Lens Major Intrinsic Protein (MIP)/Aquaporin 0 Expression in Rat Lens Epithelia Explants Requires Fibroblast Growth Factor-induced ERK and JNK Signaling

Lens major intrinsic protein (MIP), exclusive to the vertebrate lens, otherwise known as MIP26 and Aquaporin 0, is abundantly expressed as a lens fiber membrane protein. Although relatively less efficient compared with other aquaporins, MIP is suggested to function as a water channel, as an adhesion molecule, and is required for lens transparency. Because MIP is specifically expressed in lens fiber cells, we investigated in this study the activation of Mip expression after triggering differentiation of rat lens epithelia explants by fibroblast growth factor (FGF)-2. Here, we show that Mip expression in the lens cells is regulated by FGF-2. Using Real time PCR we demonstrate that endogenous Mip levels in the explants were up-regulated upon FGF-2 stimulation, in a concentration-dependent manner. Up-regulation of Mip at the transcriptional level was simultaneous with the activation of the FGF down-stream signaling components, ERK1/2 and JNK. Specific inhibitors, UO126 for ERK1/2 and SP600125 for JNK, abrogated Mip expression in response to FGF-2 in the explants. This inhibition pattern was recapitulated in reporter assays for transfection of the rat lens epithelia explants, driven by the Mip promoter (-1648/+44). Our studies show that ERK1/2 and JNK signaling pathways are required for Mip expression in lens epithelia explants induced to differentiate by FGF-2. Lens major intrinsic protein (MIP), exclusive to the vertebrate lens, otherwise known as MIP26 and Aquaporin 0, is abundantly expressed as a lens fiber membrane protein. Although relatively less efficient compared with other aquaporins, MIP is suggested to function as a water channel, as an adhesion molecule, and is required for lens transparency. Because MIP is specifically expressed in lens fiber cells, we investigated in this study the activation of Mip expression after triggering differentiation of rat lens epithelia explants by fibroblast growth factor (FGF)-2. Here, we show that Mip expression in the lens cells is regulated by FGF-2. Using Real time PCR we demonstrate that endogenous Mip levels in the explants were up-regulated upon FGF-2 stimulation, in a concentration-dependent manner. Up-regulation of Mip at the transcriptional level was simultaneous with the activation of the FGF down-stream signaling components, ERK1/2 and JNK. Specific inhibitors, UO126 for ERK1/2 and SP600125 for JNK, abrogated Mip expression in response to FGF-2 in the explants. This inhibition pattern was recapitulated in reporter assays for transfection of the rat lens epithelia explants, driven by the Mip promoter (-1648/+44). Our studies show that ERK1/2 and JNK signaling pathways are required for Mip expression in lens epithelia explants induced to differentiate by FGF-2. The vertebrate lens is composed by an anterior layer of cuboidal epithelial cells and differentiated fiber cells that comprise the bulk of the tissue (1McAvoy J.W. Chamberlain C.G. de Iongh R.U. Hales A.M. Lovicu F.J. Eye. 1999; 13: 425-437Google Scholar). During embryonic development and throughout the life of the organism, fiber cells are added via differentiation of the lens epithelial cells. Spatial and temporal regulation of gene expression is required to maintain the transparency and correct refractive index of the ocular lens. Major intrinsic protein (MIP) 1The abbreviations used are: MIP, major intrinsic protein; AQP1, aquaporin 1; FGF, fibroblast growth factor; ERK, extracellular signal-regulated kinase; JNK,c-Jun NH2-terminal kinase; RT, reverse transcriptase; MAPK, mitogen-activated protein kinse; SAPK, stress-activated protein kinase. 