The basement membrane communicates with the adjacent cells through cell surface proteins and can alter gene expression and transcript splicing. Pelisch et al. now provide evidence for the involvement of the c-Jun N-terminal kinase (JNK) pathway and the phosphatidyinositol 3-kinase (PI3K) pathway in the alternative splicing of fibronectin transcripts in a mammary epithelial cell line (EpH4) in response to laminin-rich basement membrane (lr-BM). Twelve isoforms of fibronectin are generated by alternative splicing of three discrete regions: EDII (extra domain II), EDI (extra domain I), and IIICS (type III connecting segment). EpH4 cells plated on lr-BM showed decreased inclusion of the EDI exon, no change in the inclusion of the EDII exon, and decreased inclusion of the IIICS exon that contains the integrin-binding motif LDV. Cells plated with lr-BM showed activation of JNK and extracellular signal-regulated kinase (ERK) but not of p38 or PI3K. Inhibition of JNK pharmacologically with silencing RNA (siRNA) or by expression of a dominant-negative blocked the effect of lr-BM on fibronectin splicing. Although PI3K inhibition or p38 inhibition did decrease the ratio of EDI+:EDI− and LDV+:LDV− transcripts in untreated cells, exposure to lr-BM further decreased these ratios, indicating that these two kinases are not involved in the lr-BM regulation of fibronectin splicing. Exposure of cells to lr-BM produced a sustained activation of JNK, but a transient activation of ERK was followed by dephosphorylation below basal levels. Pharmacological inhibition of ERK in untreated cells suggested that ERK (like p38 and PI3K) is involved in the basal inclusion of EDI- and LDV-containing IIICS exons. Overexpression of active JNK was not sufficient to alter splicing in the absence of lr-BM, which suggests that the interplay between JNK and ERK signaling may be important for the changes in splicing of fibronectin.
F. Pelisch, M. Blaustein, A. R. Kornblihtt, A. Srebrow, Cross-talk between signaling pathways regulates alternative splicing: A novel role for JNK. J. Biol. Chem. 280, 25461-25469 (2005). [Abstract] [Full Text]