Connective tissue activation. XXXI. Identification of two molecular forms of a human mesenchymal cell-derived growth factor, connective tissue activating peptide-V.

Abstract

We previously identified, in normal urine, a growth factor that stimulated monolayer cultures of human synovial, cartilage, and dermal fibroblasts to synthesize incremental amounts of hyaluronic acid, proteoglycans, and DNA. An isolation procedure guided by bioassays and immunologic methods disclosed 2 anionic bioactive polypeptides with Mr of 28,000 and 16,000, respectively, as judged by single bands with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in reduced and nonreduced samples. Rabbit antibodies raised against each purified protein were shown to react, on immunodiffusion and Western blot, with both antigens. Immunohistochemical and immunobinding studies detected the protein in normal human synovial, dermal, and cartilage fibroblasts and in human saphenous vein endothelial cells. The mesenchymal cell-derived growth factor is now designated connective tissue activating peptide-V (CTAP-V). Monospecific polyclonal anti-CTAP-V antibodies were used in a radial immunodiffusion assay for quantitative determination of the antigen in biologic fluids. In normal human plasma the concentration of CTAP-V was below the limit of detection. The CTAP-V concentration in normal urine was 4.5 +/- 2.0 micrograms/ml, calculated from measurements of 5-18-fold concentrated samples. Joint fluid from patients with rheumatic diseases and normal renal function had CTAP-V levels similar to those found in plasma; 2-15-fold increases were detected in plasma and joint fluid of patients with chronic renal failure. Immunodiffusion or dot-blot analysis revealed a CTAP-V-like material in the plasma or serum of 10 mammalian species. It was not detectable in 2 avian species.