Characterization of the Activation of Latent TGF-/5 by Co-cultures of Endothelial Cells and Pericytes or Smooth Muscle Cells: A Self-regulating System

The conversion of latent transforming growth factor beta (LTGF-/~) to the active species, transforming growth factor beta (TGF-/5), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-B in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-~ as determined by this assay. The concentration of TGF-B in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A + RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-/31 and TGF-B2, while SMCs produced primarily TGF-J51. No change in the expression of these two forms of TGF-/3 was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-/3 indicated that most of the active TGF-/5 was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-/3 in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-/3 in the coculture medium blocked the observed increase in PAIl levels. The increased expression of PAI-1 subsequent to "I'GF-/5 formation blocked the activation of the protease required for conversion of LTGF-~3 to TGF-/~ as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-B. This effect was lost upon removal of the PAId antibodies. Thus, the activation of LTGF-~ appears to be a self-regulating system. T RANSFORMING growth factor B (TGF-/~) ~ is a homodimeric protein with a molecular weight of 25,000. TGF-/~ is produced by a variety of cells in vitro (16, 34) and has been isolated from a number of tissue sources (2, 8, 24). The purified protein displays a variety of activities in a cell specific manner. These include the induction of matrix macromolecule synthesis (3, 6, 12), decreases in the synthesis of the proteases collagenase and plasminogen activator (PA) (5, 15, 27) and stimulation of the synthesis of irthibitors of both collagenase and PA (5, 14, 30). TGF-B is a strong chemoattractant and mitogen for some cell types (3, 25, 36), although it blocks the migration and growth of other cells (7, 10, 21, 26, 28, 35). These activities suggest that a major physiological role of TGF-B may be in wound healing. Although TGF-/5 has a widespread distribution, the molecule usually is isolated as an inactive high molecular mass 1. Abbreviations used in this paper: BAE, bovine aortic endothelial; PAI-I, plasminogen activator inhibitor 1; SMC, smooth muscle cell; TGF-B, transforming growth factor beta; LTGF-B, latent transforming growth factor beta. complex (16, 37). This inactive complex has been shown to contain the 25,000-D TGF-/3 homodimer in noncovalent association with the amino-terminal region of the TGF-/~ precursor (17a, 19, 38). The latent TGF-B complex isolated from platelets contains an additional, unrelated molecule that is covalently bound (19, 38). The latent form of TGF-/5 (LTGF-/~) does not bind to the TGF-B receptor (16, 22). However, LTGF-B can be converted into the active form by strong acid (pH 2) treatment. Although mildly acidic environments occur in vivo, activation of LTGF-B by acid pH has not been demonstrated to occur in either in vivo or in vitro biological systems. Purified LTGF-/~ can be activated by plasmin or cathepsin D (17). Recent studies suggest that proteases, specifically plasmin, act by cleaving within the amino-terminal region of the TGF-B precursor, thereby destabilizing the latent complex and releasing active TGF-B (17a). Recently, two reports described the conversion of LTGF-/~ to the active species in co-cultures of endothelial cells and pericytes or smooth muscle cells (SMCs) (1, 29). Antonelli© The Rockefeller University Press, 0021-9525/90/08/757/7 $2.00 The Journal of Cell Biology, Volume 111, August 1990 757-763 757 on Jne 2, 2017 D ow nladed fom Published August 1, 1990