Regulation of fibronectin splicing in sinusoidal endothelial cells from normal or injured liver.
Fn containing an extra type III domain (EIIIA in the rat, ED1 or EDA in humans) is commonly termed "fetal" fibronectin, but it is prominent during the injury response of adult tissues and mediates important early events in the response. This form is particularly apparent in acute liver injury, where it has been shown that sinusoidal endothelial cells produce EIIIA-fibronectin. This fibronectin isoform arises by alternative splicing of the primary transcript. In the present experiments, we have studied the regulation of fibronectin splicing in primary sinusoidal endothelial cells by transfecting a minigene containing the EIIIA exon and its flanking introns, driven by various promoters. The results indicate that fibronectin splicing in endothelial cells from normal liver is in part promoter-dependent. However, in cells from injured liver in which expression of both total and EIIIA-fibronectin is strikingly increased, promoter effects disappear. Because fibronectin splicing is known to be regulated in part by TGFbeta, we also examined the effect of a soluble inhibitor of the TGFbeta type 2 receptor. This agent had no effect on splicing by normal endothelial cells. By contrast, for endothelial cells from the injured liver, the splicing pattern reverted to that of normal cells, i.e., it became promoter-dependent. We conclude that, in the setting of injury in vivo, TGFbeta overrides the promoter dependence of fibronectin splicing in normal cells. The data suggest that TGFbeta modifies the spliceosome, if not through its known signaling intermediates, then through the products of genes regulated by this cytokine.
Chang
Ming-Ling
ML
Liver Center and Department of Medicine and Fetal Treatment Center, Department of Surgery, University of California, San Francisco, CA 94143, USA.
Chen
Jeng-Chang
JC
Alonso
Claudio R
CR
Kornblihtt
Alberto R
AR
Bissell
D Montgomery
DM
D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004727:Endothelium / Q000473:pathology; D005353:Fibronectins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051379:Mice; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D017381:Spliceosomes / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism
D005353:Fibronectins; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta