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• W2046249747 abstract "Interferon signaling is mediated by STATs and interferon regulatory factor (IRF) families of transcription factors. Ten distinct IRFs have been described and most are expressed in a variety of cells except for interferon consensus sequence-binding protein (ICSBP) and lymphoid-specific IRF/Pip that are thought to be exclusively expressed in lymphoid cells. We show here for the first time that ICSBP is constitutively and inducibly expressed in the mouse lens. In contrast to lymphoid cells with exclusive expression of ICSBP in the nucleus, ICSBP is present in both the cytoplasm and nucleus of the lens cell. However, ICSBP in the nucleus is of lower apparent molecular weight. We further show that the ICSBP promoter is constitutively bound by lens nuclear factors and that its activation requires binding of additional factors including STAT1. Furthermore, transcriptional activation of ICSBP gene by interferon γ is accompanied by selective nuclear localization of ICSBP in proliferating epithelial cells but not in the nuclei of nondividing cells in the lens fiber compartment. Constitutive and inducible expression of ICSBP in the ocular lens and differential regulation of its subcellular localization in the developing lens suggest that ICSBP may have nonimmunity related functions and that the commonly held view that it is lymphoid-specific be modified. Interferon signaling is mediated by STATs and interferon regulatory factor (IRF) families of transcription factors. Ten distinct IRFs have been described and most are expressed in a variety of cells except for interferon consensus sequence-binding protein (ICSBP) and lymphoid-specific IRF/Pip that are thought to be exclusively expressed in lymphoid cells. We show here for the first time that ICSBP is constitutively and inducibly expressed in the mouse lens. In contrast to lymphoid cells with exclusive expression of ICSBP in the nucleus, ICSBP is present in both the cytoplasm and nucleus of the lens cell. However, ICSBP in the nucleus is of lower apparent molecular weight. We further show that the ICSBP promoter is constitutively bound by lens nuclear factors and that its activation requires binding of additional factors including STAT1. Furthermore, transcriptional activation of ICSBP gene by interferon γ is accompanied by selective nuclear localization of ICSBP in proliferating epithelial cells but not in the nuclei of nondividing cells in the lens fiber compartment. Constitutive and inducible expression of ICSBP in the ocular lens and differential regulation of its subcellular localization in the developing lens suggest that ICSBP may have nonimmunity related functions and that the commonly held view that it is lymphoid-specific be modified. interferon interferon regulatory factor interferon consensus sequence-binding protein wild type transgenic cycloheximide polymerase chain reaction Interferons (IFNs)1 are a family of secreted proteins that are involved in the regulation of diverse cellular processes (1Boehm U. Klamp T. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar). In addition to their well defined roles in host defense against infectious agents, they have been associated with the regulation of cellular immunity, cell growth (1Boehm U. Klamp T. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar, 2Taniguchi T. Harada H. Lamphier M. L. Cancer Res. Clin. Oncol. 1995; 121: 516-520Crossref PubMed Scopus (117) Google Scholar, 3Vaughan P.S. van Wijnen A.J. Stein J.L. Stein G.S. J. Mol. Med. 1997; 75: 348-359Crossref PubMed Scopus (44) Google Scholar), and epithelial cell differentiation (4Saunders N.A. Jetten A.M. J. Biol. Chem. 1994; 269: 2016-2022Abstract Full Text PDF PubMed Google Scholar). The importance of IFNs is underscored by the expression of their receptors in virtually all mammalian cell types and by the fact that they regulate the expression of more than 50 cellular genes (1Boehm U. Klamp T. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar, 5Valente G. Ozmen L. Novelli F. Geuna M. Palestro G. Forni G. Garotta G. Eur. J. Immunol. 