1The abbreviations used are: MIP, major intrinsic protein; AQP1, aquaporin 1; FGF, fibroblast growth factor; ERK, extracellular signal-regulated kinase; JNK,c-Jun NH2-terminal kinase; RT, reverse transcriptase; MAPK, mitogen-activated protein kinse; SAPK, stress-activated protein kinase. is a polypeptide found exclusively in the membranes of lens fiber cells. It was first isolated as the most abundant protein of the fiber cell membrane (2Chepelinsky A.B. J. Exp. Zool. Part A Comp. Exp. Biol. 2003; 300: 41-46Google Scholar). Subsequent to the identification of AQP1 as a water channel in red blood cells, MIP (also known as AQP0) was shown to be permeable to water (3Varadaraj K. Kushmerick C. Baldo G.J. Bassnett S. Shiels A. Mathias R.T. J. Membr. Biol. 1999; 170: 191-203Google Scholar, 4Mulders S.M. Preston G.M. Deen P.M. Guggino W.B. van Os C.H. Agre P. J. Biol. Chem. 1995; 270: 9010-9016Google Scholar, 5Verkman A.S. Exp. Eye Res. 2003; 76: 137-143Google Scholar), although this permeability is much lower than for most other AQPs. MIP may also have structural roles in the lens fibers, functioning as a cell to cell adhesion molecule (6Fotiadis D. Hasler L. Muller D.J. Stahlberg H. Kistler J. Engel A. J. Mol. Biol. 2000; 300: 779-789Google Scholar, 7Michea L.F. Andrinolo D. Ceppi H. Lagos N. Exp. Eye Res. 1995; 61: 293-301Google Scholar), playing a role in gap junction formation (8Yu X.S. Jiang J.X. J. Cell Sci. 2004; 117: 871-880Google Scholar, 9Dunia I. Recouvreur M. Nicolas P. Kumar N. Bloemendal H. Benedetti E.L. J. Cell Sci. 1998; 111: 2109-2120Google Scholar) and in organizing γ-crystallins (10Fan J. Donovan A.K. Ledee D.R. Zelenka P.S. Fariss R.N. Chepelinsky A.B. Investig. Ophthalmol. Vis. Sci. 2004; 45: 863-871Google Scholar). In this way, MIP may play an important role in maintaining lens transparency by reducing the inter-fiber space and is required for the normal focusing properties of the lens (11Shiels A. Bassnett S. Varadaraj K. Mathias R. Al-Ghoul K. Kuszak J. Donoviel D. Lilleberg S. Friedrich G. Zambrowicz B. Physiol. Genomics. 2001; 7: 179-186Google Scholar). During cataractogenesis and aging, MIP undergoes selective proteolysis that may modulate gating of the MIP channels (12Boyle D.L. Takemoto L.J. Exp. Eye Res. 1999; 68: 41-49Google Scholar) and its adhesive properties (13Grey A.C. Jacobs M.D. Gonen T. Kistler J. Donaldson P.J. Exp. Eye Res. 2003; 77: 567-574Google Scholar). Moreover, mutations in the Mip gene have been linked to the mouse genetic cataracts Fraser (CatFr), lens opacity (lop), Hfi, Tohm (14Shiels A. Mackay D. Bassnett S. Al-Ghoul K. Kuszak J. FASEB J. 2000; 14: 2207-2212Google Scholar, 15Sidjanin D.J. Parker-Wilson D.M. Neuhauser-Klaus A. Pretsch W. Favor J. Deen P.M. Ohtaka-Maruyama C. Lu Y. Bragin A. Skach W.R. Chepelinsky A.B. Grimes P.A. Stambolian D.E. Genomics. 2001; 74: 313-319Google Scholar, 16Okamura T. Miyoshi I. Takahashi K. Mototani Y. Ishigaki S. Kon Y. Kasai N. Genomics. 2003; 81: 361-368Google Scholar), and two human cataracts (17Francis P. Chung J.J. Yasui M. Berry V. Moore A. Wyatt M.K. Wistow G. Bhattacharya S.S. Agre P. Hum. Mol. Genet. 2000; 9: 2329-2334Google Scholar, 18Berry V. Francis P. Kaushal S. Moore A. Bhattacharya S. Nat. Genet. 2000; 25: 15-17Google Scholar). Although expression of the Mip gene in the lens is tightly regulated temporally and spatially during embryogenesis, signaling cascades involved in this process remain unidentified. Growth factors such as FGFs, insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, and insulin are shown to promote proliferation of lens epithelial cells and differentiation into fiber cells in rats and chicken (19Chamberlain C.G. McAvoy J.W. Growth Factors. 1989; 1: 125-134Google Scholar, 20McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228Google Scholar, 21Kok A. Lovicu F.J. Chamberlain C.G. McAvoy J.W. Growth Factors. 2002; 20: 27-34Google Scholar, 22Klok E. Lubsen N.H. Chamberlain C.G. McAvoy J.W. Exp. Eye Res. 1998; 67: 425-431Google Scholar, 23Le A.C. Musil L.S. Dev. Biol. 2001; 233: 394-411Google Scholar, 24Leenders W.P. van Genesen S.T. Schoenmakers J.G. van Zoelen E.J. Lubsen N.H. Mech. Dev. 1997; 67: 193-201Google Scholar). Recently, BMP was shown to participate in fiber cell differentiation (25Belecky-Adams T.L. Adler R. Beebe D.C. Development. 