Transgenic Mice Expressing Dominant-negative Osmotic-response Element-binding Protein (OREBP) in Lens Exhibit Fiber Cell Elongation Defect Associated with Increased DNA Breaks

Abstract

Osmotic-response element-binding protein (OREBP), also known as TonEBP or NFAT5, is thought to be responsible for the induction of osmolyte-accumulating genes when cells are under hypertonic stress. Recent studies suggest that OREBP also plays a role in water reabsorption in the kidney, T-cell proliferation, and embryonic development. We developed transgenic mice that express the dominant-negative OREBP (OREBPdn) specifically in the lens because our earlier studies showed that it is particularly sensitive to osmotic stress. The transgenic mice developed nuclear cataract soon after birth, suggesting defects in lens development. The developing transgenic lenses showed incomplete elongation of fiber cells and formation of vacuoles. This is accompanied by evidence of DNA strand breaks, activation of p53, and induction of checkpoint kinase, suggesting that the developing fiber cells lacking OREBP are in a similar physiological state as cells experiencing hypertonic stress. These results indicate that OREBP-mediated accumulation of osmolytes is essential during elongation of the lens fiber cells. Osmotic-response element-binding protein (OREBP), also known as TonEBP or NFAT5, is thought to be responsible for the induction of osmolyte-accumulating genes when cells are under hypertonic stress. Recent studies suggest that OREBP also plays a role in water reabsorption in the kidney, T-cell proliferation, and embryonic development. We developed transgenic mice that express the dominant-negative OREBP (OREBPdn) specifically in the lens because our earlier studies showed that it is particularly sensitive to osmotic stress. The transgenic mice developed nuclear cataract soon after birth, suggesting defects in lens development. The developing transgenic lenses showed incomplete elongation of fiber cells and formation of vacuoles. This is accompanied by evidence of DNA strand breaks, activation of p53, and induction of checkpoint kinase, suggesting that the developing fiber cells lacking OREBP are in a similar physiological state as cells experiencing hypertonic stress. These results indicate that OREBP-mediated accumulation of osmolytes is essential during elongation of the lens fiber cells. The osmotic-response element-binding protein (OREBP), 1The abbreviations used are: ORE, osmotic-response element; OREBP, ORE-binding protein; OREBPdn, dominant-negative OREBP; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; Tg, transgenic; TonE, tonicity-responsive enhancer; P, postnatal day; E, embryonic day; p-Chk2, phosphorylated Chk2; TonEBP, tonicity-responsive enhancer-binding protein. also known as TonEBP or NFAT5, is a member of the NFAT transcription factor family characterized by the presence of the Rel homology DNA-binding domain (1Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (473) Google Scholar, 2Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar). Unlike other members in the NFAT family that are responsible for effective immune response, OREBP/TonEBP is primarily involved in the genetic program that is essential for cellular survival in response to hypertonic stress (3Woo S.K. Kwon H.M. Int. Rev. Cytol. 2002; 215: 189-202Crossref PubMed Scopus (45) Google Scholar). Through binding to the osmotic-response element (ORE) or the tonicity-responsive enhancer (TonE), OREBP/TonEBP mediates the hypertonic induction of a set of osmoprotective genes including aldose reductase, betaine/γ-aminobutyric acid transporter, and sodium-myo-inositol cotransporter, resulting in the cellular accumulation of sorbitol, betaine, and myo-inositol, respectively (4Ko B.C. Ruepp B. Bohren K.M. Gabbay K.H. Chung S.S. J. Biol. Chem. 1997; 272: 16431-16437Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 5Miyakawa H. Woo S.K. Chen C.P. Dahl S.C. Handler J.S. Kwon H.M. Am. J. Physiol. 1998; 274: F753-F761PubMed Google Scholar, 6Rim J.S. Atta M.G. Dahl S.C. Berry G.T. Handler J.S. Kwon H.M. J. Biol. Chem. 1998; 273: 20615-20621Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). It is well established that these small molecule organic osmolytes are able to replace excess intracellular electrolytes that are otherwise deleterious to normal cell functions (7Burg M.B. Kwon E.D. Kultz D. Annu. Rev. Physiol. 