1992; 22: 2403-2412Crossref PubMed Scopus (158) Google Scholar). Interaction of IFNs with their cell surface receptor leads to activation of protein tyrosine kinases, JAK1, JAK2, or Tyk2, which in turn phosphorylate and activate members of a family of latent cytoplasmic transcription factors called STATs (signal transducers and activators of transcription) (6Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 7Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar). Phosphorylated STATs form homo- or heterodimers that translocate to the nucleus where they bind to well defined DNA sequences called GAS (gamma interferonactivation site) or ISREs (IFN-stimulated responseelements) and activate the transcription of genes coding for members of the interferon regulatory factor (IRF) family of transcription factors (8Decker T. Kovarik P. Meinke A. J. Interferon Cytokine Res. 1997; 17: 121-134Crossref PubMed Scopus (342) Google Scholar, 9Nguyen H. Hiscott J. Pitha P.M. Cytokine Growth Factor Rev. 1997; 8: 293-312Crossref PubMed Scopus (423) Google Scholar). IRFs are important mediators of transcriptional activation or repression of IFN-regulatable genes. They are characterized by a 115-amino acid N-terminal DNA-binding domain that interacts with ISRE motifs of IFN-regulatable genes (9Nguyen H. Hiscott J. Pitha P.M. Cytokine Growth Factor Rev. 1997; 8: 293-312Crossref PubMed Scopus (423) Google Scholar). Direct and indirect evidence indicate that the C-terminal portion of IRFs contains a protein-protein interaction domain able to function as transcriptional activators and/or repressors (9Nguyen H. Hiscott J. Pitha P.M. Cytokine Growth Factor Rev. 1997; 8: 293-312Crossref PubMed Scopus (423) Google Scholar, 10Sharf R. Azriel A. Lejbkowicz F. Winograd S.S. Ehrlich R. Levi B.-Z. J. Biol. Chem. 1995; 270: 13063-13069Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Ten members of the IRF family have been identified, and they include ICSBP, ISGF3γ/p48, IRF-1, IRF-2, IRF-3, IRF-4/lymphoid-specific IRF/Pip/ICSAT, IRF-5, IRF-6, IRF-7, and vIRF (9Nguyen H. Hiscott J. Pitha P.M. Cytokine Growth Factor Rev. 1997; 8: 293-312Crossref PubMed Scopus (423) Google Scholar). IRF-1 and IRF-2 are the best characterized members of this family and were initially identified by studies of the transcriptional regulation of the human IFNβ gene (11Miyamoto M. Fujita T. Kimura Y. Maruyama M. Harada H. Sudo Y. Miyata T. Taniguchi T. Cell. 1988; 54: 903-913Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 12Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Abstract Full Text PDF PubMed Scopus (807) Google Scholar). They have subsequently been shown to be key factors in the regulation of cell growth through their effects on the cell cycle (2Taniguchi T. Harada H. Lamphier M. L. Cancer Res. Clin. Oncol. 1995; 121: 516-520Crossref PubMed Scopus (117) Google Scholar, 3Vaughan P.S. van Wijnen A.J. Stein J.L. Stein G.S. J. Mol. Med. 1997; 75: 348-359Crossref PubMed Scopus (44) Google Scholar). IRF-1 is a tumor suppressor (13Taniguchi T. J. Cell. Physiol. 1997; 173: 128-130Crossref PubMed Scopus (48) Google Scholar), whereas IRF-2 is oncogenic (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar). In contrast to IRF-1 and IRF-2, which are expressed in a variety of cell types, two IRF members, interferon consensus sequence-binding protein (ICSBP) (15Driggers P.H. Ennist D.L. Gleason S.L. Mak W.-H. Marks M.S. Levi B.-Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar) and lymphoid-specific IRF/Pip (Pu.1interaction partner) (16Eisenbeis C.F. Singh H. Storb U. Genes Dev. 1995; 9: 1377-1387Crossref PubMed Scopus (417) Google Scholar, 17Matsuyama T. Grossman A. Mittrucker H.-W. Siderovski D.P. Kiefer F. Kawakami T. Richardson C.D. Taniguchi T. Yoshinaga S.K. Mak T.W. Nucleic Acids Res. 1995; 23: 2127-2136Crossref PubMed Scopus (210) Google Scholar, 18Yamagata T. Nishida J. Tanaka T. Sakai R. Mitani K. Yoshida M. Taniguchi T. Yazaki Y. Hirai H. Mol. Cell. Biol. 1996; 16: 1283-1294Crossref PubMed Scopus (187) Google Scholar) are thought to be expressed exclusively in cells of macrophage and lymphocyte lineages. Constitutive expression of ICSBP is thought to be limited to B lymphocytes, and mice with null mutation for the ICSBP gene develop myelogenous leukemia-like syndrome, suggesting that ICSBP activities may be restricted to lymphoid cells (15Driggers P.H. Ennist D.L. Gleason S.L. Mak W.