs during renal fibrosis.

Background: Liver injury transforms hepatic stellate cells into myofibroblast (MFB)-like cells. With recovery from injury, MFBs undergo apoptosis, but it is unknown whether they can also revert to quiescence., Aim: To determine whether human (h)MFBs become quiescent if cultured on a basement membrane-like substrate (Matrigel)., Methods: hMFBs obtained from cirrhotic liver were re-cultured on plastic or Matrigel. Expression of genes of collagen metabolism was assayed before and after transforming growth factor beta (TGFbeta) and Oncostatin M (OSM) stimulation., Results: hMFBs had typical MFB-like morphology, with abundant alpha-smooth muscle actin (SMA) but no cytoplasmic lipid droplets. hMFBs re-cultured on Matrigel reverted to alphaSMA-negative, lipid droplet-positive quiescent morphology. alphaSMA, collagen alpha1(1) (COL1A1) and collagen alpha2(1) (COL1A2) messages were upregulated in hMFBs cultured on plastic, but suppressed by Matrigel. The opposite was true for metalloproteinase-1 mRNA. OSM but not TGFbeta reduced alphaSMA mRNA by 30% while TGFbeta but not OSM upregulated COL1A1 mRNA by 48%, in hMFBs on plastic. TGFbeta and OSM stimulated COL1A1 gene expression in Matrigel by 50 and 60%, respectively., Conclusions: Matrigel culture de-activates hMFBs yet collagen gene expression still responds to fibrogenic cytokines. The responses of hMFB gene expression to TGFbeta and OSM, are regulated differently by the extracellular matrix.

Extracellular matrix (ECM) production and turnover are tightly controlled under normal physiological conditions. Ets factors regulate matrix turnover by activating transcription of several metalloproteinases (MMPs) and are frequently overexpressed in aggressive tumors and arthritis. Because of the prominent role of transforming growth factor beta (TGF-beta) in ECM synthesis, this study was undertaken to determine the possible interactions between Ets1 and the TGF-beta pathway. Experiments using adenoviral delivery of Ets1 in human fibroblasts have established that Ets1 strongly suppresses TGF-beta induction of collagen type I and other matrix-related genes and reverses TGF-beta-dependent inhibition of MMP-1. Subsequent experiments utilizing COL1A2 promoter demonstrated that Ets1 in the presence of TGF-beta signaling interferes with the stimulatory role of p300. To gain further insight into the mechanism of Ets1 inhibition of the TGF-beta signaling, the protein levels and post-translational modifications of Ets1 after TGF-beta treatment were analyzed. The level of total Ets1 protein was not affected after 24 h of TGF-beta stimulation. Moreover, TGF-beta did not affect either serine or threonine phosphorylation levels of Ets1. However, TGF-beta induced rapid and prolonged lysine acetylation of Ets1. In addition, analyses of endogenous p300.Ets1 complexes revealed that acetylated Ets1 is preferentially associated with the p300/CBP complexes. TGF-beta treatment leads to dissociation of Ets1 from the CBP/p300 complexes. Together, these findings suggest that elevated expression of Ets1 in fibroblasts fundamentally alters their responses to TGF-beta in favor of matrix degradation and away from matrix deposition as exemplified by arthritis and cancer.

Smad7, a protein induced by transforming growth factor-beta1 (TGF-beta1) in many target cells, inhibits TGF-beta1 signal transduction and is thought to mediate an intracellular negative feedback response that limits TGF-beta1 effects. It is possible that overexpression of Smad7 could block specified effects of TGF-beta1 on mesangial cells, a TGF-beta target in glomerular disease. Smad7 mRNA was induced by TGF-beta1 within 1 h in a concentration-dependent manner in a transformed mouse mesangial cell (MMC) line. Uptake of (14)C-spermidine from the medium by MMC and the transcriptional activity of a segment of the human collagen pro-alpha2 type 1 chain (COL1A2) promoter fused to a luciferase reporter gene were used as indices of TGF-beta1. Treatment with TGF-beta1 increased (14)C-spermidine uptake rate in a time-, concentration-, and temperature-dependent manner. For example, exposure to 1 ng/ml TGF-beta1 for 15 h increased uptake approximately twofold, a response that was attenuated by cycloheximide. Transfection of Smad7 expression vector into MMC abrogated both TGF-beta1-dependent stimulation of spermidine uptake and COL1A2 promoter activity. It is concluded that: (1) TGF-beta1 induces Smad7 in MMC; (2) (14)C-spermidine uptake is a convenient quantitative index of TGF-beta1 effect in these cells; and (3) overexpression of Smad7 is a highly effective method of blocking at least some mesangial cell effects of TGF-beta1 that may warrant evaluation in vivo in experimental glomerular disease.

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.

Scleroderma is a complex disease characterized by activation of the immune system, small-vessel vasculopathy, and fibrosis of the skin and other organs. This review is limited to the discussion of scleroderma fibroblast biology and the cellular and molecular mechanisms that contribute to the abnormal deposition of collagen. Selected aspects of abnormal extracellular matrix (ECM) regulation by scleroderma fibroblasts are critically reviewed. These include the role of transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) and their receptors in the fibrotic process in scleroderma and the overview of the transcription factors involved in regulation of the human alpha2 (I) collagen (COL1A2) gene.