1997; 59: 437-455Crossref PubMed Scopus (330) Google Scholar). This adaptive mechanism is particularly important in the kidney medulla, where cells are constantly exposed to steep osmotic gradient due to the urine concentration mechanism. Besides, OREBP is also responsible for the hypertonic induction of HSP70 (8Woo S.K. Lee S.D. Na K.Y. Park W.K. Kwon H.M. Mol. Cell. Biol. 2002; 22: 5753-5760Crossref PubMed Scopus (177) Google Scholar), a molecular chaperone that plays a vital role in cellular adaptation to osmotic stress (9Shim E.H. Kim J.I. Bang E.S. Heo J.S. Lee J.S. Kim E.Y. Lee J.E. Park W.Y. Kim S.H. Kim H.S. Smithies O. Jang J.J. Jin D.I. Seo J.S. EMBO Rep. 2002; 3: 857-861Crossref PubMed Scopus (81) Google Scholar). There are growing evidences suggesting that OREBP is involved in diverse cellular responses in addition to osmoprotection. In Drosophila, OREBP orthologue MESR1 was identified as a modifier of RAS1 signaling involved in eye development (10Huang A.M. Rubin G.M. Genetics. 2000; 156: 1219-1230Crossref PubMed Google Scholar). On the other hand, OREBP was also implicated in cancer cell migration and cancer metastasis (11Jauliac S. Lopez-Rodriguez C. Shaw L.M. Brown L.F. Rao A. Toker A. Nat. Cell Biol. 2002; 4: 540-544Crossref PubMed Scopus (349) Google Scholar). In mouse embryo, OREBP is expressed in most developing tissues including brain, eye, heart, kidney, colon, and muscle. Since these developing organs are not expected to experience hypertonic stress, the physiological role of OREBP in these tissues remains elusive (12Maouyo D. Kim J.Y. Lee S.D. Wu Y. Woo S.K. Kwon H.M. Am. J. Physiol. 2002; 282: F802-F809Crossref PubMed Scopus (61) Google Scholar). Nevertheless, OREBP homozygous knock-out mice manifested mid-embryonic lethality, indicating the important role of OREBP during development. A few OREBP null mutant mice that managed to survive through parturition displayed growth retardation and perinatal lethality associated with severe renal abnormalities (13Lopez-Rodriguez C. Antos C.L. Shelton J.M. Richardson J.A. Lin F. Novobrantseva T.I. Bronson R.T. Igarashi P. Rao A. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2392-2397Crossref PubMed Scopus (226) Google Scholar). We have created transgenic (Tg) mice expressing a dominant-negative form of OREBP (OREBPdn) specifically in the collecting duct epithelial cells of the kidney and found that OREBP also plays an essential role in urine-concentrating mechanism by regulating the expression of aquaporin-2 and urea transporter genes (14Lam A.K. Ko B.C. Tam S. Morris R. Yang J.Y. Chung S.K. Chung S.S. J. Biol. Chem. 2004; 279: 48048-48054Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Furthermore, OREBP was also suggested to be vital for optimal T cell proliferation and function as Tg mice expressing OREBPdn in the T cells (15Trama J. Go W.Y. Ho S.N. J. Immunol. 2002; 169: 5477-5488Crossref PubMed Scopus (87) Google Scholar) or thymus isolated from the OREBP heterozygous knock-out mice (16Go W.Y. Liu X. Roti M.A. Liu F. Ho S.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10673-10678Crossref PubMed Scopus (234) Google Scholar) exhibit a dramatic reduction in thymic cell proliferation and T cell-mediated antibody response. The apparent correlation between OREBP activity and cell proliferation revealed by the studies from the OREBPdn transgenic and OREBP null mice suggests that an intact cellular osmotic-response pathway is central to cell proliferation and function in some tissues. However, the underlining mechanism remains unclear. More importantly, the physiological function of OREBP in developing tissues other than kidney and lymphoid remains unexplored. We have previously shown that developing mouse lens is sensitive to osmotic imbalance (17Jiang Z. Chung S.K. Zhou C. Cammarata P.R. Chung S.S. Investig. Ophthalmol. Vis. Sci. 2000; 41: 1467-1472PubMed Google Scholar). Therefore we developed Tg mice that express OREBPdn specifically in the lens fiber cells to determine the role of OREBP in lens development. Our data demonstrate, for the first time in vivo, that inhibition of OREBP activity is associated with increase in DNA breaks and activation of DNA damage-response pathway, leading to defect in lens fiber cells elongation and the development of nuclear cataract. Our findings suggest that, during lens development, OREBP-mediated osmolyte accumulation is essential for fiber cell elongation and maintenance of the integrity of their DNA. Mice were housed under diurnal lighting condition and allowed free access to food and water. The protocol of this study was reviewed and approved by the Committee on the Use of Live Animals in Teaching and Research in The University of Hong Kong. Generation of OREBPdn Transgenic Mice—To develop Tg mice, the 390-bp fragment of mouse α A-crystallin promoter (from –341 to +49) (17Jiang Z. Chung S.K. Zhou C. Cammarata P.R. Chung S.S. Investig. Ophthalmol. Vis. Sci. 2000; 41: 1467-1472PubMed Google