-H. Marks M.S. Levi B.-Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar, 19Holtschke E. Lohler J. Kanno Y. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K. Gabriele L. Waring J.F. Bachmann M.F. Zinkernagel R.M. Morse III, H.C. Ozato K. Horak I. Cell. 1996; 87: 307-317Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). We have previously reported the generation of transgenic mice with targeted ectopic expression of IFNγ in the lens under the direction of the αA-crystallin promoter (20Egwuagu C.E. Sztein J. Chan C.C. Reid W. Mahdi R. Nussenblatt R.B. Chepelinsky A.B. Invest. Ophthalmol. Visual Sci. 1994; 35: 332-341PubMed Google Scholar, 21Egwuagu C.E. Sztein J. Chan C.C. Mahdi R. Nussenblatt R.B. Chepelinsky A.B. Dev. Biol. 1994; 166: 557-568Crossref PubMed Scopus (34) Google Scholar). In these mice, the normal pattern of endogenous lens gene expression is perturbed, and the developmental fate of cells destined to become lens fiber cells is altered. It was during the course of studies to establish a biological link between expression of IFNγ and the observed developmental defects that we discovered that several IRFs are constitutively expressed in the mouse lens. In this report, we present evidence that ICSBP is constitutively and inducibly expressed in the mouse lens. BALB/c wild type (WT) mice were purchased from Jackson Laboratories (Bar Harbor, ME). CD-1 WT mice were from Charles River (Raleigh, NC). Generation of the BALB/c IFNγ transgenic (TR) mice has previously been described (20Egwuagu C.E. Sztein J. Chan C.C. Reid W. Mahdi R. Nussenblatt R.B. Chepelinsky A.B. Invest. Ophthalmol. Visual Sci. 1994; 35: 332-341PubMed Google Scholar, 21Egwuagu C.E. Sztein J. Chan C.C. Mahdi R. Nussenblatt R.B. Chepelinsky A.B. Dev. Biol. 1994; 166: 557-568Crossref PubMed Scopus (34) Google Scholar). All animal procedures conformed to Institutional Guidelines and the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. The murine lens epithelial cell line, αTN4–1 (22Yamada T. Nakamura T. Westphal H. Russell P. Curr. Eye Res. 1990; 9: 31-37Crossref PubMed Scopus (74) Google Scholar), kindly provided by Dr. Paul Russell (NEI, NIH, Bethesda, MD), was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. The CRLE2 and 1AMLE 6 mouse epithelial cell lines (23Sax C.M. Dziedzic D.C. Piatigorsky J. Reddan J.R. Exp. Eye Res. 1995; 61: 125-127Crossref PubMed Scopus (11) Google Scholar), kind gifts from Dr. Christina M. Sax (NEI, NIH), were propagated in minimum essential medium supplemented with 5% rabbit serum, 5% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were treated with murine recombinant IFNγ (Life Technologies, Inc.) at a concentration of 100 units/ml for 2 h at 37 °C, 5% CO2. Some cells were propagated in medium containing the protein synthesis inhibitor, cycloheximide (CHX) (Sigma) at 35 μg/ml for 30 min followed by addition of IFNγ and incubation for 2 h. Lenses from 6-week-old WT or TR mouse littermates were carefully dissected and washed before RNA isolation to avoid any possible contamination by other tissues. Total RNA was isolated from the lenses or cultured lens cells as recommended for the TRIzol Reagent (Life Technologies, Inc.). All RNA samples were digested with RNase-free DNase 1 (Life Technologies, Inc.) for 30 min and purified by phenol/chloroform extraction and precipitation in 0.4m LiCl. cDNA synthesis was performed at 42 °C for 1 h with 10 μg of total RNA, 0.3 μg of oligo(dT)(12–16) and 1000 units Superscript Reverse Transcriptase II (Life Technologies, Inc.) in a final volume of 50 μl. For each RNA preparation, a negative control reaction was performed without reverse transcriptase. After purification of the cDNA, hot start PCR assays were performed with AmpliTaq Gold DNA polymerase (Perkin-Elmer). Samples were incubated at 95 °C for 10 