Mammary Epithelial-Mesenchymal Interaction Regulates Fibronectin Alternative Splicing via Phosphatidylinositol 3-Kinase
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m314260200
ISSN1083-351X
AutoresMatı́as Blaustein, Federico Pelisch, Omar A. Coso, Mina J. Bissell, Alberto R. Kornblihtt, Anabella Srebrow,
Tópico(s)Cellular Mechanics and Interactions
ResumoThe 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. 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 (1Graveley B.R. Trends Genet. 2001; 17: 100-107Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar). Fibronectin (FN), 1The abbreviations used are: FN, fibronectin; Act D, actinomycin D; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; CHX, cycloheximide; ECM, extracellular matrix; EDI, extra domain I; EDII, extra domain II; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; g6CM, conditioned medium from SCg6 cells; HGF/SF, hepatocyte growth factor/scatter factor; IIICS, type III repeat connecting segment; JNK, c-Jun N-terminal kinase; KGF, keratinocyte growth factor; LUC, luciferase; LY, LY294002; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; p2CM, conditioned medium from SCp2 cells; PD, PD98059; pAKT, phospho-AKT; pERK, phospho-ERK; PI, phosphatidylinositol; pJNK, phospho-JNK; TGFβ1, transforming growth factor β1; mFN, mutant FN; RT, reverse transcriptase; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; SR, serine/arginine-rich. 1The abbreviations used are: FN, fibronectin; Act D, actinomycin D; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; CHX, cycloheximide; ECM, extracellular matrix; EDI, extra domain I; EDII, extra domain II; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; g6CM, conditioned medium from SCg6 cells; HGF/SF, hepatocyte growth factor/scatter factor; IIICS, type III repeat connecting segment; JNK, c-Jun N-terminal kinase; KGF, keratinocyte growth factor; LUC, luciferase; LY, LY294002; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; p2CM, conditioned medium from SCp2 cells; PD, PD98059; pAKT, phospho-AKT; pERK, phospho-ERK; PI, phosphatidylinositol; pJNK, phospho-JNK; TGFβ1, transforming growth factor β1; mFN, mutant FN; RT, reverse transcriptase; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; SR, serine/arginine-rich. 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 5′ to 3′) 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 (2Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar). 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 3′ splice site within this exon. In vivo EDI+ 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 (2Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar, 3Colman-Lerner A. Fischman M.L. Lanuza G.M. Bissell D.M. Kornblihtt A.R. Barañao J.L. Endocrinology. 1999; 140: 2541-2548Crossref PubMed Scopus (20) Google Scholar). It has been proposed that EDI inclusion may mediate a conformational change in the whole molecule that would result in an increased exposure of the RGD motif, therefore increasing cell-ECM interaction through the binding to α5β1 integrin. Furthermore EDI inclusion potentiates the ability of FN to promote cell cycle progression (4Manabe R. Oh-e N. Sekiguchi K. J. Biol. Chem. 1999; 274: 5919-5924Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, a growing body of data suggests that EDI per se is active. It has been shown that this region is responsible for the conversion of lipocytes into myofibroblasts in fibrotic liver (5Jarnagin W.R. Rockey D.C. Koteliansky V.E. Wang S.S. Bissell D.M. J. Cell Biol. 1994; 127: 2037-2048Crossref PubMed Scopus (377) Google Scholar) as well as for the induction of several matrix metalloproteinases (MMP-1, -3, and -9) required for cell migration and tissue remodeling (6Saito S. Yamaji N. Yasunaga K. Saito T. Matsumoto S. Katoh M. Kobayashi S. Masuho Y. J. Biol. Chem. 1999; 274: 30756-30763Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Recently, Muro et al. (7Muro A.F. Chauhan A.K. Gajovic S. Iaconcig A. Porro F. Stanta G. Baralle F.E. J. Cell Biol. 2003; 162: 149-160Crossref PubMed Scopus (239) Google Scholar) generated mice devoid of EDI exon-regulated splicing and demonstrated that EDI splicing regulation is required for proper skin wound healing and normal