PURIFICATION AND IDENTIFICATION OF A HUMAN DERMAL EXTRACT COMPONENT INHIBITORY TO FIBROBLAST PROLIFERATION

Abstract

It was previously shown that a citric acid buffer extract of human dermis (extract D) inhibited growth of human diploid fibroblasts in monolayer culture (Muir et al., 1997). Further fractionation has shown that the active principle is probably a proteoglycan, and that retention of its inhibitory activity is dependent on the use protease inhibitors throughout the extraction procedure. Elution of extract D from a DEAE-cellulose column produced four major peaks, each of which was subjected to SDS-PAGE as well as being tested for inhibitory activity on the growth of fibroblasts in culture. Peaks III and IV had no inhibitory effect, but peak I contained highly active material. Gels of this peak showed prominent bands of 120kDa (corresponding to dermatan sulphate proteoglycan II, DS-PG II) and at 45kDa (corresponding to the core protein). The latter band became more prominent when extract D which had been treated with chrondroitinase ABC was electrophoresed. Their identities were verified by Western blotting. Peak II also contained some slower-acting inhibitory material which has as yet to be identified, but contains little or no protein corresponding to the decorin core-protein. The data indicate that the intact decorin molecule, DS-PG II, is the main inhibitory principle in human skin.