Mammary Epithelial-Mesenchymal Interaction Regulates Fibronectin Alternative Splicing via Phosphatidylinositol 3-Kinase

Abstract

The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.Pre-mRNA alternative splicing is a widespread process that regulates gene expression and is the most important source of protein diversity in vertebrates (1). Fibronectin (FN), 1 the best characterized extracellular matrix (ECM) glycoprotein, plays a key role in cell adhesive and migratory behavior related to fundamental processes such as embryogenesis, wound healing, maintenance of tissue integrity, and malignancy. Different FN polypeptides arise through an intricate pattern of alternative splicing in three regions of the single primary transcript, (from 5Ј to 3Ј) extra domain II (EDII), extra domain I (EDI), and type III connecting segment (IIICS) (also called EDB or EIIIB, EDA or EIIIA, and V region, respectively), resulting in up to 12 variants in rodents. FN alternative splicing is modulated in a cell type-, development-, and age-specific manner and therefore constitutes a paradigm for studying the regulation of this complex process (2). EDI and EDII are cassette exons, either excluded from or included into the mature FN mRNA. The third site of alternative splicing, IIICS, is subject to total inclusion, partial inclusion, or total exclusion due to the presence of an internal 3Ј splice site within this exon.In vivo EDIϩ FN is poorly represented in the ECM of adult normal tissues. However, this variant is over-expressed in developing embryos, wound healing, liver fibrosis, ovary granulosa cell proliferation, and some tumors (2, 3). It has been proposed that EDI inclusion may mediate a conformational change in the whole m...

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Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

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Splicing-factor alterations in cancers

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