Scholar) was fused to the human OREBPdn to direct expression of the transgene, specifically in the mouse lens. The FLAG epitope was added to the amino terminus of OREBPdn to facilitate detection of transgene expression. The DNA fragment containing the OREBPdn transgene was microinjected into mouse oocytes. Tg mice were identified by PCR analysis of DNA obtained by tail biopsy using a pair of PCR primers, pFLAG1 (5′-TAAGCTAGCATGGACTACAAAGACGATGACGACAAG-3′) and Rp4 (5′-GGATATTGTCCACACAACATAGGGC-3′), and confirmed by Southern blot hybridization using the transgene as a probe. All Tg mice used in the experiments were heterozygous Tg mice that had been backcrossed with the C57BL/6J strain for four generations. Northern Blot—Mice were sacrificed by cervical dislocation and dissected under the dissection microscope. Total RNA (15 μg/lane) extracted from the lens and other tissues of 4-week-old mice using TRI reagent (Molecular Research Centre, Inc.) was separated by agarose-formaldehyde gel electrophoresis and transferred onto a Hybond-N+ nylon membrane. Hybridization was carried out using 32P-labeled OREBPdn and glyceraldehyde-3-phosphate dehydrogenase cDNA, respectively. Immunocytochemistry—For histological analysis, heads and whole eyes from embryos at different stages of development were dissected and fixed in 4% paraformaldehyde overnight prior to paraffin embedding. Seven-μm axial sections were prepared for immunocytochemistry. Rabbit polyclonal TonEBP antiserum (a kind gift from Dr. H. M. Kwon) was used for detection of the endogenous OREBP. Monoclonal anti-FLAG mouse IgG (Sigma, F4042) was applied for detection of transgenic expression. For detection of DNA damage checkpoint proteins, antiserum of p53 (FL-393, Santa Cruz Biotechnology, SC-6243) and phospho-Chk2 (Thr-68, Cell Signaling, 2661) were used for immunocytochemistry staining (Vector Laboratories, Inc.). In all cases, the appropriate biotinylated secondary antibodies (anti-mouse, or anti-rabbit) were used as secondary antibodies according to the instructions of the avidin:biotinylated enzyme complex kit and M.O.M. kit (Vector Laboratories, Inc.) followed by 3,3′-diaminobenzidine tetrahydrochloride substrate (Zymed Laboratories Inc.). Hematoxylin was used as counter staining. Cataract Monitoring and Gross Morphology—Pupils of mice, starting from 2 weeks old, were dilated with 1% tropicamide (Alcon, Puurs, Belgium), and lenses were monitored with a slit-lamp microscope and photographed. Lenses were and with a dissection microscope with and and from mouse embryos at different stages of development and postnatal mice were The of the lenses was and the lenses were at for and the was The of lens water was blot analysis, lenses from mice were and in containing and at for at the was in an containing of were to blot analysis using antibodies p53, and (Sigma, terminal deoxynucleotidyltransferase-mediated dUTP nick was to DNA strand breaks in on lens sections on previously Biol. 2004; PubMed Scopus Google Scholar, A. 1998; PubMed Scopus Google Scholar). sections were and were a with terminal for at The terminal was by containing of terminal of with the of and of and allowed to for at followed by the for in to the The was by the avidin:biotinylated enzyme complex (Vector Laboratories, Inc.) followed by substrate (Zymed Laboratories Inc.). were with and photographed. of OREBP during is the and expression of OREBP in the developing experiments showed that OREBP is in the lens and the of the developing mouse starting at embryonic (12Maouyo D. Kim J.Y. Lee S.D. Wu Y. Woo S.K. Kwon H.M. Am. J. Physiol. 2002; 282: F802-F809Crossref PubMed Scopus (61) Google Scholar). the expression of OREBP during mouse lens development, we prepared paraffin sections of mouse lenses from embryonic to postnatal analysis of these sections revealed that OREBP was expressed in the epithelial cells from to at In OREBP expression was to the fiber cells at and a of expression was found at OREBP was also in the secondary fiber cells On the other hand, OREBP expression was in fiber cells at Furthermore, in postnatal OREBP expression was and was to the secondary fiber cells the Generation of Transgenic Mice OREBPdn in the and of OREBP expression suggests that it a role in lens development. Since we and have shown that mutant OREBPdn, lacking the the function of endogenous OREBP (1Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (473) Google Scholar, A.K. Ko B.C. Tam S. Morris R. Yang J.Y. Chung S.K. Chung S.S. J. Biol. Chem. 2004; 279: 48048-48054Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, J. Go W.Y. Ho S.N. J. Immunol. 