life span. EDII inclusion correlates with that of EDI in several cases. EDII is excluded from FN in normal adult tissue and included during development and pathological conditions such as inflammation, major trauma, wound healing, and tumors (8Oyama F. Hirohashi S. Sakamoto M. Titani K. Sekiguchi K. Cancer Res. 1993; 53: 2005-2011PubMed Google Scholar, 9Peters J.H. Loredo G.A. Chen G. Maunder R. Hahn T.J. Willits N.H. Hynes R.O. J. Lab. Clin. Med. 2003; 141: 401-410Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Nevertheless, the biological function of EDII is unknown, and the mechanisms that regulate its alternative splicing differ from those for EDI (10Lim L.P. Sharp P.A. Mol. Cell. Biol. 1998; 18: 3900-3906Crossref PubMed Scopus (92) Google Scholar, 11Cramer P. Cáceres J.F. Cazalla D. Kadener S. Muro A.F. Baralle F.E. Kornblihtt A.R. Mol. Cell. 1999; 4: 251-258Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 12Kadener S. Cramer P. Nogués G. Cazalla D. de la Mata M. Fededa J.P. Werbajh S.E. Srebrow A. Kornblihtt A.R. EMBO J. 2001; 20: 5759-5768Crossref PubMed Scopus (109) Google Scholar). Splicing of the IIICS can generate three different variants in rodents, referred to as IIICS-0, IIICS-95, and IIICS-120 according to their lengths in the encoded amino acid residues. IIICS inclusion is higher in all fetal versus adult tissues, and this region is required for secretion of FN dimers during biosynthesis (13Schwarzbauer J.E. Spencer C.S. Wilson C.L. J. Cell Biol. 1989; 109: 3445-3453Crossref PubMed Scopus (70) Google Scholar, 14Mardon H.J. Sebastio G. J. Cell Sci. 1992; 103: 423-433PubMed Google Scholar). Alternative splicing of several pre-mRNAs can be regulated by extracellular signals such as growth factors, cytokines, hormones, and stress stimuli (15Stamm S. Hum. Mol. Genet. 2002; 11: 2409-2416Crossref PubMed Scopus (174) Google Scholar). Accordingly, we have shown previously that a basement membrane-like ECM, as well as its two main protein components, modulates FN EDI alternative splicing in a hepatoma cell line (16Srebrow A. Blaustein M. Kornblihtt A.R. FEBS Lett. 2002; 514: 285-289Crossref PubMed Scopus (19) Google Scholar). Nevertheless, only a few cases of the transducing components linking the cell surface with the nuclear splicing machinery have been investigated. For example, it has been demonstrated that the MAPK kinase 3/6-p38 pathway modifies the subcellular distribution of the heterogeneous nuclear ribonucleoprotein A1 and modulates the alternative splicing of transcripts derived from an adenovirus E1A reporter minigene. There is evidence suggesting a role for protein kinase C, Ras, and phosphatidylinositol 3-kinase (PI 3-kinase) in alternative splicing regulation (15Stamm S. Hum. Mol. Genet. 2002; 11: 2409-2416Crossref PubMed Scopus (174) Google Scholar). Recent reports linked the activation of the extracellular signal-regulated kinase (ERK) 1/2 to the regulation of CD44 alternative splicing triggered by T-cell activation. The STAR (signal transduction and activation of RNA) protein, SAM68, has been postulated as the final step of this cascade (17Matter N. Herrlich P. König H. Nature. 2002; 420: 691-695Crossref PubMed Scopus (368) Google Scholar). The mammary gland comprises stromal and epithelial cells that communicate with each other through the ECM. Disruption of this dynamic communication can both induce and promote breast cancer. Cross-talk between the mammary epithelium and stroma is also crucial for the proper patterning and function of the normal mammary gland. Unlike other organs, the mammary gland undergoes most of its growth and morphogenesis in the adult animal, and certain stages of the postnatal gland exhibit embryonic- and tumor-like features (18Cunha G.R. Young P. Hamamoto S. Guzman R. Nandi S. Epithelial Cell Biol. 1992; 1: 105-118PubMed Google Scholar, 19Wiseman B.S. Werb Z. Science. 