Background/aims: We previously showed that in cultured human myofibroblasts (hMFBs), Oncostatin M (OSM)-stimulated collagen accumulation is associated with increased tissue inhibitor of metalloproteinase (TIMP)1 message. However, the mechanism is unknown., Methods: hMFBs were isolated by outgrowth from cirrhotic liver explants and cultured. Using OSM (10 ng/ml) stimulation, with and without PD98059 (PD, a specific mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor), we measured: TIMP-1 protein in culture medium by Western blot, TIMP-1 mRNA levels and stability by Northern analysis, TIMP-1 promoter activity (including transcription site mutation analysis), DNA binding activity to nuclear proteins by electrophoretic mobility shift assay (EMSA), and total and phosphorylated MAP kinase in hMFB extracts by Western blot., Results: OSM stimulation of hMFBs increased TIMP-1 protein production 1.69-fold, TIMP-1 mRNA levels 2.36-fold, promoter activity 2.22-fold, TIMP-1 message stability, and phosphorylation of mitogen-activated protein kinase (MAPK). PD inhibited OSM-mediated stimulation of TIMP-1 protein, mRNA, promoter activity, phosphorylation of MAPK, and TIMP-1 message stability. An SP-1 transcription site of the TIMP-1 promoter is essential for OSM induction of TIMP-1 promoter activity. EMSA demonstrates that this site binds to transcriptional factors SP-1 and SP-3., Conclusions: OSM stimulates the TIMP-1 axis in hMFBs in vitro via a MEK-MAP kinase cascade.

Many different plant species synthesise triterpenoid saponins as part of their normal programme of growth and development. Examples include plants that are exploited as sources of drugs, such as liquorice and ginseng, and also crop plants such as legumes and oats. Interest in these molecules stems from their medicinal properties, antimicrobial activity, and their likely role as determinants of plant disease resistance. Triterpenoid saponins are synthesised via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterised, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. Considerable advances have recently been made, however, in the area of 2,3-oxidosqualene cyclisation, and a number of genes encoding the enzymes that give rise to the diverse array of plant triterpenoid skeletons have been cloned. Progress has also been made in the characterisation of saponin glucosyltransferases. This review outlines these developments, with particular emphasis on triterpenoid saponins.

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.

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.

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.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Objective: Systemic sclerosis (scleroderma, SSc) frequently affects the lungs, and interstitial pulmonary fibrosis is one of its major complications. The pathophysiology of SSc lung disease s poorly understood, but recent studies document an inflammatory process resembling that of idiopathic pulmonary fibrosis with increased numbers of activated alveolar macrophages and granulocytes in bronchoalveolar lavage (BAL) fluid). We determined levels of 2 potentially important mediators of fibroproliferative repair in BAL fluid from patients with SSc., Methods: Using Western blot and ELISA techniques we measured levels of platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta in BAL fluid from patients with SSc and healthy controls. The mitogenic effect of these cytokines on SSc lung myofibroblasts was determined by [3H]thymidine incorporation., Results: SSc BAL fluid contains significantly elevated levels of PDGF-AA and PDGF-BB. Where TGF-beta 1 was significantly elevated in SSc lavage fluid, the amount of TGF-beta 2 was significantly less than that observed in normal lavage fluid. Myofibroblasts cultured from SSc lavage fluid exhibited enhanced [3H]thymidine incorporation upon exposure to the growth factors present in SSc BAL fluid: PDGF and TGF-beta 1. SSc lung myofibroblasts pretreated with TGF-beta 1 exhibited an enhanced mitogenic effect upon stimulation by PDGF, due in part to the induction of the PDGF alpha receptor., Conclusion: Our studies support a role for PDGF and TGF-beta 1 in the pathogenesis of SSc lung disease.

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.

On the basis of 7 cases of temporal bone paraganglioma diagnosis of this tumor is presented. Apart from aural symptoms cranial nerves palsy may appear. Computer tomography, magnetic resonance imaging and subtraction angiography are essential investigations.

Previous studies have demonstrated that platelet-derived growth factor (PDGF) alpha receptor expression is up-regulated by transforming growth factor-beta 1 (TGF-beta 1) in scleroderma dermal fibroblasts, but not in healthy control fibroblasts. We asked whether this selective effect in scleroderma cells was TGF-beta 1-specific or a general response by studying responses to other growth factors. In this study, we compared the expression of alpha and beta PDGF receptor subunits (mRNA and protein levels) in these two cell types in response to basic fibroblast growth factor (bFGF) and TGF-beta 1. bFGF coordinately stimulated mRNA levels of alpha and beta receptor subunits in healthy fibroblasts, but did not change PDGF receptor expression in scleroderma fibroblasts. Conversely, and in agreement with previous observations, TGF-beta 1 induced PDGF alpha receptor expression in scleroderma fibroblasts, but not in healthy fibroblasts. PDGF beta receptor mRNA levels were induced to similar degrees by TGF-beta 1 in both cell types. PDGF alpha receptor protein levels correlated directly with mRNA levels, induced by bFGF only in healthy fibroblasts and by TGF-beta 1 only in scleroderma fibroblasts. However, PDGF beta receptor protein levels were not altered by either growth factor in either cell type. Thus, the activated state of scleroderma fibroblasts does not include receptor-signaling pathways to bFGF. This distinct pattern of expression of PDGF alpha receptors in scleroderma fibroblasts suggests a possible role for the coordinately expressed PDGF AA ligand/alpha receptor system in the development of fibrosis.