2002; 129: 3795-3802Google Scholar, 26Faber S.C. Robinson M.L. Makarenkova H.P. Lang R.A. Development. 2002; 129: 3727-3737Google Scholar). The binding of growth factors to their cell surface receptors leads to the activation of the receptor tyrosine kinase eventually transducing the signals into cellular targets via distinct downstream signaling molecules (27Cadena D.L. Gill G.N. FASEB J. 1992; 6: 2332-2337Google Scholar). FGFs regulate a multitude of biological phenomena including adhesion, migration, proliferation, and differentiation (19Chamberlain C.G. McAvoy J.W. Growth Factors. 1989; 1: 125-134Google Scholar, 20McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228Google Scholar, 28Boilly B. Vercoutter-Edouart A.S. Hondermarck H. Nurcombe V. Le B.X Cytokine Growth Factor Rev. 2000; 11: 295-302Google Scholar). Overexpression of secreted FGF-1, -3, -4, -7, -8, and -9 in the lens of transgenic mice causes the premature differentiation of anterior epithelial cells into fiber cells (29Robinson M.L. MacMillan-Crow L.A. Thompson J.A. Overbeek P.A. Development. 1995; 121: 3959-3967Google Scholar, 30Robinson M.L. Ohtaka-Maruyama C. Chan C.C. Jamieson S. Dickson C. Overbeek P.A. Chepelinsky A.B. Dev. Biol. 1998; 198: 13-31Google Scholar, 31Lovicu F.J. Overbeek P.A. Development. 1998; 125: 3365-3377Google Scholar). However, overexpression of secreted FGF-2 suggests a role in modulating fiber cell differentiation and survival (32Stolen C.M. Jackson M.W. Griep A.E. Development. 1997; 124: 4009-4017Google Scholar). Experiments using dominant-negative FGF receptors also provide insights into the role of FGFs in lens cell survival (29Robinson M.L. MacMillan-Crow L.A. Thompson J.A. Overbeek P.A. Development. 1995; 121: 3959-3967Google Scholar, 33Chow R.L. Roux G.D. Roghani M. Palmer M.A. Rifkin D.B. Moscatelli D.A. Lang R.A. Development. 1995; 121: 4383-4393Google Scholar, 34Stolen C.M. Griep A.E. Dev. Biol. 2000; 217: 205-220Google Scholar). It has been demonstrated that FGF-1 or FGF-2 can stimulate either proliferation or differentiation of explanted rat lens epithelia in a dose-dependent manner (19Chamberlain C.G. McAvoy J.W. Growth Factors. 1989; 1: 125-134Google Scholar, 20McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228Google Scholar). In this study, we have used explanted rat lens epithelia induced to differentiate by FGF-2 to address the molecular mechanisms that determine how FGF signaling regulates Mip expression. Here we show that FGF-2 induces Mip expression in explanted lens epithelia. We provide evidence that both FGF-induced ERK1/2 and JNK activation result in the up-regulation of Mip at the transcriptional level. FGF-2 ability to up-regulate Mip gene expression is obliterated in the presence of a specific JNK inhibitor. We further demonstrate that an Mip active promoter (-1648/+44 bp) responds to ERK and JNK inhibitors in a similar way as the endogenous Mip gene. Our results suggest that both ERK and JNK signaling pathways downstream of FGF activation are required for Mip expression and JNK activation is pivotal in addition to ERK activation. Reagents—The MIP-specific polyclonal antiserum was obtained from Alpha Diagnostic (San Antonio, TX). Antibodies to total ERK (rabbit polyclonal), total JNK (rabbit polyclonal), phospho-ERK (mouse monoclonal), and phospho-JNK (mouse were from Cell an of ERK was from and an of JNK was obtained from (San and reporter were by the the or of a mouse Mip Y. and A. B. into the of the gene. and and were used of was with a and an to the Lens were from rats The lens with epithelial cells was from the lens and its with the surface of a The lens epithelia explants explants were to the (19Chamberlain C.G. McAvoy J.W. Growth Factors. 1989; 1: 125-134Google Scholar), in and FGF-2 to differentiation J.W. Chamberlain C.G. Development. 