2002; 296: 1046-1049Crossref PubMed Scopus (613) Google Scholar). As EDI exon inclusion is linked to development, tissue remodeling, and tumorigenesis, the mammary gland represents a useful physiological context in which to study the regulation of FN alternative splicing by the cellular microenvironment, in particular by cell-cell interaction. In this study we investigated the effects of different growth factors and cytokines on FN alternative splicing in a functionally normal mouse mammary epithelial cell line (SCp2) and show that hepatocyte growth factor/scatter factor (HGF/SF), keratinocyte growth factor (KGF), and acidic FGF (aFGF) stimulate EDI and IIICS inclusion but not EDII into mature FN mRNA. Furthermore, we demonstrate that soluble factors secreted by a mouse mammary mesenchymal cell line (SCg6) present in the conditioned medium from these cells (g6CM) also regulate FN EDI and IIICS alternative splicing in SCp2 cells, indicating that epithelial-mesenchymal interaction influences pre-mRNA alternative splicing. We explored the signaling pathways involved in this regulatory phenomenon, showing that g6CM as well as HGF/SF act through a PI 3-kinase-dependent cascade to alter FN splicing pattern in this cellular context. This work defines different extracellular cues that regulate FN alternative splicing, increasing FN isoforms preferentially involved in cell adhesion, proliferation, migration, metastasis, and tissue remodeling. Furthermore, the data presented here provide new insights into how the cellular microenvironment can influence gene expression. Cell Culture—SCp2 and SCg6 cells were grown in Dulbecco's modified Eagle's medium:F-12 (DMEM:F-12; Invitrogen) supplemented with 2% fetal bovine serum, insulin (5 μg/ml, Sigma) and gentamicin (50 μg/ml, Invitrogen). Cell co-culture was performed using Falcon Tissue Culture Inserts bearing a 0.4 μm-pore size P.E.T membrane (Becton Dickinson Labware). Approximately, 1–2 × 105 cells were plated in DMEM:F-12 supplemented with 2% fetal bovine serum into 35-mm tissue culture wells. After 24 h, cell monolayers were rinsed, an insert was placed inside each well, and 1–2 × 105 cells were plated on top of the insert filter. Cells in the bottom well and in the insert were cultured together for 48 h. Cell Treatments—Approximately 1–2 × 105 cells were plated in DMEM:F-12 supplemented with 2% fetal bovine serum into 35-mm tissue culture wells. After 24 h, the medium was replaced by serum-free DMEM:F-12, and cells were grown for another 24 h before treatment with growth factors (20 ng/ml), g6CM, SCp2 conditioned medium (p2CM) or serum-free DMEM:F-12 (control). g6CM was obtained by plating 1 × 106 cells into 100-mm tissue culture dishes in 2% fetal bovine serum medium and replacing it with serum-free DMEM:F-12 after 24 h. Cell supernatant was collected 24 h later and centrifuged to discard cell debris. Alternatively, g6CM was filtered through a 0.45-μm porous filter to obtain identical results. The same protocol was carried out with SCp2 cells to obtain p2CM. Reagents—Growth factors and cytokines utilized were: epidermal growth factor (EGF), KGF, basic fibroblast growth factor (bFGF), aFGF, and HGF/SF from Sigma and transforming growth factor β1 (TGFβ1) and leukemia inhibitory factor (LIF) from R&D Systems, Inc. Kinase inhibitors were from Calbiochem. actinomycin D (Act D) was from Invitrogen and cycloheximide (CHX) from Sigma. Plasmids and Transfections—EDI minigene plasmids were as follows: pSVEDATot (α-globin promoter (20Caputi M. Casari G. Guenzi S. Tagliabue R. Sidoli A. Melo C.A. Baralle F.E. Nucleic Acids Res. 1994; 22: 1018-1022Crossref PubMed Scopus (137) Google Scholar)); pSVEDA/wtFN (wild type FN promoter (21Cramer P. Pesce C.G. Baralle F.E. Kornblihtt A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11456-11460Crossref PubMed Scopus (268) Google Scholar)); pSVEDA/mFN (mutant FN promoter (21Cramer P. Pesce C.G. Baralle F.E. Kornblihtt A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11456-11460Crossref PubMed Scopus (268) Google Scholar)); pSVEDA/CMV (CMV promoter (21Cramer P. Pesce C.G. Baralle F.E. Kornblihtt A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11456-11460Crossref PubMed Scopus (268) Google Scholar)); pSVEDA ΔStu (α-globin promoter, mutated ESS (20Caputi M. Casari G. Guenzi S. Tagliabue R. Sidoli A. Melo C.A. Baralle F.E. Nucleic Acids Res. 1994; 22: 1018-1022Crossref PubMed Scopus (137) Google Scholar)); pSVEDA ΔStu/mFN (mutant FN promoter, mutated ESS (11Cramer P. Cáceres J.F. Cazalla D. Kadener S. Muro A.F. Baralle F.E. Kornblihtt A.R. Mol. Cell. 1999; 4: 