HETEROGENEITY OF LATENT TRANSFORMING GROWTH FACTOR-BETA ISOLATED FROM BONE-MATRIX PROTEINS

Transforming growth factor- β(TGFβ) is a family of 25-kDa peptides that appear to be important regulators of cell proliferation, differentiation, and differentiated function in many tissues. TGFβ is present in platelets and serum and is released by cultured cells as several distinct large mol wt complexes of TGFβ with other proteins. These complexes are biologically inactive and are generically called latent TGFβ(L-TGFβ). Large quantities of TGFβ are present in bone matrix. This study was undertaken to determine whether the TGFβ in bone matrix was present as a free 25-kDa peptide or a large mol wt L-TGFβ complex. TGFβ activity was determined by inhibition of [3H]methylthymidine incorporation in mink lung epithelial cells. The specificity of inhibition was determined by treating fractions with a polyclonal rabbit antiporcine TGFβ-blocking antibody before assay. Latency was examined by assaying untreated and acid-treated fractions for TGFβactivity. Acid treatment of EDTA extracts of the bovine bone matrix proteins increased TGFβ activity from a mean of 0.8 pg/µg protein to 56 pg/µg. Under native conditions L-TGFβ eluted from S400 between the 600-400 kDa mol wt standards. No activity eluted in the fractions with authentic 25-kDa TGFβ.Eighty-five percent of the L-TGFβ bound to lentil lectin, and this separated into four discrete L-TGFβ peaks (I-IV) at 0.22, 0.25, 0.35, and 0.42 M NaCl with Mono-Q anion exchange chromatography. Mono-Q pools II and III were reseparated by molecular size on Superose-12 under native and dissociative conditions. Under native conditions TGFβ activity was latent and eluted in the large mol wt fractions. No 25-kDa TGFβ was present. With dissociating conditions (4 M GuHCl) all TGFβ activity eluted in the small mol wt fractions identical to the elution position of authentic 25-kDa TGFβ. The active fractions from the dissociative separation of Mono-Q pool III were separated by C4 reverse phase HPLC. There were three discrete peaks of TGF? activity corresponding to TGFβ1, TGFβ2, and an unidentified form of TGF. Maximum activation of L-TGFβ in each Mono-Q peak occurred at pH 3-3.5. There was partial activation at pH 4.5, but no additional activation at pH 1.5. Plasmin treatment activated the L-TGFβ only in Mono-Q pools III and IV, and the maximum plasmin activation was only 30% of that seen at pH 3-3.5. We conclude that 1) TGFβ is present in bone matrix as a large mol wt glycoprotein complex with reduced biological activity, latent TGFβ; 2) based upon molecular size, relative intrinsic to acid-enhanced activity, plasmin activation, and charge separation, there appear to be multiple forms of bone matrix-derived L-TGFβ. © 1990 by The Endocrine Society.