The irreversible and inexorable accumulation of extracellular matrix is characteristic of a variety of acquired and hereditary renal disorders, leading to fixed organ dysfunction or failure. Recent evidence from several laboratories suggests that TGF-beta, a pleiotropic cicatricial mediator, plays an important role in the pathogenesis of glomerular scarring (sclerosis). alpha1(I) and alpha1(IV) mRNA are acutely increased in the extraglomerular and glomerular compartments early in the course of a crescentic model of glomerulonephritis in the rabbit. Concomitant with this change, was expression of TGF-beta mRNA and active protein. Anti-TGF-beta antibodies neutralized this activity and have been shown by others to block or ameliorate the development of glomerular sclerosis in a model of immune mesangial injury. Thus, TGF- beta likely plays an important role in the development of glomerular sclerosis and interstitial fibrosis. While the action of TGF-beta on synthesis of interstitial collagens has been well studied, little is known about the action of TGF-beta on the metabolism of basement membrane components which accumulate in sclerotic glomerular matrices. Collagen IV, the major structural component of glomerular basement membranes, is assembled from at least 5 genetically distinct polypeptides. The alpha1(IV) and alpha2(IV) chains are synthesized by glomerular cells in vitro. TGF-beta increases collagen IV biosynthesis independent of cell proliferation due to increased transcription of the alpha1(IV) and alpha2(IV) genes leading to a 3-5 fold increase in the steady-state level of collagen IV mRNA. The mechanism of TGF-beta- induced changes in collagen IV gene transcription are unknown. Collagen IV gene transcription depends on two independent mechanisms, transcript initiation and elongation. The alpha1(IV) and alpha2(IV) genes are separated by a 130 bp bidirectional promoter which initiates transcripts under the regulation of enhancer sequences embedded in the structural genes. The mechanism of transcript attenuation is not known. Since TGF-beta increases collagen IV mRNA in cells already initiating transcripts at a near maximal level, it is postulated to alter transcript elongation efficiency (attenuation). In order to test this hypothesis, the following studies are proposed: 1) Collagen IV transcript initiation and elongation will be assessed by nuclear run-on transcription in quiescent and TGF-beta stimulated murine glomerular mesangial cells. The site(s) of transcript attenuation will be mapped using fragments of the alpha1(IV) gene; 2) The function of cis-acting sequences which are postulated to attenuate transcription will be assessed by transfection of chimeric gene constructs in quiescent and TGF-beta stimulated cells; 3) Nuclear proteins which specifically interact with the cis-acting sequence will be identified in crude extracts of quiescent and TGF-beta stimulated cells and their binding site characterized; 4) Unique DNA-binding proteins will be characterized by cDNA cloning from an expression library. These studies will provide insights into the molecular mechanism of TGF-beta action on collagen IV gene transcription and aberrant extracellular matrix accumulation in renal diseases. With this information therapeutic strategies may be developed to alter the course of renal disease.
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