251-258Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar)), and pSVEDA Δ2e/mFN (mutant FN promoter, mutated ESE (11Cramer P. Cáceres J.F. Cazalla D. Kadener S. Muro A.F. Baralle F.E. Kornblihtt A.R. Mol. Cell. 1999; 4: 251-258Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar)). Plasmids used for luciferase assays were pG5-Luc, pGal4-Elk1, and pGBDXGal4-Atf2. Experiments with RasV12 included pCEV-RasV12 or empty vector (pBluescript, Stratagene). Transfections were performed 24 h after plating using FuGENE 6 (Roche Applied Science). Approximately 2 × 105 cells were transfected with 3 μl of FuGENE 6 and 2 μg of total plasmid DNA in 35-mm tissue culture wells. Co-transfection of pCMVβgal allowed standardization by transfection efficiencies. Cells were stimulated 24 h after transfection. Luciferase Assays—Luciferase (LUC) activity in cell lysates was measured using the Luciferase Assay System (Promega). Cells were washed with phosphate-buffered saline before lysis with 100 μl of reporter lysis buffer (Promega). Cell extracts were centrifuged, and 30 μl of the supernatant was mixed with 100 μl of luciferase assay buffer II (Promega). LUC activity was tested with a junior luminometer (Berlthold, Bad Wildbad, Germany). RNA Isolation and Radioactive RT-PCR Amplification—Total RNA purification from cultured cells and RT-PCR analysis were carried out as described (11Cramer P. Cáceres J.F. Cazalla D. Kadener S. Muro A.F. Baralle F.E. Kornblihtt A.R. Mol. Cell. 1999; 4: 251-258Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). The set of primers used for FN EDI (22Werbajh S.E. Urtreger A.J. Puricelli L.I. de Lustig E.S. Bal de Kier Joffe E. Kornblihtt A.R. FEBS Lett. 1998; 440: 277-281Crossref PubMed Scopus (30) Google Scholar), FN EDI minigene (20Caputi M. Casari G. Guenzi S. Tagliabue R. Sidoli A. Melo C.A. Baralle F.E. Nucleic Acids Res. 1994; 22: 1018-1022Crossref PubMed Scopus (137) Google Scholar), and FN IIICS (23Chauhan A.K. Iaconcig A. Baralle F.E. Muro A.F. Gene. 2004; 324: 55-63Crossref PubMed Scopus (29) Google Scholar) were described previously. The set of primers used for EDII amplification was hEDB-dir (16Srebrow A. Blaustein M. Kornblihtt A.R. FEBS Lett. 2002; 514: 285-289Crossref PubMed Scopus (19) Google Scholar) and mEDB-rev (5′-CAGTGGACAGTGAATGAGTTGG-3′). Radioactive RT-PCR products were electrophoresed in 6% (w/v) polyacrylamide native gels and detected by autoradiography. Radioactivity in the bands was measured in a scintillation counter according to the Cerenkov method (11Cramer P. Cáceres J.F. Cazalla D. Kadener S. Muro A.F. Baralle F.E. Kornblihtt A.R. Mol. Cell. 1999; 4: 251-258Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Western Blot Analysis—After treatment, cells were lysed in boiling 2× sample buffer (4% SDS, 20% glycerol, 120 mm Tris, pH 6.8, 0.002% bromphenol blue, 200 mm β-mercaptoethanol) at the time points indicated. Proteins were separated (12% acrylamide SDS-PAGE), blotted, probed with specific antibodies, and visualized by enhanced chemiluminescence detection using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and Luminol (Sigma). Antiphospho-ERK (pERK), anti-phospho-JNK (pJNK), anti ERK2, anti-JNK, and anti-AKT antibodies were from Santa Cruz Biotechnology. Anti-phospho-AKT (pAKT) was from Cell Signaling Technology. Differential Regulation of FN Alternatively Spliced Exons in Response to Growth Factors in Mammary Epithelial Cells—A number of growth factors (HGF/SF, TGFβ1, and EGF) stimulate EDI inclusion into mature mRNA in different cultured cell lines (16Srebrow A. Blaustein M. Kornblihtt A.R. FEBS Lett. 2002; 514: 285-289Crossref PubMed Scopus (19) Google Scholar, 24Inoue T. Nabeshima K. Shimao Y. Koono M. Biochem. Biophys. Res. Commun. 1999; 260: 225-231Crossref PubMed Scopus (20) Google Scholar, 25Shimao Y. Nabeshima K. Inoue T. Koono M. Int. J. Cancer. 