Induction of stable microtubules in 3T3 fibroblasts by TGF-β and serum

Abstract

Previous studies have shown that fibroblasts induced to migrate into an in vitro wound rapidly generate an array of stable, post-translationally detyrosinated microtubules (Glu MTs) oriented toward the direction of migration. To understand how cells generate a stable array of MTs at a specific location, we have analyzed the contribution of media components 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 generating Glu MTs in response to wounding. Such SFM-treated monolayers were capable of generating oriented Glu MTs within 1 hour of wounding, if calf serum (CS) was added back to the medium. The Glu MTs in the CS refed cells were oriented toward the wound in cells at the wound edge, and were juxtanuclear in cells within the monolayer, demonstrating that CS restored the Glu MT array characteristic of each cell type. To determine the nature of the ‘Glu MT-inducing’ factor in CS, we subjected CS to different treatments and found that the CS factor was nondialyzable, resistant to heat, mild acid and trypsin, but inactivated by treatment with dithiothreitol. The factor was not absorbed by charcoal and was present in lipoprotein-deficient serum. These properties are consistent with the properties of a number of polypep-tide growth factors, so we screened purified growth factors for their ability to induce Glu MTs in wounded SFM-treated monolayers. Of all the growth factors tested, only TGF-β1 and TGF-β2 induced a significant level (> 70% of the CS response) of oriented Glu MTs. The SFM-treated cells were exquisitely sensitive to TGF-β1, with significant induction of Glu MTs observed at 0.01 ng/ml TGF-β1. Induction of Glu MTs observed by immunofluorescence after CS or TGF-β treatments were paralleled by increases in Glu tubulin detected on western blots. The Glu MTs formed after either CS or TGF-β1 treatment showed enhanced resistance to nocodazole, confirming that both treatments increased the level of stable MTs in cells. The TGF-β1 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-β1 was even greater than that in CS. Unlike CS, TGF-β1 did not stimulate the migration of SFM-treated cells into the wound or the entry of SFM-treated cells into the cell cycle, showing that MT stabilization is independent of these events. These results demonstrate that MT stabilization can be regulated by external factors and that TGF-β is a potent inducing factor for stable MTs.