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• W2002079757 abstract "Fibroblast growth factor (FGF) signaling is necessary for both proliferation and differentiation of lens cells. However, the molecular mechanisms by which FGFs exert their effects on the lens remain poorly understood. In this study, we show that FGF-2 repressed the expression of lens-specific genes at the proliferative phase in primary cultured lens cells. Using transfected cells, we also found that the activity of L-Maf, a lens differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK target sites on L-Maf promotes its stabilization. The stable L-Maf mutant protein promotes the differentiation of lens cells from neural retina cells. Taken together, these results indicate that FGF/ERK signaling negatively regulates the function of L-Maf in proliferative lens cells and that stabilization of the L-Maf protein is important for lens fiber differentiation. Fibroblast growth factor (FGF) signaling is necessary for both proliferation and differentiation of lens cells. However, the molecular mechanisms by which FGFs exert their effects on the lens remain poorly understood. In this study, we show that FGF-2 repressed the expression of lens-specific genes at the proliferative phase in primary cultured lens cells. Using transfected cells, we also found that the activity of L-Maf, a lens differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK target sites on L-Maf promotes its stabilization. The stable L-Maf mutant protein promotes the differentiation of lens cells from neural retina cells. Taken together, these results indicate that FGF/ERK signaling negatively regulates the function of L-Maf in proliferative lens cells and that stabilization of the L-Maf protein is important for lens fiber differentiation. fibroblast growth factor FGF receptor extracellular signal-regulated kinase mitogen-activated protein kinase MAPK/ERK kinase glutathione S-transferase lens-specific Maf β-galactosidase luciferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The vertebrate lens consists of only two cell populations, and because of this simple tissue organization, it provides an excellent model of cell differentiation. The anterior surface of the lens is covered by a simple epithelium, whereas the remainder and bulk of the lens is composed of elongated lens fibers (1McAvoy J.W. Chamberlain C.G. de Iongh R.U. Hales A.M. Lovicu F.J. Eye. 1999; 13: 425-437Crossref PubMed Scopus (220) Google Scholar). During embryonic development and throughout the life of the organism, fiber cells are added via differentiation of the lens epithelial cells. The proliferative epithelial cells in the anterior germinative zone move across the lens equator to the posterior transitional zone followed by withdrawal of cell cycling and differentiation into fiber cells. Fiber cell differentiation is characterized by cell elongation and the eventual degradation of all cellular organelles including the nucleus. These morphological changes are accompanied by specific activation of crystallin proteins as well as intermediate filament components such as filensin and CP49 (reviewed in Ref. 2Graw J. Dev. Genet. 1996; 18: 181-197Crossref PubMed Scopus (125) Google Scholar). Molecular genetic approaches have also recently identified a number of signaling molecules and transcription factors that are critical for lens development. Fibroblast growth factors (FGFs)1 regulate a diverse range of biological activities including adhesion, migration, proliferation, and differentiation (reviewed in Ref. 3Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1057) Google Scholar). FGF signaling is one of the most important regulators of differentiation from lens epithelial cells into fiber cells. Using lens explant cultures, it has been demonstrated that FGF-1 and FGF-2 can stimulate both proliferation of lens epithelial cells and differentiation of lens fiber cells (4McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228PubMed Google Scholar,5Schulz M.W. Chamberlain C.G. de Iongh R.U. McAvoy J.W. Development. 