min to activate the AmpliTaq Gold, and amplification was carried out for 25 cycles at 94 °C for 45 s, 63 °C for 45 s, and 72 °C for 45 s, and this was followed by a final 10-min extension at 72 °C. All the primer pairs used for PCR amplifications spanned at least one intron, making it possible to distinguish between amplification products derived from cDNA and those resulting from any contaminating genomic DNA templates. The sequence of the PCR primers used are: for mouse G3PDH, 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ and 5′-CATGTAGGCCATGAGGTCCACCAC-3′ (24Sabath D.E. Broome H.E. Prystowsky M.B. Gene (Amst.). 1990; 91: 185-191Crossref PubMed Scopus (380) Google Scholar), and for mouse ICSBP, 5′-GCTGCGG CAGTGGCTGATCGAACAGATCG-3′ and 5′-AGTGGCAGGCCTGCACTGGGCTGCTG-3′ (25Kanno Y. Kozak C.A. Schindler C. Driggers P.H. Ennist D.L. Gleason S.L. Darnell Jr., J.E. Ozato K. Mol. Cell. Biol. 1993; 3: 3951-3963Crossref Google Scholar). For Southern blot analysis, the amplified fragments were electrophoresed in agarose gels, transferred onto Hybond N+ nylon membrane (Amersham Pharmacia Biotech), and probed with fluorescein-dUTP 3′-end-labeled oligonucleotide probe, internal to the corresponding PCR primers. Probe labeling and signal detection were performed with the ECL 3′-oligolabeling and detection system (Amersham Pharmacia Biotech). Total RNA (30 μg) was fractionated on a 0.8% agarose-formaldehyde gel, transferred to Hybond N+ membrane (Amersham Pharmacia Biotech), and hybridized for 12 h at 65 °C in hybridization solution containing 5 × 106 cpm/ml of probe as described (26Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Stuhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995: 4.9-4.10Google Scholar). ICSBP or β-actin-specific cDNA fragments were labeled to high specific activity (>108 cpm/μg) with [α-32P]dCTP by random priming (oligolabeling kit; Amersham Pharmacia Biotech) and used as hybridization probes. After two high stringency washes in 0.1 × SSC, 0.1% SDS at 65 °C, signals were detected by autoradiography at −70 °C. with Kodak X-Omat AR film and Cronex intensifying screens. Lenses derived from 6-week-old WT or TR mouse littermates were disrupted in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 μm leupeptin, 2 μmpepstatin, 0.1 μm aprotinin, 1 mm4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 0.5 mm phenylmethylsulfonyl fluoride, and 1 μmE-64 on ice. Extracts were clarified by centrifugation, and protein levels were determined by Coomassie Blue dye binding method as recommended for Coomassie Plus Protein Assay Reagent (Pierce). For analysis of the lens epithelial cell lines, cells were cultured for 2 h in medium alone or medium containing 100 units/ml IFNγ. The cells were lysed and fractionated to cytosolic and nuclear fractions as described previously (27Politis A.D. Ozato K. Coligan J.E. Vogel S.N. J. Immunol. 1994; 152: 2270-2278PubMed Google Scholar). Nuclear and cytosolic fractions were also obtained from the EL4 lymphoma cell line (ATCC TIB-39) (American Type Culture Collection, Manassas, VA) and BALB/c mouse spleen cells. All samples were heated for 10 min at 95 °C in 1× sample buffer and electrophoresed in 10% SDS/polyacrylamide gel. The gel was electroblotted onto polyvinylidene fluoride membrane, blocked with 5% nonfat milk, and probed with either goat anti-mouse ICSBP polyclonal antibodies (1:2000) from Santa Cruz Biotech (Santa Cruz, CA) or a rabbit anti-mouse ICSBP polyclonal antibody (1:2000) fromZymed Laboratories Inc. (San Francisco, CA). Mouse αA-crystallin-specific antibody was kindly provided by Sam Zigler (NEI, NIH). Preimmune serum was also used in parallel as control. Signals were detected with horseradish peroxidase-conjugated secondary F(ab′)2 antibodies using the ECL system (Amersham Pharmacia Biotech). Seventeen day mouse embryos were fixed in 4% paraformaldehyde and embedded in Ameraffin tissue embedding medium (Baxter). Tissue sections (5 μm) were deparaffinized in xylene, rehydrated through a graded alcohol series, and used for immunostaining by the avidin-biotin-peroxidase complex method (Vector