|Molecular Vision 2004;
Received 24 November 2003 | Accepted 19 February 2004 | Published 19 February 2004
Characterization of the roles of STAT1 and STAT3 signal transduction pathways in mammalian lens development
Samuel Ebong,1 Ana B. Chepelinsky,2 Michael L.
Robinson,3 Haotian Zhao,3 Cheng-Rong Yu,1
Charles E. Egwuagu1
Laboratories of 1Immunology and 2Molecular & Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD; 3Children Research Institute, Columbus, OH
Correspondence to: Charles E. Egwuagu, MPH, PhD, Molecular Immunology Section, National Eye Institute, National Institutes of Health, Building 10, Room 10N116, 10 Center Drive, Bethesda, MD, 20892-1857; Phone: (301) 496-0049; FAX: (301) 480-3914; email: email@example.com
Purpose: IGF-1 and PDGF are implicated in regulating lens proliferation and/or providing spatial cues that restrict lens proliferation to germinative and transition zones of the lens. However, very little is known about how IGF-1- or PDGF-induced signals are transduced and coupled to gene transcription in lens cells. Therefore, we examined whether these growth factors mediate their effects in the lens through the evolutionarily conserved JAK/STAT signal transduction pathway and if STAT signaling is essential for mammalian lens development.
Methods: Expression of STAT1 and STAT3 was analyzed in mouse lens and lens epithelial cells by RT-PCR and western blot analysis. Activation of the STAT signaling pathway was examined by a combination of gel-shift, super-shift, and western blotting assays. Regulation of lens proliferation and gene transcription by STAT pathways was assessed by 3H-Thymidine incorporation or RT-PCR assays with lens explants treated or untreated with Genistein or the JAK2 and STAT3 inhibitor, AG-490. Mice with targeted deletion of STAT3 in the lens were generated by Cre/lox recombination and STAT1-/-, STAT3-/- deficient as well as normal lenses were characterized by histology.
Results: We show that PDGF and IGF1 induce proliferation in 1AMLE6 lens cells and couple their extracellular signals to gene transcription, in part through activation of STAT3 and to a lesser extent STAT1 signal transduction pathways. We further show that targeted deletion of STAT3 in E10.5 lens does not produce overt developmental lens defects. STAT1 knockout mice also exhibit a normal lens phenotype.
Conclusions: Our results showing that deletion of either STAT1 or STAT3 does not affect the normal development of the lens is surprising in view of the fact that STAT pathways are activated in developing chick or mouse lens and inappropriate activation of STAT1 pathway in the lens by ectopic lens expression of IFNγ inhibits lens differentiation and induces cataract in transgenic mice. Our data thus suggest that although STAT-signaling pathways may contribute to activation of gene transcription in the lens, it may not be essential for normal lens development or STAT proteins may be functionally redundant during lens development. However, because several growth factors and cytokines present in the lens activate STATs in mouse lens explants and 1AMLE6 lens epithelial cells, it may well be that this evolutionarily conserved signaling pathway is under stringent control in the mammalian lens. Whereas deficiency in any particular STAT pathway can be compensated for by any of the functionally redundant STAT proteins induced by a wide array of growth factors in the lens, chronic or prolonged activation of a particular STAT protein may perturb homeostatic balance in STAT-dependent growth factor signaling, culminating in pathologic lens changes.
The JAK/STAT signal transduction pathway is an evolutionarily conserved signaling mechanism in species as diverse as insects, slime molds, and mammals . JAKs (janus kinases) are comprised of four non-receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) while STATs (signal transducers and activators of transcription) consist of seven structurally and functionally related latent cytoplasmic transcription factors (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) . Signaling through the JAK/STAT pathway is initiated when a cytokine or growth factor binds to its cognate receptor on a target cell and induces a cascade of events that result in phosphorylation of the receptor and one or more JAKs . The activated receptor/JAK complex then serves as a docking site for recruitment of specific STATs, leading to phosphorylation at a tyrosine residue in the STAT c-terminal domain. Homo- and/or hetero-dimers of activated STATs translocate to the nucleus where they bind to members of the GAS (gamma-activated site) family of enhancers to activate or repress gene transcription . However, activation of a particular STAT member is highly dependent on the particular growth factor or cytokine and one or more STATs can be activated by the same agonist depending on the developmental stage or physiologic state of the cell . For example, the only factor known to activate STAT2 is IFNα/β while IL-4 selectively activates STAT1 , STAT6 , and IL-12 can activate STAT3 and STAT4 . STATs regulate diverse cellular functions including proliferation, differentiation, apoptosis, and embryonic development. The importance of STAT signaling is underscored by the diverse array of pathologic conditions that arise from disruption or aberrant activation of STATs. These include a number of human neoplastic and autoimmune diseases and developmental anomalies as well as dwarfism . Contribution of individual STAT proteins to development has been studied using mice deficient in specific STAT proteins [6-8] and these studies have revealed that while STAT family members may have overlapping functions, they also have unique physiologic roles. For example a major function of STAT1 is in regulating IFNγ activities and host immunity while STAT3 deletion results in embryonic lethality.
