Developmental Biology 210, 44–55 (1999) Article ID dbio.1999.9267, available online at http://www.idealibrary.com on Interferon Regulatory Transcription Factors Are Constitutively Expressed and Spatially Regulated in the Mouse Lens Wenmei Li,* Chandrasekharam N. Nagineni,* Bassey Efiok,† Ana B. Chepelinsky,‡ and Charles E. Egwuagu*,1 *Laboratory of Immunology and ‡Laboratory of Molecular & Developmental Biology, National Eye Institute, and †Laboratory of Molecular Hematology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulation of cell growth and immunological responses. Nine IRFs have been described and they are expressed in a variety of cells, except for ICSBP and LSIRF/Pip, which are thought to be expressed exclusively in immune cells. Here, we show that IRF-1, IRF-2, ICSBP, and LSIRF/Pip are constitutively expressed in the mouse lens. These IRFs are present in both the cytoplasm and the nuclei of lens cells. However, the nuclear and cytoplasmic proteins exhibit distinct mobilities on SDS/PAGE. We further show that in the developing mouse lens, IRF-1 and IRF-2 are expressed at high levels in differentiated lens fiber cells with very low and barely detectable levels in undifferentiated lens epithelial cells. Although the level of ICSBP expression is very low in the normal mouse lens, in transgenic mice with constitutive expression of interferon g in the lens, its level is markedly elevated and ICSBP expression is detected exclusively in the nuclei of undifferentiated lens cells. Taken together, our data suggest that expression of IRF transcription factors is spatially regulated in the lens and that distinct IRFs may contribute to differential gene regulation in the epithelial and fiber compartments of the vertebrate lens. Key Words: IFNg; ICSBP; LSIRF/Pip; IRF-1; IRF-2; STAT1; lens differentiation. INTRODUCTION called STATs (signal transducers and activators of tran- scription) (Darnell, 1997). Phosphorylated STATs form Interferons (IFNs) are multifunctional cytokines induced homo- or heterodimers that translocate to the nucleus in almost all vertebrate cells in response to infection or where they bind to well-defined DNA sequences called immunological stimuli. There are two main types: Type 1 GAS (gamma IFN activation site) or ISREs (IFN-stimulated IFN comprises leukocyte (IFNa) and fibroblast (IFNb) IFNs response elements) and activate transcription of genes cod- while Type 2 or immune IFN is IFNg (Vilcek and Sen, ing for members of the interferon regulatory factors (IRFs) 1996). Although the two types of IFNs differ in the cell family of transcription factors (Decker et al., 1997; Boehm surface receptors they bind to and their physiological in- et al., 1997). ducers, their biological activities are mediated through IRFs interact with ISRE motifs in the promoters of activation of the JAK/STAT signaling pathway (Schindler IFN-regulatable genes and they mediate transcriptional and Darnell, 1995). Interaction of IFNs with their receptors activation or repression of these genes. Ten members of the leads to activation of protein tyrosine kinases, JAK1, JAK2, IRF family have been identified and they include ICSBP, or Tyk2, which in turn phosphorylate and activate mem- ISGF3g/p48, IRF-1, IRF-2, IRF-3, IRF-4/LSIRF/Pip/ICSAT, bers of a family of latent cytoplasmic transcription factors IRF-5, IRF-6, IRF-7, and vIRF (Nguyen et al., 1997). IRFs differ in the range of cell types they are normally expressed 1 To whom correspondence should be addressed at the Labora- in, their physiological inducers, and the biological processes tory of Immunology, National Eye Institute, National Institutes of they affect (Nguyen et al., 1997). With the recent demon- Health, 10/10N116, 10 Center Drive MCS 1858, Bethesda, MD stration of a virally encoded homologue of cellular IRFs 20892. Fax: (301) 480-3914. E-mail: [email protected]. (Moore et al., 1996), it is likely that more IRFs will be 44 0012-1606/99 Differential Expression of IRFs in the Lens 45 identified and previously described members would be and Ophthalmology Resolution on the Use of Animals in Re- found to possess new functions. search. IRF-1 and IRF-2 are the best characterized members of this family and were initially identified as regulators of the Cell Culture and IFNg Treatment IFN system (Miyamoto et al., 1988; Harada et al., 1989). They have subsequently been shown to be key factors in the The murine lens epithelial cell line, aTN4-1 (Yamada et al., regulation of cell growth through their effects on the cell 1990), kindly provided by Dr. Paul Russell (NEI, NIH, Bethesda, cycle (Taniguchi et al., 1995; Vaughan et al., 1997). IRF-1 is MD), was grown in Dulbecco’s modified Eagle’s medium supple- mented with 10% fetal bovine serum, 2 mM glutamine, penicillin thought to function in a manner analogous to the tumor (100 U/ml), and streptomycin (100 mg/ml). The