Matrix composition and transforming growth factor-beta modulate sinusoidalendothelial cell EIIIA fibronectin expression in vitro.

The EIIIA splice variant of fibronectin is minimally present in normal adul t tissue, but is markedly increased following injury. We have shown that se cretion of EIIIA fibronectin ([EIIIA]Fn) by sinusoidal endothelial cells (S EC) precedes and directly contributes to the activation of the hepatic stel late cell. To characterize those factors which are likely to regulate [EIII A]Fn expression in vivo, we examined the influence of TGF-beta and matrix c omposition on SEC-derived [EIIIA]Fn in vitro. SECs were isolated from sham operated and bile duct ligated (BDL) rats, placed in culture and exposed to the putative regulatory factors. During short term culture, [EIIIA]Fn mRNA expression decreased by (similar to)40% in SECs from both sham operated an d BDL rats. By contrast, [EIIIA]Fn splicing increased in favour of the [EII IA]Fn form (61% [Sham] vs 57% [BDL]). To examine the role of matrix on [EII IA]Fn expression, SECs were plated on either collagen I or EHS matrix. [EII IA]Fn expression declined only 15% in SECs on HS matrix, whereas it decreas ed 40% in cells plated on collagen I; splicing was unaffected by matrix phe notype. To examine the role of TGF-P, exogenous TGF-beta 1 was added to SEC s in culture. The cytokine produced a dose-dependent increase in [EIIIA]Fn mRNA expression up to 3-fold. At doses of 7.5 ng/ml, [EIIIA]Fn splicing inc reased 15-228. By run-on analysis, this increase in fibronectin expression was transcriptional, at least in part. TGF-beta 1 also increased synthesis of [EIIIA]Fn. To confirm these results, we used an inhibitor of TGF-beta, s oluble TGF-beta type II receptor (sTGF-beta R). The inhibitor when added to SECs in culture abolished the effect of exogenous TGF-beta 1 on [EIIIA]Fn expression. It also inhibited autocrine TGF-P secretion by SECs from BDL ra ts in that it reduced both [EIIIA]Fn expression and splicing (p < 0.001). A t the protein level, sTGF-beta R reduced [EIIIA]Fn secretion by SECs from B DL animals. The data indicate that (a) TGF-beta is a major regulator of [EI IIA]Fn expression and splicing, and (b) the cellular substratum influences some aspects of SEC function in vitro, including expression of [EIIIA]Fn. T he latter may prove useful in studies of the molecular regulation of [EIIIA ]Fn splicing.