Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-␤ (TGF-␤) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-␤ in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-␤ is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-␤ stimulation, (2) activation of the promoter by both exogenous TGF-␤ and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-␤ blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-␤ not possible in Smad3؊/؊ mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-␤/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smaddependent TGF-␤ target genes in fibroblasts.

Transforming growth factor (TGF)-beta represents a prototype of multifunctional cytokine. Its broad activities include, among others, context-specific inhibition or stimulation of cell proliferation, control of extracellular matrix (ECM) synthesis and degradation, control of mesenchymal-epithelial interactions during embryogenesis, mediation of cell and tissue responses to injury, control of carcinogenesis, and modulation of immune functions. Regulation of production and turnover of ECM components is essential for tissue homeostasis and function. TGF-beta exerts its effects on cell proliferation, differentiation, and migration in part through its capacity to modulate the deposition of ECM components. Specifically, TGF-beta isoforms have the ability to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Deregulation of these functions is associated with abnormal connective tissue deposition, as observed, for example, during scarring or fibrotic processes. In this review we discuss the current understanding of the signaling mechanisms used by TGF-beta to elicit its effects on target genes, focusing primarily on Smad proteins and their role in the transcriptional regulation of ECM gene expression. Other signaling mechanisms, such as the MAP/SAP kinase or Ras pathways, although potentially important for transmission of some of the TGF-beta signals, will not be described. Transforming growth factor-beta (TGF-beta) plays a critical role in the regulation of extracellular matrix gene expression. Its overexpression is believed to contribute to the development of tissue fibrosis. The recent identification of Smad proteins, TGF-beta receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, has increased our understanding of the molecular mechanisms underlying TGF-beta action. This review focuses primarily on the mechanisms underlying Smad modulation of gene expression and how they relate to wound healing. Potential implications for the development of therapeutic approaches against tissue fibrosis are discussed.

Hedgehog (Hh) and transforming growth factor-B (TGF-B) family members are involved in numerous overlapping processes during embryonic development, hair cycle, and cancer. Herein, we show that TGF

No abstract

Transforming growth factor-b (TGF-b) superfamily members signal via membrane-bound heteromeric serinethreonine kinase receptor complexes. Upon ligand-binding, receptor activation leads to phosphorylation of cytoplasmic protein substrates of the Smad family. Following phosphorylation and oligomerization, the latter move into the nucleus to act as transcription factors to regulate target gene expression. TGF-b responses are not solely the result of the activation Smad cascade, but are highly cell-type specific and dependent upon interactions of Smad signaling with a variety of other intracellular signaling mechanisms, initiated or not by TGF-b itself, that may either potentiate, synergize, or antagonize, the rather linear TGF-b/Smad pathway. These include, (a), regulation of Smad activity by mitogen-activated protein kinases (MAPKs), (b), nuclear interaction of activated Smads with transcriptional cofactors, whether coactivators or corepressors, that may be themselves be regulated by diverse signaling mechanisms, and (c), negative feedback loops exerted by inhibitory Smads, transcriptional targets of the Smad cascade. This review focuses on how MAPKs modulate the outcome of Smad activation by TGF-b, and how cross-signaling mechanisms between the Smad and MAPK pathways may take place and affect cell fate in the context of carcinogenesis.

Members of the transforming growth factor-beta (TGF-beta) superfamily are pleiotropic cytokines that have the ability to regulate numerous cell functions, including proliferation, differentiation, apoptosis, epithelial-mesenchymal transition, and production of extracellular matrix, allowing them to play an important role during embryonic development and for maintenance of tissue homeostasis. Three TGF-beta isoforms have been identified in mammals. They propagate their signal via a signal transduction network involving receptor serine/threonine kinases at the cell surface and their substrates, the SMAD proteins. Upon phosphorylation and oligomerization, the latter move into the nucleus to regulate transcription of target genes. This review will summarize recent advances in the understanding of the mechanisms underlying SMAD modulation of extracellular matrix gene expression in the context of wound healing and tissue fibrosis.

Matrix metalloproteinases belong to a family of zinc-dependent enzymes capable of degrading extracellular matrix and basement membrane components. Their expression is greatly modulated by cytokines and growth factors and involves the gene products of the Fos and Jun families of oncogenes. After extra(peri)cellular activation, their activity can be further controlled by specific tissue inhibitors of metalloproteinases. A correct balance between these regulatory mechanisms is necessary to ensure matrix remodeling in normal physiological processes such as embryonic development, but the overexpression of these enzymes may initiate or contribute to pathological situations such as cartilage degradation in rheumatoid arthritis or to tumor progression and metastasis. Delineation of the mechanisms of metalloproteinase and metalloproteinase inhibitors gene expression, understanding of their mode of interactions, and characterization of their patterns of expression in various tissues in normal and pathological states will lead to new therapeutic strategies to counteract the deleterious effects of matrix metalloproteinases in human disease. Q 1993 WiIey-Liss, Inc.

Summary There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression–based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype‐specific signatures with measurements of characteristics of melanoma phenotype‐specific biology in different melanoma cell lines and short‐term cultures. We further show that 86% of 536 tested melanoma lines and short‐term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two‐state system, as described by the phenotype switching model, underlies melanoma progression.