TGF-beta 1 up-regulates paxillin protein expression in malignant astrocytoma cells: requirement for a fibronectin substrate

Cytokines can influence the interactions between members of the integrin ce ll adhesion receptor family and the extracellular matrix thereby potentiall y affecting cell function and promoting cell adhesion, growth and migration of malignant astrocytoma tumor cells. As malignant astrocytoma cells synth esize TGF-beta1 in vivo, we analysed the effects of TGF-beta1 on signaling events associated with integrin receptor ligation, focusing on the effects on paxillin, a phosphorylated adaptor protein, that acts as a scaffold for signaling molecules recruited to focal adhesions. TGF-beta1 -stimulation of primary astrocytes and serum-starved U-251MG malignant astrocytoma cells a ttached to fibronectin induced a substantial increase in the levels of paxi llin protein (fivefold increase at 2.0 ng/ml) in a dose- and time-dependent manner compared to the levels observed on plating onto fibronectin in the absence of stimulation. In the astrocytoma cells, this resulted in an incre ase in the pool of tyrosine-phosphorylated paxillin, although it did not ap pear to alter the extent of phosphorylation of the paxillin molecules. In c ontrast, in primary astrocytes the protein levels were upregulated in the a bsence of a parallel increase in phosphorylation. The TGF-beta1-stimulated increase in paxillin levels required ligation of the fibronectin receptor, as it was not induced when the cells were plated onto vitronectin, collagen or laminin. The increase in the pool of paxillin on TGF-beta1 stimulation of the fibronectin-plated astrocytoma cells was associated with an increase in translation, but was not associated with an increase in the steady-stat e levels of paxillin mRNA. Stimulation with TGF-beta1 on a fibronectin subs trate increased subsequent attachment and spreading of U-251MG cells onto f ibronectin and, to a lesser extent, vitronectin, but not collagen. Our resu lts indicate that physiologic levels of TGF-beta1 stimulate the expression of paxillin protein at the level of translation through a process that requ ires engagement of the fibronectin receptor, and promotes attachment and sp reading of malignant astrocytoma cells on fibronectin.