1989; 107: 221-228Google Scholar) or at the FGF-2 were in with the of the for and of The study was by the of and of and the explants were with reporter with the using the transfection to the a transfection in the explants were with the and The was using the reporter and a The was to the by the The reporter was by its to the and was isolated by using the to the was with the for of Using Real of Mip and that of the gene were by time PCR using the and for the time PCR was with from The was and and the specific for Mip was for mouse were and and the for was The Mip the to of MIP were in to the PCR for for and of for and for or the for for and of for and for were for that the from be of Mip was with expression. was compared and expressed in and cell of total from the lens explants were by using and to membranes were with a of in for at the were in for total for total JNK, for phospho-ERK and for at the were in the at a of for at The were using an and to The were lens explants were in in for at of MIP, and phospho-JNK in the explants was by the explants were in and for at The explants were in with or for at The explants were in by for in and or the explants were with and by a The explants were with a were for using lens epithelia explants in were in in at for were in for at was of the with a of the epithelia explants was in a by The were and with of epithelial cells were from the surface in of the explants using a at FGF-2 Mip in Lens studies have shown that FGF-1 and FGF-2 promote lens epithelial cell proliferation and differentiation in lens (19Chamberlain C.G. McAvoy J.W. Growth Factors. 1989; 1: 125-134Google Scholar, 20McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228Google Scholar). We used the rat lens to determine the of FGF-2 MIP protein expression A lens epithelia in is shown in Because the expression of fiber cell in the explants in of with we MIP protein expression in the explants by after in A polyclonal specific to the MIP protein the expression of MIP in the explants MIP expression is at time A of a of the demonstrated that MIP cells as to the cuboidal of the epithelial cells we the Mip levels in the and explants. demonstrated the up-regulation of Mip as by the of a of the Mip gene in the explants with FGF-2. Mip gene were in the explants FGF-2 In the of the lens epithelia explants were as by the expression of a factor expressed in lens epithelia and in lens C. Chepelinsky A.B. Dev. Biol. 1998; Scholar) FGF-2 Mip in Lens the of the that the Mip level was up-regulated in the explants with a study was to determine the Mip gene promoter the response required to by FGF-2. Mip promoter and of the Mip and of to the reporter gene were used for transfection In the presence of the lens explants with the reporter activation of the Mip promoter The Mip promoter a in reporter to the However, the Mip promoter of Mip are the of the Mip promoter response to FGF-2 in lens epithelia explants, we also the Mip promoter in the presence of FGF-2 in two cell that Mip and cell that in the presence of the Mip an active a promoter is active in rat cells. We obtained similar results using a mouse lens epithelial cell C.M. D.C. J. Exp. Eye Res. 1995; 61: Scholar) that endogenous of either the or reporter in a response to transcriptional activation by FGF-2 driven reporter was results that the at to of the Mip gene response to FGF also that this response is specific to lens epithelia cells. FGF-2 for Lens Cell and Chamberlain J.W. Chamberlain C.G. Development. 1989; 107: 221-228Google Scholar) have demonstrated that FGF induces proliferation and differentiation of rat lens epithelia explants. this differentiation study by FGF-2 in lens explants, was to first demonstrate the of FGF-2 to differentiation in We this by using explants in the presence of and FGF, in by an fiber cell in the explants with of the explants with of FGF-2 and their epithelial In A and cuboidal of lens epithelial cells can be with the of in the fiber cells in and cells eventually a fiber cell after of results that in lens explants are of differentiation to fiber cells the of FGF-2. Mip in Lens FGF-2 that of induces lens fiber cell differentiation were by to determine the Mip expression pattern in the presence of of Real time PCR was to determine