Interleukin 4 (also known as "B cell stimulatory factor-l"), a cytokine product of T lymphocytes and mast cells, stimulates synthesis of the extracellular matrix proteins, types I and III collagen and fibronectin, by human dermal fibroblasts in vitro. Stimulation of collagen by human recombinant (hr)IL-4 was also demonstrated in several fibroblastic synovial cell lines obtained from patients with rheumatoid arthritis and osteoarthritis. The stimulatory effect of hrIL-4 on fibroblast collagen synthesis was specifically neutralized by rabbit anti-hrIL-4 Ig. IL-4 specifically increased the steady-state levels of types I and III procollagen and fibronectin mRNAs, with no effect on cytoplasmic fl-actin mRNA. Quantitative analysis of the levels of Pro al (I) collagen transcripts in IL-4-treated fibroblast cultures was also corroborated by antisense RNA-mRNA hybridization and RNAse resistant hybrids which showed that IL-4-treated fibroblasts expressed higher levels of Pro al (I) collagen transcripts. Nuclear run-off transcription experiments indicated that IL4 stimulated the rates of mRNA biogenesis. Based on these observations we conclude that IL4 exerts its effect on collagen and fibronectin synthesis at the pretranslational level, resulting in synthesis of these extracellular matrix proteins. These and other data suggest that IL4 may be a "fibrogenic cytokine" that could be important in promoting biogenesis of extracellular matrix proteins in normal wound healing and in pathological fibrosis in which mast cells and T lymphocytes play a central role. (J. Clin. Invest. 1992.

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Massive cell migration, proliferation, phenotypic differentiation, and enhanced biosynthetic activities characterize the sites of wound healing and fibrosis. Regulation of cellular functions by extracellular matrix, which consists of a dynamic assemblage of a variety of interacting molecules capable of reorganization in response to endogenous and exogenous stimuli, represents a fundamental epigenetic mechanism regulating cellular behavior and phenotype. Interactions of the individual components of extracellular matrix with specific cell surface molecules, integrin receptors, and proteoglycans initiate a cascade of signal transduction leading to varied short-term or persistent cellular responses. Extracellular matrix also serves as an important reservoir of cytokines and growth factors, thus modulating the action of a host of potent biological response modifiers by their selective, local accumulation and release. Currently known mechanisms by which extracellular matrix modulates different facets of the process of tissue remodeling after injury, which culminate either in normal wound repair or fibrosis, are discussed.

Transforming growth factor-jB (TGF,8), when injected subcutaneously into newborn mice, induces a rapid fibrotic response, stimulates chemotaxis, and elevates the rates of biosynthesis of collagen and fibronectin by fibroblasts in vitro. We explored the molecular mechanisms of TGFft-mediated stimulation of collagen and fibronectin synthesis in cultured human foreskin fibroblasts.TGF,/ preferentially stimulated the synthesis of fibronectin and type I procollagen chains 3-5-fold as shown by polypeptide analysis. Concomitant elevation in the steady state levels of messenger RNAs (mRNAs) coding for type I procollagen and fibronectin also occurred but without a net increase in the rate of transcription of either of these genes. The preferential stabilization of mRNAs specifying type I procollagen and fibronectin provides a partial explanation for the mechanisms by which TGFft enhances the synthesis of type I procollagen and fibronectin in mesenchymal cells.

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The enhanced synthesis of fatty acids in the liver and adipose tissue in response to insulin is critically dependent on the transcription factor SREBP-1c (sterol-regulatory-element-binding protein 1c). Insulin increases the expression of the SREBP-1c gene in intact liver and in hepatocytes cultured in vitro. To learn the mechanism of this stimulation, we analysed the activation of the rat SREBP-1c promoter and its truncated or mutated congeners driving a luciferase reporter gene in transiently transfected rat hepatocytes. The rat SREBP-1c promoter contains binding sites for LXR (liver X receptor), Sp1, NF-Y (nuclear factor-Y) and SREBP itself. We have found that each of these sites is required for the full stimulatory response of the SREBP-1c promoter to insulin. Mutation of either the putative LXREs (LXR response elements) or the SRE (sterol response element) in the proximal SREBP-1c promoter reduced the stimulatory effect of insulin by about 50%. Insulin and the LXR agonist TO901317 increased the association of SREBP-1 with the SREBP-1c promoter. Ectopic expression of LXRalpha or SREBP-1c increased activity of the SREBP-1c promoter, and this effect is further enhanced by insulin. The Sp1 and NF-Y sites adjacent to the SRE are also required for full activation of the SREBP-1c promoter by insulin. We propose that the combined actions of the SRE, LXREs, Sp1 and NF-Y elements constitute an insulin-responsive cis-acting unit of the SREBP-1c gene in the liver.