1999; 82: 449-458Crossref PubMed Scopus (28) Google Scholar). We tested whether these and other growth factors and cytokines could regulate EDI alternative splicing in the mouse mammary epithelial cell line SCp2. After cells were exposed to different stimuli for 48 h, they were harvested, and RNA was extracted to compare the relative proportions of the two mRNA isoforms (lacking or containing the EDI exon) using an RT-PCR-based method (Fig. 1A). EGF, aFGF, bFGF, KGF, and HGF/SF increased the EDI+/EDI- ratio up to 4-fold (Fig. 1B). This effect was seen as early as 5 h and peaked at 24–48 h (data not shown). Surprisingly, the widely reported regulator of EDI alternative splicing, TGFβ1, did not up-regulate EDI+ isoform in these cells, even at the wide range of concentrations tested (0.1–50 ng/ml; data not shown). As a positive control, TGFβ1 stimulated EDI inclusion in Hep3B cells. Furthermore, leukemia inhibitory factor also failed to modify the EDI+/EDI- ratio (data not shown). To explore whether the growth factors that regulate EDI inclusion also affect the splicing of the other two FN alternative regions, we performed specific RT-PCR for EDII and IIICS (Fig. 1A). Alternative splicing of IIICS was also modified upon treatment of SCp2 cells with different growth factors (Fig. 1C). Interestingly, aFGF, KGF, and HGF/SF up-regulated IIICS-120, the IIICS isoform containing the LDV motif. This motif has been shown to promote cell adhesion by interacting with α4β1 and α4β7 integrins (26Humphries M.J. Akiyama S.K. Komoriya A. Olden K. Yamada K.M. J. Cell Biol. 1986; 103: 2637-2647Crossref PubMed Scopus (305) Google Scholar). Therefore, this newly synthesized FN (EDI+, IIICS-120) would have an enhanced potential for binding to a specific subset of integrins associated with cell adhesion, proliferation, migration, and matrix metalloproteinase induction (4Manabe R. Oh-e N. Sekiguchi K. J. Biol. Chem. 1999; 274: 5919-5924Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Saito S. Yamaji N. Yasunaga K. Saito T. Matsumoto S. Katoh M. Kobayashi S. Masuho Y. J. Biol. Chem. 1999; 274: 30756-30763Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 27Huhtala P. Humphries M.J. McCarthy J.B. Tremble P.M. Werb Z. Damsky C.H. J. Cell Biol. 1995; 129: 867-879Crossref PubMed Scopus (369) Google Scholar, 28Serini G. Bochaton-Piallat M.L. Ropraz P. Geinoz A. Borsi L. Zardi L. Gabbiani G. J. Cell Biol. 1998; 142: 873-881Crossref PubMed Scopus (674) Google Scholar, 29Liao Y.F. Gotwals P.J. Koteliansky V.E. Sheppard D. Van De Water L. J. Biol. Chem. 2002; 277: 14467-14474Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Conversely, the EDII+ FN isoform was not detected in RNA from untreated cells, and none of the described stimuli altered this splicing pattern (Fig. 1D). These results extend the number of growth factors that stimulate EDI inclusion, bringing the FGF family into the scene for the first time and suggesting that a number of mitogenic agents, but not every cytokine, are key mediators of extracellular up-regulation of EDI inclusion in mammary epithelial cells. Furthermore, a strong correlation between EDI and IIICS splicing regulation emerged from these experiments, whereas EDII splicing pattern remained unaffected by these treatments. This is consistent with the idea that alternative splicing of EDI and EDII exons is controlled by different molecular mechanisms (10Lim L.P. Sharp P.A. Mol. Cell. Biol. 1998; 18: 3900-3906Crossref PubMed Scopus (92) Google Scholar, 11Cramer P. Cáceres J.F. Cazalla D. Kadener S. Muro A.F. Baralle F.E. Kornblihtt A.R. Mol. Cell. 1999; 4: 251-258Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 12Kadener S. Cramer P. Nogués G. Cazalla D. de la Mata M. Fededa J.P. Werbajh S.E. Srebrow A. Kornblihtt A.R. EMBO J. 2001; 20: 5759-5768Crossref PubMed Scopus (109) Google Scholar). Soluble Factors Secreted by Mammary Mesenchymal Cells Regulate FN Alternative Splicing in Mammary Epithelial Cells—The epithelial-mesenchymal interaction is mediated by growth factors secreted by both cell types. We have shown that many of these growth factors affect FN alternative splicing. However, these experiments performed with recombinant factors are far from representing the actual extracellular milieu. To simulate the epithelial-mesenchymal interaction and evaluate its influence on FN alternative splicing, we chose a cell culture model composed of two mouse mammary cell lines, the epithelial line SCp2 and the mesenchymal line SCg6. Both cell lines derive from the same parental cell strain, CID-9 (30Desprez P.Y. Roskelley C. Campisi J. Bissell M.J. Mol. Cell. Differ. 