the endogenous Mip level in the explants. that FGF-2 induced Mip we used this to demonstrate a time Mip levels after and are shown in The Mip expression in explants after or in The in Mip and in results that Mip as fiber cell differentiation to be We a study to determine the Mip level is induced by of FGF-2 or Real time PCR results demonstrated that at the FGF-2 is in Mip after to in results demonstrate that the of FGF-2 is both for the and Mip expression with lens fiber cell FGF-2 ERK1/2 in Lens in a is known to stimulate the signaling in of cells. In study, we to determine ERK is and by FGF-2. Using a of from to of ERK was by specific to phospho-ERK was used to the from the explants and the total and ERK in explants for with either or FGF-2. The two and are a level of total ERK1/2 in the explants a and in was and and in and and in in the and explants the and with this of the explants for with and FGF-2 and by the presence of In a phospho-ERK was in the cell and the level We in ERK in the of FGF compared with FGF However, in FGF-2 the level of ERK in the cells. In explants with the of the was than in the explants and the results obtained by The of the explants for with and FGF-2 similar results for and explants. However, in the explants with the ERK was in the compared with explants suggest a in ERK activation as the cells to a differentiation A ERK activation in explants in FGF-2 for is with the of epithelial cells to a differentiation that to results that FGF-2 in a ERK that is simultaneous with an to fiber cell differentiation and of ERK activation. the other in the presence of a lower of cells that remain epithelial the activation downstream of ERK Mip in Lens determine FGF-induced ERK activation is required for Mip expression in the lens fiber cells, we two to the cells in the explants that to differentiate in the presence of UO126 of expressed endogenous Mip to a reporter driven by the Mip promoter was by the ERK assays to ERK that in the presence of ERK activation by FGF-2 at was In the presence of UO126 cells from differentiation as by the epithelial of the cells that similar as of epithelial cells with A and In as cells were differentiation as shown by the formation of and of their epithelial Real time PCR results a in the Mip level in the presence of UO126 in explants with the Mip promoter bp) a in promoter with further in the to result in inhibition FGF-induced Mip in Lens JNK is known to the signaling Exp. Eye Res. 2002; Scholar, Exp. Eye Res. 2002; 74: Scholar). Although we and other have FGF-induced ERK activation to lens fiber cell activation to be The role of JNK in Mip expression in the lens fiber cells has been Here, in we show that FGF-2 induces to a level activation was in a lower of FGF-2 and to in the presence of a of FGF-2 and in in the and explants the FGF-2 with this of the explants for with and FGF-2 as by the presence of JNK In a phospho-JNK was in the cell However, in FGF-2 the level of JNK in the cells, in the In in the explants with JNK was in the The of the explants for with and FGF-2 JNK suggest that the JNK is required at the in the FGF signaling JNK Mip in Lens determine JNK is involved in the expression of we used a specific of JNK, for with FGF-2 in the presence of the JNK SP600125 of the phospho-JNK level show the of MIP protein expression in explants in the presence of FGF for in the of the MIP protein expression We have the of the MIP by of either or in of the demonstrate that SP600125 specifically the JNK and ERK we and The ERK expression was by the JNK either in or the role of JNK Mip expression may be at the transcriptional level was by using time PCR to the endogenous Mip level. a in the Mip level in the explants in the presence of and SP600125 for The explants with and expressed Mip to the level. with the Mip promoter a in promoter in the presence of SP600125 results suggest that JNK is required for the activation of Mip expression in lens epithelia explants induced to differentiate in by FGF-2. The activation and signaling cascades the regulation of gene expression during the differentiation process of lens epithelia into lens is specifically expressed in lens and its expression is in lens epithelia. FGF-1 and FGF-2 promote cell proliferation and differentiation in rat lens epithelia explants (19Chamberlain C.G. McAvoy J.W. Growth Factors. 