1993; 118: 117-126PubMed Google Scholar). In addition, in vivo gain-of-function studies have shown that misexpression of FGF-1, -4, -8, or -9 under the control of the lens-specific αA-crystallin promoter causes lens abnormalities and the so-called microphthalamia (small lens) phenotype (6Robinson M.L. Overbeek P.A. Verran D.J. Grizzle W.E. Stockard C.R. Friesel R. Maciag T. Thompson J.A. Development. 1995; 121: 505-514PubMed Google Scholar, 8Lovicu F.J. Overbeek P.A. Development. 1998; 125: 3365-3377Crossref PubMed Google Scholar). These FGFs effectively induce cell cycle withdrawal, loss of the characteristic cuboidal morphology, elongation of the cell shape, and accumulation of β-crystallin, resulting in premature differentiation of lens epithelial cells into fiber cells. In addition, a truncated form of FGF receptor (FGFR) lacking the cytoplasmic kinase domain inhibits fiber cell differentiation when expressed in transgenic mice under the control of a lens-specific promoter (7Robinson M.L. MacMillan-Crow L.A. Thompson J.A. Overbeek P.A. Development. 1995; 121: 3959-3967PubMed Google Scholar), and a secreted form of FGFR can cause a delay in fiber differentiation (9Govindarajan V. Overbeek P.A. Development. 2001; 128: 1617-1627PubMed Google Scholar). Together, these studies implicate FGFs as bifunctional molecules that regulate both proliferation and differentiation of the lens cells. The transcription factors that mediate FGF signaling in lens development and the molecular mechanisms used by FGFs to regulate the transcriptional factors remain to be identified. Several transcription factors, including Pax6, Sox2, Six3, c-Maf, and L-Maf, are known to be important in lens development and in regulating the expression of lens-specific genes such as crystallin genes (10Cvekl A. Sax C.M., Li, X. McDermott J.B. Piatigorsky J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4681-4685Crossref PubMed Scopus (97) Google Scholar, 11Duncan M.K. Haynes J.I., II Cvekl A. Piatigorsky J. Mol. Cell. Biol. 1998; 18: 5579-5586Crossref PubMed Google Scholar, 12Kamachi Y. Uchikawa M. Tanouchi A. Sekido R. Kondoh H. Genes Dev. 2001; 15: 1272-1286Crossref PubMed Scopus (307) Google Scholar, 13Lengler J. Krausz E. Tomarev S. Prescott A. Quinlan R.A. Graw J. Nucleic Acids Res. 2001; 29: 515-526Crossref PubMed Google Scholar, 14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar, 15Kim J.I., Li, T., Ho, I.C. Grusby M.J. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3781-3785Crossref PubMed Scopus (199) Google Scholar, 16Kawauchi S. Takahashi S. Nakajima O. Ogino H. Morita M. Nishizawa M. Yasuda K. Yamamoto M. J. Biol. Chem. 1999; 274: 19254-19260Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Ring B.Z. Cordes S.P. Overbeek P.A. Barsh G.S. Development. 2000; 127: 307-317Crossref PubMed Google Scholar). Of these factors, L-Maf is one of the most attractive candidates for mediating FGF signaling, because L-Maf has been identified as a lens-specific regulator of avian αA-crystallin expression (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar), and c-maf-deficient mice have defective lens fiber differentiation and loss of crystallin gene expression (16Kawauchi S. Takahashi S. Nakajima O. Ogino H. Morita M. Nishizawa M. Yasuda K. Yamamoto M. J. Biol. Chem. 1999; 274: 19254-19260Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Ring B.Z. Cordes S.P. Overbeek P.A. Barsh G.S. Development. 2000; 127: 307-317Crossref PubMed Google Scholar). There results show that Mafs are critical for expression of crystallin genes and lens fiber differentiation. L-Maf is a basic region/leucine zipper transcription factor and regulates the expression of αA-crystallin by binding the lens-specific enhancer element αCE2 (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar, 18Matsuo I. Kitamura M. Okazaki K. Yasuda K. Development. 1991; 113: 539-550PubMed Google Scholar). In chicken, the expression of L-mafis initiated in the lens placode and is subsequently restricted to the developing lens (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar, 19Yoshida T. Yasuda K. Genes Cells. 