Laboratories, Burlingame, CA.). After preincubation for 30 min with 2% blocking serum, sections were incubated for 2 h at room temperature with antibodies (2 μg/ml) specific to mouse ICSBP (Santa Cruz). Control sections received the appropriate normal serum. In addition, antibody specificity control experiments were carried out by incubating the primary antibody with a 10-fold excess of a blocking peptide specific for the immunogenic epitope (ICSBP amino acid 407–425) for 2 h. The neutralized antibody was then used for immunostaining reactions with control tissue sections. All sections were subsequently incubated with biotinylated secondary antibody for 30 min at room temperature, and signal was visualized with diaminobenzidine-H2O2 as recommended (Vector). In some experiments, sections were counterstained with hematoxylin. Lens nuclear extracts were prepared either from 1–3-day-old WT CD-1 mouse lenses or cultured lens epithelial cells as described previously (28Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar). Buffer used for nuclear protein extraction contained the following protease inhibitors: 2 μm leupeptin, 2 μm pepstatin, 0.1 μm aprotinin, 1 mm4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 0.5 mm phenylmethylsulfonyl fluoride, and 1 μmE-64. Protein concentration was determined by the Coomassie Blue dye binding as recommended for Coomassie Plus Protein Assay Reagen kit (Pierce), and extracts were stored at −70 °C until use. Nuclear extracts (10 μg) in binding buffer (20 mm HEPES, pH 7.9, 50 mm KCl, 10% glycerol, 0.5 mmdithiothreitol, 0.1 mm EDTA) containing 0.14 μg/μl poly(dI-dC) were incubated on ice for 10 min. 32P-Labeled double-stranded DNA probe (50,000 cpm) was added and incubated for an additional 20 min on ice. Samples were electrophoresed in 4% polyacrylamide gel in 0.5 × Tris borate-EDTA buffer. For competition experiments, the nuclear extract was preincubated with unlabeled probe and poly(dI-dC) for 20 min on ice prior to the addition of labeled probe. The sequences used for the double-stranded probes or competitors are: ICSBP pIRE/GAS, 5′-AGTGATTTCTCGGAAAGAGAGCGCTTC-3′ (−175 to −149), and ICSBP-IRE, 5′-GTAAAGAGA GAAAAGGACTC-3′ (-217 to −198) (25Kanno Y. Kozak C.A. Schindler C. Driggers P.H. Ennist D.L. Gleason S.L. Darnell Jr., J.E. Ozato K. Mol. Cell. Biol. 1993; 3: 3951-3963Crossref Google Scholar). For supershift assays, STAT1, STAT2, STAT3, or STAT4 antibody (Upstate Biotechnology Inc., Lake Placid, NY) was added to the binding buffer containing the nuclear extract and preincubated on ice for 10 min. The 32P-labeled probe was then added, and the entire mixture was incubated for an additional 20 min on ice before electrophoresis. We had previously generated TR mice with ectopic expression of IFNγ in the lens to study the paracrine effects of IFNγ in the eye (20Egwuagu C.E. Sztein J. Chan C.C. Reid W. Mahdi R. Nussenblatt R.B. Chepelinsky A.B. Invest. Ophthalmol. Visual Sci. 1994; 35: 332-341PubMed Google Scholar, 21Egwuagu C.E. Sztein J. Chan C.C. Mahdi R. Nussenblatt R.B. Chepelinsky A.B. Dev. Biol. 1994; 166: 557-568Crossref PubMed Scopus (34) Google Scholar). In this study, we examined mRNA and protein levels of IFNγ-inducible transcription factors in the lenses of WT and TR mouse littermates to determine whether there is any correlation between enhanced expression of members of the IRF family of transcription factors and the abnormal lens phenotype observed in our TR mice. We found that both IRF-1 and IRF-2 are constitutively expressed in the lens and the levels of IRF-1 is markedly enhanced in the TR mouse lens (data not shown). Most surprising, we found ICSBP to be constitutively and inducibly expressed in the mouse lens as indicated by reverse transcribed PCR and Western blot analyses (Fig.1). These results have been confirmed by six independent experiments, and the authenticity of the ICSBP transcripts has been verified by cDNA sequencing; the nucleotide sequence of the ICSBP transcripts isolated from the lens is identical to published sequences reported for ICSBP derived from mouse hematopoietic cells (15Driggers P.H. Ennist D.L. Gleason S.L. Mak W.