Mitogen-activated protein kinase (MAPK) is activated in many cell types in response to growth factors during the G0-G1 transition in the cell cycle. We investigated the effects of platelet-derived growth factor (PDGF) AA and PDGF BB on activation of MAPK in human dermal fibroblasts, and asked whether its activation correlates with proliferative responses of human fibroblasts to PDGF AA and PDGF BB. Treatment with either PDGF isoform for 20 min resulted in equal phosphorylation of MAPK as visualized by gel shifts in Western blotting with anti-MAPK polyclonal antibody. This finding was confirmed by measurements of MAPK activity in response to increasing doses (2-20 ng/ml) of PDGF AA and PDGF BB in in vitro assays with myelin basic protein as a substrate. PDGF AA was slightly less potent than PDGF BB, but both growth factors induced similar maximal activations. Kinetics of activation were also similar for both isoforms, with maximal induction of MAPK at 10-20 min after growth factor addition followed by a gradual decline to control levels at 1 h. Activation of MAPK by both PDGF isoforms was also confirmed by measuring myelin basis protein phosphorylation in MAPK immunoprecipitates. Thus both PDGF AA and PDGF BB are potent activators of MAPK in human dermal fibroblasts. In contrast, PDGF BB elicited a strong mitogenic response, while PDGF AA had no significant effect on DNA synthesis in human dermal fibroblasts. These data indicate that acute activation of MAPK is not sufficient to stimulate cells to progress through cell cycle. PDGF AA may have other biologic functions or may play a co-stimulatory role in proliferation of human dermal fibroblasts.

Differences in the responses to growth factors of normal fibroblasts and scleroderma fibroblasts have been demonstrated previously. Because human dermal fibroblasts are heterogeneous populations, whether known differences between papillary and reticular dermal fibroblasts could account for the noted differences between normal and scleroderma fibroblasts was investigated. Papillary dermal fibroblasts were grown from a dermatome section of normal skin from an adult donor. Reticular dermal fibroblasts were cultured from punch biopsy specimens taken from the same location. In vitro, papillary dermal fibroblasts proliferated more rapidly, had a higher mitotic index and reached greater density at confluence, confirming previous observations. The reticular dermal fibroblasts were more dendritic. Reticular dermal fibroblasts had higher rates of tritiated thymidine uptake and larger increases in mitotic index in response to isoforms of platelet-derived growth factor (PDGF). The characteristic response of scleroderma fibroblasts, potentiation of the mitogenicity of PDGF AA by transforming growth factor-beta (TGF-beta), was not observed in either cell type. Therefore, the phenotypic characteristics of scleroderma fibroblasts cannot be explained by an unusual admixture of papillary and reticular fibroblasts.

We describe the detection and the growth of fibroblasts with human smooth muscle cell differentiation features from bronchoalveolar lavage (BAL) fluid of 53% of patients with scleroderma, but not from healthy controls. The binding of alpha-actin, vimentin and desmin antibodies by scleroderma lung fibroblasts exceeded that of normal adult lung fibroblasts, indicating that scleroderma lung fibroblasts express some markers of human smooth muscle cell differentiation (myofibroblasts), which may account for differences in biological behavior. A mesenchymal cell phenotype was documented by mRNA analysis, showing high expression of collagen type I and fibronectin in these cells. Fibronectin is also released in significantly higher amounts by scleroderma alveolar macrophages than by macrophages from healthy donors.

Lesional fibroblasts propagated from the skin of patients with scleroderma, when compared to normal fibroblasts, show increased synthesis of several collagens and increased levels of their corresponding mRNAs. Using constructs (COL1A2/CAT) containing the promoter for the alpha 2 (I) collagen gene in transient transfection assays with matched pairs of scleroderma and normal skin fibroblasts, we observed higher transcriptional activity of the COL1A2 gene in scleroderma fibroblasts and, in contrast to normal fibroblasts, no further expression was observed in the presence of TGF beta 1. Analysis of the expression of COL1A2 promoter deletion constructs indicates that the TGF beta responsive element functional in normal fibroblasts and the sequence involved in intrinsic upregulation of COL1A2 gene expression in scleroderma fibroblasts are both located between bp-376 (Bgl II) and bp-108 (Sma I) sites. These data may indicate that intrinsic upregulation of extracellular matrix genes in scleroderma fibroblasts utilizes a TGF beta dependent pathway.