2002; 7: 693-706Crossref PubMed Scopus (34) Google Scholar). In addition, ectopic expression of L-Maf induces the expression of several crystallin and filensin genes (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar) and promotes the formation of lens-like structures called lentoid bodies in retinal primary cultures. Furthermore, a dominant-negative form of L-Maf inhibits crystallin gene expression and proper lens cell differentiation (20Reza H.M. Ogino H. Yasuda K. Mech. Dev. 2002; 116: 61-73Crossref PubMed Scopus (56) Google Scholar). Thus, L-Maf is clearly important for crystallin gene expression and for lens development. To address the molecular mechanisms that determine how FGF signaling regulates transcription of the lens-specific genes in lens development, we investigated the effects of FGF signaling on the regulation of L-Maf function. Here we show that FGF-2 promotes proliferation of lens cells in the early stages of primary culture but represses expression of lens-specific genes at the same time points. In addition, FGF/ERK signaling represses activity of an L-Maf-dependent reporter in the lens cells. We also found that residues Thr-57 and Ser-65 of the L-Maf protein are important as phosphorylation sites for extracellular-signal regulated kinase (ERK)in vitro. Mutations at either of these two sites caused an increased stability of the L-Maf protein, resulting in enhanced expression of δ-crystallin. These results indicate that FGF/ERK signaling controls terminal differentiation of the lens fiber cells through regulation of L-Maf stability. Lens cell cultures were prepared as previously described (18Matsuo I. Kitamura M. Okazaki K. Yasuda K. Development. 1991; 113: 539-550PubMed Google Scholar). Lens cells from 14-day-old chick embryos were maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% chicken serum. When the cells reached confluence, they were harvested with trypsin and centrifuged to remove nonproliferative lens fiber cells. The cells were then cultured at a density of 1 × 105/30-mm dish to establish primary cultures. Transfection with plasmids or treatment with FGF-2 (human fibroblast growth factor-2, PeproTech House) were carried out 1 day after establishment of the primary cultures. Plasmids used to express FLAG epitope-tagged L-Maf (pEFX3-fg-L-Maf) constructs in lens cells have been described previously (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar). pEFX-β-gal containing the human EF-1α promoter and β-galactosidase open reading frame inserts and pCAGGS-GFP carrying the cytomegalovirus enhancer, β-actin basal promoter, and green fluorescent protein open reading frame sequences were used as controls for normalization (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar). Single amino acid mutations of the L-Maf protein were carried out by subcloning anEcoRV-EagI fragment from pEFX3-fg-L-Maf, corresponding to the coding region of the N terminus of L-Maf, into pBluescript vector (pBsk-L-Maf). Point mutations were generated on pBsk-L-Maf using the QuikChange site-directed mutagenesis kit (Stratagene) using mutagenic oligonucleotide primers, as follows: S14A, 5′-GCT GCC CAC CGC CCC CCT CGC CAT CG-3′; T57A, 5′-CCC CTC TCA GCG CGC CGT GCT CCT CC-3′; S65A, 5′-CCG TGC CTT CCG CGC CCA GTT TCT GC-3′ (mutations are underlined). Mutations were confirmed by sequence analysis and then subcloned back into pEFX3-fg-L-Maf digested with EcoRV and EagI. pCAGGS-DN-L-Maf, an expression plasmid for a dominant-negative form of L-Maf, was described previously (20Reza H.M. Ogino H. Yasuda K. Mech. Dev. 2002; 116: 61-73Crossref PubMed Scopus (56) Google Scholar). A plasmid encoding the S218/222E Δ32–51 mutant of MEK1 was purchased from Stratagene. Reporter constructs containing six copies of an αCE2 core sequence and the β-actin basal promoter (cαALuc) were described previously (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar). tkLuc is a luciferase reporter plasmid carrying the viral thymidine kinase gene promoter (base pairs −197 to +56) (18Matsuo I. Kitamura M. Okazaki K. Yasuda K. Development. 