CRLE 2 and 1AMLE suppressor p53, activating a set of genes whose products are 6 mouse epithelial cell lines (Sax et al., 1995), kind gifts from Dr. required for negative regulation of cell growth. On the other Christina M. Sax (NEI, NIH, Bethesda, MD), were propagated in hand, IRF-2, which shares significant sequence similarity to minimum essential medium supplemented with 5% rabbit serum, IRF-1 within the DNA binding domain, represses IRF1- 5% fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml) and regulatable genes (Taniguchi, 1997). In contrast to IRF-1 and streptomycin (100 mg/ml). The cells were treated with murine IRF-2 that are expressed in a variety of cell types, two IRF recombinant IFNg (Life Technologies, Gaithersburg, MD) at a members, ICSBP (interferon consensus sequence binding concentration of 100 U/ml for2hat37°C, 5% CO2. Some cells protein) (Driggers et al., 1990) and LSIRF/Pip (lymphoid- were treated with medium containing the protein synthesis inhib- m specific IRF or Pu.1 interaction partner) (Eisenbeis et al., itor, cycloheximide (CHX) (Sigma, St. Louis, MO) at 35 g/ml for 30 min followed by addition of IFNg and incubation for 2 h. 1995; Matsuyama et al., 1995; Yamagata et al., 1996) are thought to be expressed exclusively in cells of macrophage and lymphocyte lineages. Constitutive expression of ICSBP Reverse-Transcribed Polymerase Chain Reaction is thought to be limited to B lymphocytes (Driggers et al., (RT-PCR) 1990; Politis et al., 1994; Nelson et al., 1996). However, Total RNA was isolated from 6-week-old WT or TR mouse lenses transcription of the ICSBP gene can be induced in T cells or from lens cell lines by a modification of the phenol–guanidine and macrophages by either IFNg or antigenic stimulation isothiocyanate single-step method (Chomczynski and Sacchi, 1987) as (Politis et al., 1994; Nelson et al., 1996). LSIRF/Pip expres- described for the TRIzol Reagent (Life Technologies). Lenses were sion is thought to be restricted to cells of the T and B carefully dissected and washed before RNA isolation to avoid any lymphocyte lineages. Mice with a null mutation for the possible contamination by other tissues. All RNA samples were ICSBP or LSIRF/Pip gene develop myelogenous leukemia- digested with RNase-free DNase 1 (Life Technologies) for 30 min and like syndrome (Holtschke et al., 1996) or developmental purified by phenol–chloroform extraction and precipitation in 0.4 M m defects in lymphocyte maturation (Mittrucker et al., 1997), LiCl. cDNA synthesis was performed at 42°C for 1 h with 10 gof total RNA, 0.3 mg oligo(dT) and 1000 U Superscript Reverse respectively. Taken together, these observations have pro- (12-16) Transcriptase II (Life Technologies) in a final volume of 50 ml. For each vided support for the notion that LSIRF/Pip and ICSBP are RNA preparation, a negative control reaction was performed without lymphoid-specific IRFs. reverse transcriptase. After purification of the cDNA by LiCl precipi- We have previously reported the generation of transgenic tation, hot-start PCR assays were performed with AmpliTaq Gold mice with targeted ectopic expression of IFNg in the lens DNA polymerase (Perkin–Elmer, Foster City, CA). Samples were under the direction of the aA-crystallin promoter (Egwuagu incubated at 95°C for 10 min to activate the AmpliTaq Gold and et al., 1994a,b). In these mice, eye development is inhibited amplification was carried out for 25 cycles at 94°C for 45 s, 63°C for and the developmental fate of cells destined to become lens 45 s, and 72°C for 1 min. This was followed by a final 10-min fiber cells is altered. In the present study, we show that extension at 72°C. All the primer pairs used for PCR amplifications abnormal lens differentiation observed in the IFNg trans- spanned at least one intron, making it possible to distinguish between amplification products derived from cDNA and those resulting from genic mice may derive in part from perturbations in the any contaminating genomic DNA templates. The sequences of the levels of IRF factors in the lens. We also show for the first PCR primers used are for mouse b-actin, 59-GTGGGCC- time that ICSBP, LSIRF/Pip, IRF-1, and IRF-2 are constitu- GCTCTAGGCACCAA-39 and 59-TCGTTGCCAATAGT- tively expressed in the normal mouse lens and that expres- GATGACTTGGC-39 (Alonso et al., 1986); for mouse G3PDH, sion of these IRFs is spatially regulated in the developing 59-TGAAGGTCGGTGTGAACGGATTTGGC-39 and 59- mouse lens. CATGTAGGCCATGAGGTCCACCAC-39 (Sabath et al., 1990); for mouse IRF-1, 59-TGAGACCCTGGCTAGAGATGC-39 and 59- ACTCAGAGAGACTGCTGCTGACGAC-39 (Pine et al., 1990); for mouse IRF-2, 59-AGCATCAACCAGGAATAGATAAAC-39 and 59- MATERIALS AND METHODS ATAGGTGTTCCGTGTCCCCAT-39 (Harada et al., 1989); for mouse ICSBP, 59-GCTGCGGCAGTGGCTGATCGAACAGATCG-39 and Animals 59-AGTGGCAGGCC TGCACTGGGCTGCTG-39 (Driggers et al., 1990).