1989; 1: 125-134Google Scholar, 20McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228Google Scholar, 21Kok A. Lovicu F.J. Chamberlain C.G. McAvoy J.W. Growth Factors. 2002; 20: 27-34Google Scholar, 22Klok E. Lubsen N.H. Chamberlain C.G. McAvoy J.W. Exp. Eye Res. 1998; 67: 425-431Google Scholar). the two biological epithelial cell proliferation and fiber cell are regulated by the FGFs to be In this was to FGF-2 regulates Mip expression in lens cells and to the signaling pathways that are with this We used the of the lens to how FGF-2 downstream pathways the that both ERK and JNK signaling to regulate Mip expression. It has been that a specific of ERK signaling such as cell and expression in lens cells. FGF-induced expression of is F.J. McAvoy J.W. Development. 2001; Scholar). of the of two distinct and the that ERK activation is the of lens fiber cell differentiation and the expression of important Here, in this we show that FGF-2 induces Mip expression in lens epithelia explants in a dose-dependent manner. The of Mip gene expression by is at the transcriptional in the Mip promoter is to FGF-2. Mip promoter activation was lens and the endogenous Mip expression pattern in the explants an with a of FGF-2. MIP, and factors such as are in lens epithelial cells, are expressed in differentiated fiber cells although an of ERK is found in the lens epithelial cells H. H. Y. K. J. Biol. Chem. 2003; Scholar, D.J. Mol. Vis. 2003; 9: Scholar). address the of how FGF-2 induces Mip we studies the FGF-2 concentration-dependent activation of ERK1/2 was with the expression of Mip in the lens explants. the ERK activation by UO126 in the of endogenous Mip expression in the lens explants. The ability of the Mip promoter to expression in response to FGF-2 was by results the of the for Mip the of the of In the in and in lens cell such as the and have been shown to be involved in cell proliferation and differentiation S. Beebe D.C. Dev. Biol. 1998; Scholar, R.A. Investig. Ophthalmol. Vis. Sci. 1999; Scholar, S. Y. T. Y. A. McAvoy J.W. Exp. Eye Res. 2001; Scholar, Zelenka P.S. Investig. Ophthalmol. Vis. Sci. 2001; Scholar, J. Chan C.C. R. Chepelinsky A.B. Dev. Biol. Scholar, J. I. J. Investig. Ophthalmol. Vis. Sci. 2001; Scholar). A two tightly regulated biological has been the by the growth factors is was to major the ERK is involved in Mip expression in the lens The evidence the using and are by reporter assays the regulation of Mip expression at the transcriptional level. The of the JNK a in the signaling ERK, is demonstrated in this FGF-2 in was of JNK activation to in the lens explants. Using similar assays for the ERK we demonstrate that JNK is required for Mip expression in the lens explants in FGF-2 A specific to JNK JNK activation by FGF and Mip expression. This of JNK was also at the transcriptional level Mip was by time using an Mip promoter in the presence of SP600125 the of an JNK in Mip promoter in response to FGF-2. In the of lens and are in a process cell or M.A. 61: Scholar, M.A. 2000; Scholar). may be to survival signaling mechanisms are ERK and JNK inhibitors were In by using we and cell was in the presence of the inhibitor. the inhibition of Mip expression by two inhibitors is the result of Our results that the Mip promoter has downstream of ERK and JNK It is that two their ability to regulate specific factors to the Mip promoter in the lens explants. We also demonstrate that ERK and JNK pathways are of Mip than a JNK signaling with the ERK upon FGF-2 to an efficient expression of Although how Mip expression is regulated by FGF-2 in is provide insights into the signaling pathways Mip expression in the lens studies are required to the factors the and signals and to the response in the Mip