1991; 113: 539-550PubMed Google Scholar). For UAS6βLuc, six copies of the upstream activation sequence fragment were inserted into the βLuc plasmid (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar) at the 5′ end of the β-actin basal promoter and luciferase gene. To construct the GAL4-Elk1 expression plasmid, the coding sequence for the activation domain of human Elk1 (amino acids 307–428) (kindly provided by Dr. R. Treisman (21Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1108) Google Scholar)) was amplified by PCR, subcloned into pCMX-GAL4 (kind gift from Dr. K. Umesono), and digested withBamHI and EcoRV (pCMX-GAL4-Elk1). Primary lens cultures in 12-well dishes were transfected with 0.5 μg of DNA using DEMRI-C according to the manufacturer's protocol (Invitrogen). After 4 h of transfection, the medium was replaced by fresh 10% fetal bovine serum, 1% chick serum medium. Cells were incubated for a further 48 h, and then whole cell lysates were prepared in extraction buffer (0.1 ml of 25 mm Tris-phosphate, pH 7.8, 15% glycerol, 2% CHAPS, 1% l-α-phosphatidylcholine, 1% bovine serum albumin, 4 mm EGTA, 8 mmMgCl2, 1 mm dithiothreitol, 0.4 mm p-amidinophenyl methanesulfonyl fluoride hydrochloride (APMSF, Wako)). The luciferase activities of the lysates were measured with a microtiter plate luminometer (Dynex). Transfection efficiency in each dish was normalized by the β-galactosidase activity. All luciferase activity values represent mean ± standard deviation of results obtained from duplicates of three independent transfection experiments. GST and GST-L-Maf proteins were expressed in Escherichia coli DH5α strain and then purified from the bacterial extracts using glutathione-Sepharose beads according to the manufacturer's recommendations (Amersham Pharmacia Biotech). The recombinant L-Maf protein was incubated with purified ERK (New England Biolabs) in kinase buffer (50 mm Tris-HCl, 10 mm MgCl21 mm EGTA, 2 mm dithiothreitol, 0.01% Brij35) at 30 °C for 3 h. Nuclear extracts were prepared from the lens cell cultures as described previously (18Matsuo I. Kitamura M. Okazaki K. Yasuda K. Development. 1991; 113: 539-550PubMed Google Scholar). An equal amount of protein from each extract was subjected to 10% SDS-PAGE and was subsequently transferred to a nitrocellulose membrane (Schleicher & Schuell). The membranes were incubated with the following primary antibodies diluted in blocking buffer: anti-FLAG M2 monoclonal antibody (Kodak, 1:8000), anti-GST (Amersham Pharmacia Biotech, 1:8000), anti-L-Maf (1:1000 (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar)), anti-ERK (Promega, 1:1000), anti-phospho-ERK (Promega, 1:1000), and anti-δ-crystallin (1:1 (22Sawada K. Agata K. Yoshiki A. Eguchi G. Jpn. J. Ophthalmol. 1993; 37: 355-368PubMed Google Scholar)). After three washes in PBST (phosphate-buffered saline containing 0.1% Tween 20), membranes were incubated with the following secondary antibodies conjugated to horseradish peroxidase diluted in blocking buffer: anti-mouse IgG antibody (Dako, 1:1000), anti-rabbit IgG antibody (Dako, 1:1000). The signals were detected with an ECL kit (Amersham Pharmacia Biotech). The stability of the L-Maf was analyzed by incubating the cells for 3 h in the presence of proteasome inhibitor. The inhibitors used were LLnL (Z-Leu-Leu-Leu-aldehyde, 20 μg/ml, BostonBiochem), MG132 (BostonBiochem, 50 μm), andclasto-lactacystin-β-lactone (lactastatin, BostonBiochem, 10 μm). After the treatment, cells were washed to remove inhibitors followed by further incubation in fresh Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% chick serum for various periods. Then the whole cell extracts were subjected to Western blot analysis. Northern blot analyses were carried out as described previously (23Maegawa S. Yasuda K. Inoue K. Mech. Dev. 1999; 81: 223-226Crossref PubMed Scopus (122) Google Scholar). In brief, total RNA was isolated from the cultured lens cells, and equal amounts of RNA were separated on formaldehyde-agarose gels. After electrophoresis, RNA was blotted onto Biodyne A nylon membranes (Pall) and hybridized with digoxigenin-labeled DNA probes. mRNAs were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche Molecular Biochemicals) and visualized by reaction with the chemiluminescent substrate, disodium 3-(4-methoxysprio{1,2-dioxetane 3,2′(5′-chloro)tricyclo[3.3.1.13,7] decan}-4-yl)phenyl phosphate (Applied Biosystems) (Tropix). Immunostaining of primary cultured cells was carried out as described previously (19Yoshida T. Yasuda K. Genes Cells. 