-H. Marks M.S. Levi B.-Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar). Detection of ICSBP transcripts in the WT lens was unexpected because constitutive transcription of the ICSBP gene is thought to be restricted to B-lymphocytes (15Driggers P.H. Ennist D.L. Gleason S.L. Mak W.-H. Marks M.S. Levi B.-Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar, 19Holtschke E. Lohler J. Kanno Y. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K. Gabriele L. Waring J.F. Bachmann M.F. Zinkernagel R.M. Morse III, H.C. Ozato K. Horak I. Cell. 1996; 87: 307-317Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 27Politis A.D. Ozato K. Coligan J.E. Vogel S.N. J. Immunol. 1994; 152: 2270-2278PubMed Google Scholar). Analysis of rat and bovine lenses reveal that the ICSBP protein and mRNA are also expressed in these species (data not shown). This is the first time that constitutive expression or transcriptional activation of the ICSBP gene has been demonstrated in mammalian cells that are not directly involved in immunological responses. As the vertebrate lens is comprised of undifferentiated, proliferating lens epithelial cells and terminally differentiated fiber cells, we sought to determine the spatial localization of cells expressing ICSBP in the lens. Fig.2 shows ICSBP localization in embryonic day 17 TR and WT mouse eye sections using polyclonal antibodies specific to mouse ICSBP. In these experiments, three serial sections were fixed onto the same glass slide; one section served as a negative control and was incubated with normal preimmune serum, another section received the primary antibody, and the third section received the primary antibody and 10-fold excess of a neutralizing peptide specific to an immunogenic epitope of mouse ICSBP. In all six independent experiments performed, the experimental sections showed identical antibody-staining patterns, whereas the negative control section consistently showed no immunological reactivity. The section containing 10-fold molar excess of the peptide consistently showed no significant immunoreactivity. In some experiments the amount of the peptide was varied, and the neutralizing effect of the blocking peptide was found to be dose-dependent. As shown in Fig. 2, ICSBP protein is present in both the cytoplasm and nuclei of lens cells. In the TR mouse lens, intense nuclear localization of ICSBP is observed in cells at the lens equator (Fig. 2, C and D) and anterior epithelia (Fig. 2 F) but not in nuclei of the cells at the lens fiber compartment (white arrows in Fig. 2, Dand F). In the WT lens, the amount of ICSBP in the nucleus is very low and not easily detectable. However, cytoplasmic ICSBP is easily detectable, and the level in the lens epithelia appears to be higher compared with that of the fiber compartment (arrowhead in Fig. 2 E). We obtained similar results using eye sections of mice at days 16–20 of embryonic development (data not shown). As indicated by our immunolocalization studies, a significant amount of ICSBP expression occurs in the WT lens epithelia, and there is selective accumulation of the ICSBP protein in the nuclei of epithelial cells of the TR mouse lens. To confirm these results we examined well characterized lens epithelial cell lines for ICSBP expression. Three lens cell lines, αTN4–1, CRLE2, and 1AMLE6 were therefore treated with mouse IFNγ in either the presence or absence of CHX and analyzed for constitutive or inducible expression of ICSBP. RNA was isolated from the various treatment groups and used for Northern analyses. In each of the cell lines, two ICSBP mRNAs of 3.0 and 1.7 kilobases are detected (Fig.3 A), and their sizes are similar to those of mouse ICSBP transcripts in hematopoietic cells (15Driggers P.H. Ennist D.L. Gleason S.L. Mak W.-H. Marks M.S. Levi B.-Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar,25Kanno Y. Kozak C.A. Schindler C. Driggers P.H. Ennist D.L. Gleason S.L. Darnell Jr., J.E. Ozato K. Mol. Cell. Biol. 1993; 3: 3951-3963Crossref Google Scholar). In cells treated with IFNγ, a significant increase in ICSBP is observed, indicating activation of the gene by IFNγ. Treatment with CHX prior to addition of IFNγ had no effects on the level of ICSBP transcripts, suggesting that inducible transcription of lens ICSBP mRNAs does not require de novo protein synthesis. In addition, cells that were treated for 2 h with IFNγ and untreated cells were fractionated into cytoplasmic and nuclear fractions and analyzed for the presence of the ICSBP protein by Western blotting. As indicated in Fig. 3 B, the ICSBP protein is detected in both the cytoplasm and nucleus. However, the ICSBP present in the nucleus migrates faster on SDS/polyacrylamide gel electrophoresis and appears to be of a lower apparent molecular weight. Cells that were not treated with IFNγ were found to contain more ICSBP in the cytoplasm than in the nucleus. After treatment with IFNγ, both higher and lower molecular weight ICSBP species are detected in the cytoplasm. However, the lower molecular weight species present in the nucleus is significantly increased. As shown in Fig.3 B, the ICSBP species detected in the lens cell nucleus co-migrates on SDS/polyacrylamide gel electrophoresis with the ICSBP of mouse lymphoid cells. Consistent with previous reports (27Politis A.D. Ozato K. Coligan J.E. Vogel S.N. J. Immunol. 1994; 152: 2270-2278PubMed Google Scholar), neither mouse spleen cells nor the lymphoma cell line EL4 contain ICSBP in the cytoplasm. Transcriptional activation of the ICSBP gene by IFNγ is mediated by the binding of activated STAT1 homodimers in the nucleus to the conserved cis regulatory palindromic IFNγ-responsive GAS element, pIRE/GAS, present in the ICSBP gene at positions −147 to −175 (25Kanno Y. Kozak C.A. Schindler C. Driggers P.H. Ennist D.L. Gleason S.L. Darnell Jr., J.E. Ozato K. Mol. Cell. Biol. 1993; 3: 3951-3963Crossref Google Scholar). We therefore tested by electrophoretic mobility shift assay whether endogenous lens nuclear factors are able to bind pIRE/GAS. Analysis performed using nuclear extracts derived from WT mouse lens is shown in Fig.4 A. Two prominent DNA-protein complexes are formed with the pIRE/GAS probe, and formation of the complexes is competed by the unlabeled probe (Fig. 4 A,lane 3), indicating that the interaction is specific. A DNA element located in the ICSBP gene at positions −191 to −217 and 22 base pairs upstream from the mouse pIRE/GAS site (25Kanno Y. Kozak C.A. Schindler C. Driggers P.H. Ennist D.L. Gleason S.L. Darnell Jr., J.E. Ozato K. Mol. Cell. Biol. 1993; 3: 3951-3963Crossref Google Scholar) contains a minimum ISRE motif (GAAANN) resembling the IRE or Pu box. This sequence, referred to as ICSBP-IRE (25Kanno Y. Kozak C.A. Schindler C. Driggers P.H. Ennist D.L. Gleason S.L. Darnell Jr., J.E. Ozato K. Mol. Cell. Biol. 1993; 3: 3951-3963Crossref Google Scholar), was used as a competitor to further characterize the ICSBP/GAS binding activities in the lens. The ICSBP-IRE probe allowed us to detect lens factors that bind to other DNA elements in the ICSBP promoter besides its GAS site. As shown in Fig. 4 A, the IRE probe competed for the complex labeledb but not with the a complex (lane 4), indicating that there are factors in the lens that constitutively interact with GAS, as well as, non-GAS elements of the ICSBP promoter. To further characterize the interaction between the ICSBP/pIRE and ICSBP-IRE elements with lens nuclear factors, we analyzed nuclear extracts derived from the various lens cell lines before and after treatment with IFNγ. Typical results obtained from these studies are shown in Fig. 4 B. Either probe formed a common complex (d) that is competed away by a 100-fold excess of either probe. Similar to results obtained using lens nuclear extracts, formation of this complex is also observed in extracts from cells that were not treated with IFNγ (lanes 2 and 6), confirming that lens nuclear factors constitutively bind to these elements. An additional retarded band (c) was detected with the pIRE/GAS probe after treatment of the cells with IFNγ (Fig.4 B, compare lanes 6 and 7), suggesting that these DNA binding activities are" @default.
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