2002; 169: 5477-5488Crossref PubMed Scopus (87) Google Scholar), we the role of OREBP in the lens by Tg mice with the human OREBPdn (14Lam A.K. Ko B.C. Tam S. Morris R. Yang J.Y. Chung S.K. Chung S.S. J. Biol. Chem. 2004; 279: 48048-48054Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) fused to the α A-crystallin promoter that been shown to direct the expression of genes specifically in the lens fiber cells (17Jiang Z. Chung S.K. Zhou C. Cammarata P.R. Chung S.S. Investig. Ophthalmol. Vis. Sci. 2000; 41: 1467-1472PubMed Google Scholar, Chung S.K. Chung S.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). A FLAG epitope was at the amino terminus of the OREBPdn to facilitate the detection of transgene expression The Tg mice were identified by PCR and confirmed by Southern blot hybridization not of Tg mice were and were found to express of the transgene and Northern blot analysis showed that the of transgene expression in is than that in In the transgene expression was in the lens not in other tissues including the brain, and of mice similar nuclear cataract in the and embryonic lens we to in a of transgene expression. of the Transgenic Tg mice showed nuclear cataract 2 weeks after birth, the time suggesting defect in their determine the of we the expression of the transgene and the of the lens in the stages of development. FLAG was not in the epithelial cells lens development expression was in the lens fiber cells starting at The transgene expressed at this FLAG was in the fiber where the of the was found in the as well as in the secondary fiber where the was in the and the The secondary fiber cells not express the transgene of the transgene was associated with cellular were in the lens and and secondary fiber cells showed elongation leading to an in the lens the fiber cell the transgene expression was and was to the secondary fiber cells. to the lenses at were The of transgene expression was in postnatal lens and the transgene was expressed at the secondary fiber cells and to the fiber cells However, or fiber cell defect was in all postnatal mouse lens the lens in the fiber cells were of the lenses of the Tg mice showed the presence of in the fiber cells suggesting a in the determine the of OREBP inhibition on postnatal lens development, mouse lenses were by slit-lamp All Tg mice nuclear cataract starting at 2 weeks after and for the of their revealed on the lens of the Tg mice lens in lenses from mice were and under dissection microscope. In the the secondary fiber cell were to the to form at to and were not under microscope and J.I. E.S. and of B. Scholar). However, of the Tg lenses were and In was at the of the and The and at the of a that was revealed by the of the lens under dissection microscope Since the is by the of fiber cell the suggest that OREBPdn expression to fiber cell with the in embryonic lens. Since defects in fiber cell development in we the eye of the Tg shown in OREBPdn expression not the growth of the mice the were similar between the Tg mice and their as from weeks to However, eyes from the Tg mice were than from their starting from and at through weeks In when the of the lenses from postnatal mice was the lenses of the Tg mice were also found to be than of their at all stages by were displayed in mice was not due to the of the lens because the water of the lens was similar in Tg mice and not of DNA and of DNA in Tg been shown that hypertonic stress DNA breaks D. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and cell proliferation that is associated with cell in the and M.B. Am. J. Physiol. 2000; PubMed Google Scholar). of with addition the of p53 protein as well as in p53 phosphorylated on Kultz D. J. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We inhibition of the osmotic-response pathway by OREBPdn expression the p53 increase in p53 protein in the lenses of the Tg mice was revealed by the expression of be by D. Investig. Ophthalmol. Vis. Sci. Google Scholar), was not analysis revealed that p53 at when Tg expression is A and and to the and the secondary fiber cells of the Tg mice, suggesting that p53 expression is associated with OREBPdn expression and Furthermore, phosphorylated checkpoint 2 the for the activation of p53, was also expressed in the fiber cells of the Tg mice and Since DNA damage is of the leading to and p53 resulting in cell and A. S. Annu. Rev. 2004; PubMed Scopus Google Scholar), we in the lens of the Tg the we to fiber cells from to not Nevertheless, when a that is sensitive in DNA strand breaks was used Biol. 2004; PubMed Scopus Google Scholar, A. 1998; PubMed Scopus Google Scholar), we a increase in the of dUTP into the of DNA in the fiber cells of the Tg mice, suggesting DNA breaks in lens and these data suggest that OREBPdn expression results in DNA