Regulation of Collagen Synthesis in Scleroderma

Fleischmajer, Raul

Abstract

Collagen is synthesized in a precursor form known as procollagen with extension propeptides at the amino and carboxyl ends of the collagen molecule. It is classically accpeted that both propeptides are cleaved by specific proteases before the collagen molecule polymerizes to form fibrils. Recently, we showed that the amino propeptide (AP) of type I collagen plays a role in collagen fibril formation up to 35 nm fibrils, at which time, the propeptide is cleaved from the fibril. In addition, the free AP by a feedback mechanism appears to have a role in the regulation of collagen synthesis. Preliminary data in our laboratory suggest that the excess deposition of collagen in scleroderma may be due to a faulty mechanism in the utilization of the AP of type I collagen, in fibril formation and regulation of collagen synthesis. To extend these preliminary observations, skin specimens will be obtained from patients at early and late stages of the disease. Immunofluorescence microscopy will be performed with antibodies directed against the AP of type I and type III procollagens and fibronectin. Those areas that show a positive reaction will be dissected and studied at the ultrastructural level by immunoelectron microscopy with ferritin tagged antibodies. This technique will allow us to define the location of the AP in collagen fibrils, ground substance and on cell surfaces. The AP will also be demonstrated by extracting collagens from scleroderma skin and performing SDS-PAGE followed by immunoblotting. Scleroderma fibroblast cultures will be obtained from various levels of affected and normal skin. Cell strains will be divided into high and low collagen producers by measuring the synthesis of collagenase-sensitive polypeptides. The effect of the AP of type I and type III collagen will be tested """"""""in vitro"""""""" by measuring its effect on collagen synthesis. In addition, we will study the """"""""in vitro"""""""" release of the amino propeptide in scleroderma fibroblasts. This will be achieved by using a radioimmune assay and molecular sieve chromatography. All cell pellets will be studied by immunoelectron microscopy for the presence and distribution of the amino propeptide.