2002; 7: 693-706Crossref PubMed Scopus (34) Google Scholar). The cells were fixed and incubated with the appropriate antibody: purified rabbit anti-L-Maf IgG (4McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228PubMed Google Scholar), 1:1000; AlexaFluor-488 goat anti-mouse IgG (Molecular Probes), 1:1000; AlexaFluor-594 goat anti-rabbit IgG (Molecular Probes), 1:1000. An Olympus AX70 confocal microscope was used to determine the localization of L-Maf, δ-crystallin, and phospho-ERK in lentoid bodies in primary cultured lens cells. The probe used for the electrophoretic mobility shift assay was 5′-CATTTCTGCTGACCACGTTGCCTTC-3′ (cαA). Double-stranded oligonucleotide probes were labeled at the 3′ end using the Klenow fragment and [α-32P]dATP. Then, 5 pmol of probe was incubated with 1 pmol of L-Maf protein for 30 min at 30 °C in reaction buffer (10 mm Hepes-KOH, pH 7.9, 0.1 mm EDTA, 2 mm dithiothreitol, 100 mm KCl, 5 mm MgCl2, bovine serum albumin, 10% glycerol, 5 μg/μl poly (dI-dC)-poly (dI-dC)). The reaction mixture was loaded onto a 4% polyacrylamide gel. The gel was transferred to DEAE paper, dried, and subjected to autoradiography. Previous studies had shown that FGF-1 and FGF-2 promote proliferation and differentiation of lens epithelial cells in explant cultures (4McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228PubMed Google Scholar, 24Li L. Zhou J. James G. Heller-Harrison R. Czech M.P. Olson E.N. Cell. 1992; 71: 1181-1194Abstract Full Text PDF PubMed Scopus (282) Google Scholar). However, the molecular mechanisms underlying FGF signaling in lens development are yet to be elucidated. To address these mechanisms, we used primary cultures enriched for proliferating lens epithelial cells. Initially, we aimed to confirm whether FGF-2 regulates both proliferation and differentiation of the lens cells. Cell counts were performed at different times during culture with or without FGF-2 treatment. The number of FGF-2-treated lens cells was greatly increased on days 1 and 2 compared with the untreated control cells (Fig. 1 A). However, by days 3–6, neither the FGF-2-treated nor untreated lens cells showed significant proliferation. After 6 days of culture, we found abundant and large lentoid bodies in the FGF-2-treated lens cell cultures (Fig. 1 B). Lentoid bodies are clusters of differentiated fiber cells. These results demonstrate that FGF-2 promotes both proliferation and differentiation of primary cultured lens cells, which is consistent with previous results using lens explants (4McAvoy J.W. Chamberlain C.G. Development. 1989; 107: 221-228PubMed Google Scholar, 24Li L. Zhou J. James G. Heller-Harrison R. Czech M.P. Olson E.N. Cell. 1992; 71: 1181-1194Abstract Full Text PDF PubMed Scopus (282) Google Scholar). Next we examined the effect of FGF-2 on the expression of marker genes for lens fiber differentiation, crystallin genes and filensin gene. Primary lens cells were incubated with or without FGF-2, and then total RNA was isolated and subjected to Northern blot analysis. After day 3, expression of both crystallin and filensin genes was enhanced by the addition of FGF-2 (Fig. 1 C). Surprisingly, FGF-2 efficiently reduced the expression of δ-, αA-, βB1-crystallin, and filensin genes at earlier culture points (Fig. 1 C, days 1–2), which coincided with the period of lens cell proliferation (Fig. 1 A). These results suggest that FGF-2 represses lens-specific genes at early stages of culture when the lens epithelial cells are proliferating, whereas the same growth factor promotes lens fiber differentiation to augment crystallin gene expression and lentoid body formation at later stages of cell culture. On the basis of FGF-2 treatment, we then proposed that transcription factors regulating the expression of crystallins should function under the control of FGF-2. One of the attractive candidates for this regulation is lens-specific Maf (L-Maf), which binds to chicken αA-crystallin enhancer (αCE2) and regulates expression of the αA-, δ-, βB1-crystallin, and filensin genes (14Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (234) Google Scholar, 18Matsuo I. Kitamura M. Okazaki K. Yasuda K. Development. 