Characterization of the latent transforming growth factor beta complex in bone

Transforming growth factor beta (TGF-beta) is a 25 kD multifunctional polypeptide with pronounced effects on the proliferation and differentiation of a variety of cells in vitro. TGF-beta is a potent regulator of the activity of cells with the osteoblast phenotype and of isolated osteoclasts. It is released in increased amounts by bone cultures stimulated to resorb. Organ cultures of neonatal mouse calvaria produce TGF-beta as an inert large-molecular-weight complex that must be dissociated to release biologically active TGF-beta (5-8 ng/ml). We have shown recently that stimulated isolated avian osteoclasts release active TGF-beta from this bone-derived biologically latent form. In this report we have characterized this bone latent form of TGF-beta. Only small amounts of active TGF-beta (less than 0.5 ng/ml) and no free binding protein are detectable in conditioned medium from bone cultures. Active TGF-beta can be detected in acid-treated calvarial conditioned media in which none or only minute amounts could previously be detected. Following incubation at 37 degrees C, this activated TGF-beta gradually loses activity. Cross-linking studies using 125I-labeled TGF-beta show that this loss of activity is due to TGF-beta binding to a protein of approximately 300 kD. The TGF-beta latent complex accumulates in a linear manner and is stable in the presence of serum and the protease trypsin. Increases in temperature and pH extremes dissociate the complex to release active TGF-beta. Decreases in pH result in an exponential increase in TGF-beta activity. Significant activation of the latent TGF-beta was detectable at pH values as high as 4 and 5. Since the osteoclastic microenvironment is acidic during bone resorption, these data suggest that this acidic microenvironment may regulate TGF-beta activity by releasing active TGF-beta from its latent complex.