Sterol regulatory element-binding proteins (SREBPs) 3 are transcription factors that regulate expression of genes controlling cholesterol homeostasis and de novo fatty acid synthesis (1-7). SREBP-1a and SREBP-1c, which differ only in their first exon, are derived from a single gene through the use of alternative promoters, whereas SREBP-2 is encoded by a separate gene (8). Although there is clearly some functional overlap among the three SREBP isoforms (5), these proteins regulate different metabolic pathways. SREBP-1c preferentially affects transcription of genes that regulate de novo lipid synthesis, whereas SREBP-2 regulates genes involved in cholesterol biosynthesis and metabolism. The SREBP-1a isoform transactivates both lipogenic and cholesterogenic genes (9). In addition, the three SREBP isoforms exhibit differential tissue-specific expression. In replicating tumor cell lines, SREBP-1a constitutes greater than 90% of the SREBP-1 pool; conversely, SREBP-1c is the predominant isoform in liver and adipose tissue (9). Increased hepatic levels of nuclear SREBP-1c are thought to mediate the development of hyperlipidemia in type II diabetes and hyperinsulinemia (10 -12). Nutritional and hormonal factors have been shown to regulate expression of SREBP-1c and its downstream regulatory targets (10,(13)(14)(15). Insulin induces the expression of SREBP-1c mRNA and nascent precursor protein (10,16,17). Glucagon opposes this effect of insulin via its second messenger cAMP (18). Newly synthesized SREBPs contain two transmembrane domains that are embedded in the endoplasmic reticulum (ER) with the NH 2 -and COOH-terminal sequences exposed to the cytoplasm. Following transport from ER to Golgi, the transcriptionally active NH 2 -terminal segments of SREBPs are liberated by two successive cleavages; the first cleavage in the loop extending into the vesicular lumen is carried out by site 1 protease (S1P), and the second cleavage is executed within the NH 2 -proximal transmembrane domain by site 2 protease (S2P).Regulation of post-translational proteolysis has been studied most extensively in the case of SREBP-2 and SREBP-1a, both of which are regulated primarily by sterols. Within the ER, the

Pax9 and Msx1 encode transcription factors that are known to be essential for the switch in odontogenic potential from the epithelium to the mesenchyme. Multiple lines of evidence suggest that these molecules play an important role in the maintenance of mesenchymal Bmp4 expression, which ultimately drives morphogenesis of the dental organ. Here we demonstrate that Pax9 is able to directly regulate Msx1 expression and interact with Msx1 at the protein level to enhance its ability to transactivate Msx1 and Bmp4 expression during tooth development. In addition, we tested how a missense mutation (T62C) in the paired domain of PAX9 that is responsible for human tooth agenesis (1) affects its functions. Our data indicate that although the mutant Pax9 protein (L21P) can bind to the Msx1 protein, it fails to transactivate the Msx1 and Bmp4 promoter, presumably because of its inability to bind cognate paired domain recognition sequences. In addition, synergistic transcriptional activation of the Bmp4 promoter was lost with coexpression of mutant Pax9 and wild-type Msx1. This suggests that Pax9 is critical for the regulation of Bmp4 expression through its paired domain rather than Msx1. Our findings demonstrate the partnership of Pax9 and Msx1 in a signaling pathway that involves Bmp4. Furthermore, the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 at the level of transcription and through formation of a protein complex determines the fate of the transition from bud to cap stage during tooth development.