1991; 113: 539-550PubMed Google Scholar). In addition, a dominant-negative form of L-Maf that lacks its acidic domain (DN-L-Maf (20Reza H.M. Ogino H. Yasuda K. Mech. Dev. 2002; 116: 61-73Crossref PubMed Scopus (56) Google Scholar)) repressed expression of the αA-, δ-, βB1-crystallin, and filensin genes in primary cultured lens cells (Fig. 2 A) and chick embryos (20Reza H.M. Ogino H. Yasuda K. Mech. Dev. 2002; 116: 61-73Crossref PubMed Scopus (56) Google Scholar), showing that L-Maf is essential for the expression of the lens-specific genes. To assess the possibility that FGF signaling regulates the L-maf gene, we first examined the effect of FGF-2 on expression of L-maf mRNA. Lens cell primary cultures were incubated for 48 h with or without FGF-2, and then total RNA was isolated and subjected to Northern blot analysis. L-mafmRNA levels in the FGF-2-treated cells were comparable to those in the untreated control cells (Fig. 2 B), indicating that FGF-2 does not significantly affect transcription of the L-mafgene. We next examined the effect of FGF-2 on regulation of L-Maf at the protein level. Since we could not detect l-Maf protein by Western blot analysis (data not shown), we measured the transcriptional activity of L-Maf protein in primary cultured lens cells using a reporter gene construct containing six copies of a 25-bp L-Maf binding site in αCE2, β-actin basal promoter, and the luciferase coding sequence (cαALuc, Fig. 2 C). This construct was transiently transfected into the lens cells, which were incubated with or without FGF-2 for 48 h. Luciferase activities provided a measure of transcriptional activity. FGF-2 clearly repressed the expression of the reporter gene (Fig. 2 C), an effect that was dose-dependent (Fig. 2 D, left panel). This repression was not due to a general effect on transcription because a luciferase reporter driven by either a thymidine kinase promoter or a β-actin basal promoter alone was not affected by the addition of FGF-2 (tkLuc and βLuc, Fig. 2 C). We also examined the effect of a specific FGF receptor inhibitor, SU5402 (25Mohammadi M. McMahon G. Sun L. Tang C. Hirth P. Yeh B.K. Hubbard S.R. Schlessinger J. Science. 1997; 276: 955-960Crossref PubMed Scopus (1015) Google Scholar). The reporter activity significantly increased in the presence of SU5402 (Fig. 2 D, right panel), indicating that inhibition of FGF signaling promotes expression of the reporter gene. Taken together, these results indicate that FGF signaling inhibits L-Maf-dependent transcription in the primary cultured lens cell, resulting in repression of the lens-specific genes at the early culture stage. ERK1 and ERK2, members of the mitogen-activated protein kinase (MAPK) family, play major roles in a variety of developmental processes and biological actions induced by FGF signaling (26Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar, 27Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar). Because activated ERK is detected in lens in vivo (28Lovicu F.J. McAvoy J.W. Development. 2001; 128: 5075-5084PubMed Google Scholar), FGF-2 may regulate L-Maf activity through ERK. To test this possibility, we first examined whether FGF signaling involves the ERK pathway in primary cultured lens cells. Because serum is a potent activator of the MAPK cascade, lens cells were serum-starved for 4 h followed by FGF-2 treatment for 1 h. In the whole cell extracts of unstimulated cultured lens cells, phosphorylated ERK was detected only as a weak band by Western blot analysis with an anti-phospho-ERK antibody (Fig. 3 A, lane 1). In contrast, phosphorylation of ERK was increased in the FGF-2-treated cells (Fig. 3 A, lane 2). This phosphorylation of ERK was clearly inhibited by addition of PD98059, a specific inhibitor of MEK1, the ERK kinase (Fig. 3 A, lane 3). These results demonstrate that FGF-2 promotes phosphorylation of ERK through MEK1 in lens cells. We next examined the effect of constitutively active MEK1 on the activity of L-Maf in the primary cultured lens cells using the cαALuc reporter (Fig. 3 B, left). Introduction of the constitutively active MEK1 caused a decrease in expression of the reporter gene. In contrast, MEK1 inhibitor PD98059 promoted expression of the reporter gene (Fig. 3 B), indicating that L-Maf activity in lens cells is regulated through the MEK1/ERK pathway. We also confirmed that the constitutively active MEK1 could