1993; 1: 99-110Google Scholar). As shown in Fig. 2, SCg6 cells express higher levels of EDI+, EDII+, and IIICS-120 FN than SCp2 cells. Co-culture experiments were performed with tissue culture inserts that allow the interchange of soluble factors without direct cell-cell contact. The presence of SCg6 cells stimulated EDI exon inclusion in SCp2 cells, increasing the EDI+/EDI- ratio by 3–4-fold compared with that of SCp2 cells cultured with SCp2 cells on the other side of the filter (Fig. 3A). In contrast, EDI splicing in SCg6 was not affected by the co-culture with SCp2 cells (data not shown). To assess whether a dynamic cell-cell communication was essential for this effect, we tested the influence of g6CM on EDI alternative splicing in SCp2 cells. Although treatment with conditioned medium from SCp2 (p2CM) did not change EDI inclusion, treatment with g6CM elicited an increase in the EDI+/EDI- ratio already observed after 5 h and more pronounced after 24 and 48 h (Fig. 3B). Furthermore, IIICS regulation was again correlated with that of EDI, showing a marked preference for the IIICS-120 isoform (Fig. 3B), whereas EDII alternative splicing was not affected by this treatment (data not shown). These results show a novel regulation of FN alternative splicing by cell-cell interaction and provide a physiological context for studying the regulation of alternative splicing by extracellular stimuli. Signal Transduction Pathways Activated by g6CM and HGF/SF—A number of studies have associated extracellular stimuli and the regulation of FN alternative splicing. Nevertheless, the signaling cascades that lead to these effects remain unexplored (15Stamm S. Hum. Mol. Genet. 2002; 11: 2409-2416Crossref PubMed Scopus (174) Google Scholar). The pathways that link extracellular stimuli and cellular effects are classically grouped into kinase cascades activated by either mitogenic (MAPK pathways) or stress (SAPK (stress-activated protein kinase) pathways) stimuli (31Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). It is well known that most growth factors activate the Ras-Raf-MAPK kinase (MEK)-ERK1/2 pathway (32Paumelle R. Tulasne D. Leroy C. Coll J. Vandenbunder B. Fafeur V. Mol. Biol. Cell. 2000; 11: 3751-3763Crossref PubMed Scopus (62) Google Scholar). To confirm that this pathway was activated in response to g6CM and/or growth factors, we first performed a luciferase-based GAL-ELK1 reporter assay. SCp2 cells were transiently transfected with two plasmids. One plasmid carried the LUC reporter gene driven by a minimal promoter fused to binding sites for the yeast transcription factor GAL4, and the other coded for a fusion protein containing the GAL4 DNA binding domain plus the activation domain from ELK1 transcription factor, which is known to be phosphorylated and therefore activated by ERK1/2. Approximately a 10-fold activation of the reporter system was observed in cells treated with both g6CM and HGF/SF (Fig. 4A). The stress kinases p38 and c-Jun N-terminal kinase (JNK), which are occasionally activated by growth factors (33Recio J.A. Merlino G. Oncogene. 2002; 21: 1000-1008Crossref PubMed Scopus (145) Google Scholar), can also activate the transcription factor ELK1 in some cell lines. Therefore, we performed a LUC-based GAL-ATF2 reporter assay that only differs from the one described above in that the second plasmid codes for GAL4 binding domain fused to the activation domain of ATF2 transcription factor. ATF2 is known to be activated by JNK and p38 but not by ERK1/2. Upon treatment of SCp2 cells with g6CM or HGF/SF, a 1.5-fold activation of the reporter was observed, indicating that the JNK and/or p38 pathway are activated but to a lesser extent compared with the ERK1/2 pathway (Fig. 4B). To confirm and dissect these results, we performed Western blots for pERK and pJNK. We observed a robust increase in pERK and a clear augmentation in pJNK after 30 min of treatment (Fig. 4C). These activations were sustained in time, with high levels of pJNK and pERK still seen at 2 and 5 h, respectively (data not shown). Conversely, we detected little or no phosphorylation of p38 after 30 min of treatment with g6CM or HGF/SF, which contrasted with the strong activation induced by anisomycin, a known activator
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