INDUCTION OF STABLE MICROTUBULES IN 3T3 FIBROBLASTS BY TGF-BETA AND SERUM

Previous studies have shown that fibroblasts induced to migrate into a n in vitro wound rapidly generate an array of stable, post-translation ally detyrosinated microtubules (Glu MTs) oriented toward the directio n of migration. To understand how cells generate a stable array of MTs at a specific location, we have analyzed the contribution of media co mponents to the formation of oriented Glu MTs in wounded monolayers of 3T3 fibroblasts. When confluent monolayers were placed in serum-free medium (SFM) for 2 days before wounding, the cells contained virtually no Glu MTs or nocodazole-resistant MTs and were incapable of generati ng Glu MTs in response to wounding. Such SFM-treated monolayers were c apable of generating oriented Glu MTs within 1 hour of wounding, if ca lf serum (CS) was added back to the medium. The Glu MTs in the CS refe d cells were oriented toward the wound in cells at the wound edge, and were juxtanuclear in cells within the monolayer, demonstrating that C S restored the Glu MT array characteristic of each cell type. To deter mine the nature of the 'Glu MT-inducing' factor in CS, we subjected CS to different treatments and found that the CS factor was nondialyzabl e, resistant to heat, mild acid and trypsin, but inactivated by treatm ent with dithiothreitol. The factor was not absorbed by charcoal and w as present in lipoprotein-deficient serum. These properties are consis tent with the properties of a number of polypeptide growth factors, so we screened purified growth factors for their ability to induce Glu M Ts in wounded SFM-treated monolayers. Of atl the growth factors tested , only TGF-beta1 and TGF-beta2 induced a significant level (greater-th an-or-equal-to 70% of the CS response) of oriented Glu MTs. The SFM-tr eated cells were exquisitely sensitive to TGF-beta1, with significant induction of Glu MTs observed at 0.01 ng/ml TGF-beta1. Induction of Gl u MTs observed by immunofluorescence after CS or TGF-beta treatments w ere paralleled by increases in Glu tubulin detected on western blots. The Glu MTs formed after either CS or TGF-beta1 treatment showed enhan ced resistance to nocodazole, confirming that both treatments increase d the level of stable MTs in celts. The TGF-beta1 induction of stable MTs was slower than that of CS (2-4 hours onset versus 1 hour onset), but by 24 hours the level of MT stabilization in TGF-beta1 was even gr eater than that in CS. Unlike CS, TGF-beta1 did not stimulate the migr ation of SFM-treated cells into the wound or the entry of SFM-treated cells into the cell cycle, showing that MT stabilization is independen t of these events. These results demonstrate that MT stabilization can be regulated by external factors and that TGF-beta is a potent induci ng factor for stable MTs.