activate Elk considerably, an ERK-regulated transcription factor, using the GAL4-Elk1 and UAS6βLuc reporter gene assay (Fig. 3 B,right), indicating that MEK1 is active in the cultured lens cells and the inhibitory effect of MEK1 is not ubiquitous to all transcription. These results indicate that MEK1/ERK mediates FGF-2 signaling to repress L-Maf-dependent transcription. We identified three putative MAPK target sites in the L-Maf acidic region. To test whether L-Maf is directly phosphorylated by ERK, we first performed in vitro phosphorylation analysis. When incubated with purified ERK, recombinant L-Maf protein was clearly phosphorylated (Fig. 4 A). To examine phosphorylation of L-Maf in living cells, we introduced an expression plasmid encoding wild-type L-Maf into the COS-7 monkey kidney cell line. Ectopically expressed L-Maf protein was detected as a 42-kDa band in COS-7 cells by Western blot analysis using an anti-FLAG antibody (Fig. 4 C). A 34-kDa band, which is approximately the same molecular size as the in vitro translated L-Maf protein, appeared when the cell lysate was treated with calf intestine alkaline phosphatase, indicating that L-Maf is phosphorylated in living cells. To identify the ERK phosphorylation site, a series of L-Maf mutant proteins was made with substitutions to alanine residues at the three potential sites of Ser-14, Thr-57, or Ser-65 (designated as S14A, T57A, and S65A, respectively). The wild-type and mutant proteins were subjected to in vitro phosphorylation. Incorporation of [γ-32P]ATP was detected with the wild-type L-Maf protein and mutant S14A, suggesting that Ser-14 is not involved in phosphorylation by ERK (Fig. 4 B). In contrast, T57A and S65A mutants showed a significantly reduced phosphorylation signal. These results implicate Thr-57 and Ser-65 as important in the phosphorylation of L-Maf by ERK. We next evaluated the effects of mutating the phosphorylation sites on the transcriptional activation of L-Maf. The mutation on Ser-14 did not significantly affect the transcriptional activity of L-Maf (Fig. 5 A), whereas the T57A and S65A L-Maf mutants showed ∼5- and 7-fold higher levels of reporter activity than the wild-type protein, respectively. We also examined the importance of Thr-57 and Ser-65 for the ability of L-Maf to induce the expression of its in vivo target gene, δ-crystallin. When either T57A or S65A mutant L-Maf was introduced into primary cultured retinal cells, expression of endogenous δ-crystallin was significantly induced (Fig. 5 C), and the number of δ-crystallin-positive cells increased 3–4-fold compared with wild-type L-Maf (Fig. 5 C, TableI). To confirm that these mutational effects involve MEK1/ERK signaling, constitutively active MEK1 was cotransfected into cultured lens cells together with either wild-type or S65A mutant L-Maf. The transcriptional activity of endogenous L-Maf and overexpressed wild-type L-Maf were decreased by the co-expression of constitutively active MEK1 (Fig. 5 B), whereas S65A mutant L-Maf was not sensitive to MEK1 signaling. Taken together, these results indicate that the ERK phosphorylation sites at Thr-57 and Ser-65 are important for the transcriptional activity of L-Maf and that phosphorylation at these sites negatively regulates L-Maf function in lens cells.Table IMutation of MAPK target sites of L-Maf promotes lens differentiationOverexpressed L-MafNo. of δ-crystallin-positive cells/2.4 mm2Exp. 1Exp. 2Exp. 3Wild type8135S14A122614T57A204018S65A294713Mock000 Open table in a new tab To elucidate how L-Maf function is regulated by phosphorylation, we measured the DNA binding activity of phosphorylated or unphosphorylated L-Maf protein by an electrophoretic" @default.
• W2002079757 created "2016-06-24" @default.
• W2002079757 creator A5002256460 @default.
• W2002079757 creator A5018399011 @default.
• W2002079757 creator A5073214988 @default.
• W2002079757 creator A5075824931 @default.
• W2002079757 date "2003-01-01" @default.
• W2002079757 modified "2023-09-28" @default.
• W2002079757 title "The Stability of the Lens-specific Maf Protein is Regulated by Fibroblast Growth Factor (FGF)/ERK Signaling in Lens Fiber Differentiation" @default.
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