leading to the activation of of p53 and and and by and detection of DNA breaks by and of lens The lens a to study cell growth and because it of a of fiber cells under an of epithelial cells. from other cell the lens fiber cells are and these cells are to elongation to lens development. We showed that OREBP is expressed in the epithelial and fiber cells during lens development this the fiber cells are and to the of the lens OREBP expression was also found to be associated with the secondary fiber cells at a of embryonic development and in postnatal lens. These data suggest that OREBP a role in lens development. The OREBPdn Tg mice showed nuclear cataract This is the of lens development. the embryonic the lenses of the Tg mice fiber and incomplete elongation of fiber cells. are a in cataract The of formation is not It is most due to fiber cells. the in the fiber cells in the as a of cell during lens However, the damage to the fiber cells to of the and nuclear cataract. Since OREBPdn was expressed in the fiber cells that were our findings the role of OREBP in cell growth and of fiber cells is an essential in lens development. It is by intracellular leading to osmotic water and the increase in cell J. Cell. Physiol. PubMed Scopus Google Scholar). The of the fiber cells to their elongation a to the function of OREBP. OREBP, by program than hypertonic mediates accumulation of osmolytes to intracellular osmotic to facilitate water On the other hand, it been suggested that during the induction of cell the of and the of amino intracellular osmotic osmotic stress to cells to that of of cells to S.N. PubMed Scopus Google Scholar). cells in is by a dramatic accumulation of lens S.N. PubMed Scopus Google Scholar, Am. J. Physiol. 1999; PubMed Google Scholar), suggesting that OREBP is to the increase in osmolyte accumulation not to in the created by amino acid also to increase osmotic for cell the intracellular osmotic or the of osmolytes in the fiber cells in the embryonic lenses is that the lens fiber cells from the OREBPdn Tg mouse are experiencing hypertonic stress is the that have DNA strand breaks similar to in cells under hypertonic stress. It been shown that of kidney cells to hypertonic stress DNA breaks D. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and DNA strand breaks were also in the kidney in M.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar). This is with the induction of p53 and cell Kultz D. J. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. M. M. Y. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.F. G.M. M.B. Physiol. A Mol. Physiol. PubMed Scopus Google Scholar). In lens fiber cells of the OREBPdn Tg mice, DNA damage was accompanied by the activation of the DNA damage checkpoint pathway, as by the of This is different from cells under hypertonic stress in DNA damage response, as by is M.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar). In the protein plays an important role in the DNA damage response including of p53 and of and proteins, resulting in cell at or or J. M. C. DNA 2004; 3: PubMed Scopus Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the expression of p53 in fiber cells and the of in the Tg lens the role of p53 in cell of these cells. It is that the activation of DNA damage-response pathway also for the lymphoid in the OREBP knock-out mice (16Go W.Y. Liu X. Roti M.A. Liu F. Ho S.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10673-10678Crossref PubMed Scopus (234) Google Scholar), as well as the development of in the transgenic mice that express OREBPdn in the kidney collecting duct epithelial cells (14Lam A.K. Ko B.C. Tam S. Morris R. Yang J.Y. Chung S.K. Chung S.S. J. Biol. Chem. 2004; 279: 48048-48054Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In the lens of the mice, p53 was found in the epithelial cells of the central and as well as in the lens fiber cells at the R. Y. A. O. Investig. Ophthalmol. Vis. Sci. 2002; Google Scholar). However, the physiological function of p53 in these cells not been transgenic mice p53 developed associated with fiber cell and nuclear cataract H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). our transgenic mice also p53 and developed fiber cell and nuclear The between these mouse suggests that p53 induction a role in the of our Tg the activation of p53 also the program of lens fiber as suggested by the presence of nuclear in the lenses of the Tg However, the role of p53 leading to these abnormalities in the lenses of these Tg mice is not In our study that OREBP is important for fiber cell growth and elongation and a mechanism a osmotic-response pathway to DNA integrity and cell We Dr. H. M. Kwon for TonEBP and and for the with DNA