Growth factors and cytokines present in the ocular media and in contact with the lens have profound effects on development of the ocular lens [9-11]. Although several in vitro and in vivo studies suggest that growth factors such as FGFs, IGFs, and PDGF are involved in mechanisms of lens polarization, proliferation, and differentiation [9-11], very little is known of the mechanisms by which the signals induced by these growth factors are coupled to gene transcription in the lens cell. In vitro studies using rat lens explants suggest a potential role of mitogen-activated protein kinase/extracellular regulated kinases 1/2 (MAPK/ERK1/2)  in the regulation of lens proliferation and early morphological events associated with fiber differentiation but not in later events of lens maturation and terminal differentiation . However, it is not clear whether lens growth and differentiation are regulated in vivo by this pathway. Proteins such as STAT1 and STAT3 that are involved in the JAK/STAT signal transduction pathway are expressed in chick  and mouse [14-16] lenses and constitutive activation of STAT1 in the mouse lens induces aberrant proliferation of lens epithelia and inhibits lens differentiation in transgenic mice [14-16]. It is interesting that FGFs, IGF-1, and PDGF activate both MAPK and STAT pathways and the intensity and duration of STAT pathway has been shown to be regulated in part via phosphorylation of critical serine residues in STAT proteins by MAP kinases [1,17]. Thus, cross talk between these two major signaling pathways may form the basis of the synergistic effects of these diverse growth factors in the ocular lens.
In this study, we have restricted our focus on the JAK/STAT pathway and have examined whether IGF-1 and PDGF mediate their effects in lens through activation of STAT proteins and if this signaling pathway is essential for normal development of the mammalian lens.
Cell culture and cytokine/growth factor treatment
Murine lens epithelial cell lines CRLE2 and 1AMLE6 were propagated in Minimum Essential Medium (MEM) supplemented with 5% rabbit serum, 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). For cytokine/growth factor treatment, cells were grown to 70-80% confluency, washed, and incubated in serum-free media overnight prior to stimulation. Cells were treated with either IGF1 or PDGFaa (both from PeproTech Inc., Rocky Hill, NJ) at a concentration of 50 ng/ml each. Treatment with murine recombinant IFNγ (PeproTech) was at a concentration of 100 U/ml and control cells were cultured in serum-free media alone.
Lens organ culture
Lens organ cultures were prepared from 6 week old Balb/c mouse (Charles River Laboratories, Wilmington, MA) eyes as previously described . The lenses were cultured for 24 h and examined for integrity and transparency. Lens epithelial explants were then harvested under a dissecting microscope.
Mouse lens epithelial cell explants were incubated for 24 h in serum free M199 medium or medium supplemented with IGF-1 or PDGF in the presence or absence of Genistein (100 μM) or AG-490 (100 μM). For inhibition studies, cells were pretreated with inhibitors for 30 min and stimulated for 24 h. 3H-thymidine (0.5 μCi/well) was added to cultures after 6 h of stimulation, for a total of 18 h. Each data point represents average of quadruplet cultures.
Reverse transcribed polymerase chain reaction (RT-PCR)
Total RNA was isolated as previously described . cDNA synthesis was performed at 42 °C for 1 h with 10 μg of total RNA, 0.3 μg oligo dT(12-16), and 1,000 U Superscript Reverse Transcriptase II (both from Invitrogen, Carlsbad, CA). Hot start PCR assays were performed with AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA) and amplification was carried out for 25 or 30 cycles at 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 1 min. This was followed by a final 10 min extension at 72 °C. The sequence of the PCR primers used were: β-actin, 5'-GTG GGC CGC TCT AGG CAC CAA-3' and 5'-TCG TTG CCA ATA GTG ATG ACT TGG C-3'; STAT1, 5'-TGG GAG CAC GCT GCC TAT GAT GTC-3' and 5'-CCT TCG CTT CCA CTC CAC GAG CTC-3'; STAT3, 5'-CTT GGG CAT CAA TCC TGT GG-3' and 5'-TGC TGC TTG GTG TAT GGC TCT AC-3'; IRF-1, 5'-TGA GAC CCT GGC TAG AGA TGC-3' and 5'-ACT CAG AGA GAC TGC TGC TGA CGA C-3'; ICSBP, 5'-GCT GCG GCA GTG GCT GAT CGA ACA GAT CG-3' and 5'-AGT GGC AGG CCT GCA CTG GGC TGC TG-3'.
Western blot analysis
Mouse lenses or cultured lens cells were disrupted in RIPA buffer supplemented as previously described . Extracts were clarified by centrifugation and protein levels were determined by the BCA method as recommended by the manufacturer (Pierce, Rockford, IL). Samples were heated for 10 min at 95 °C in 1X sample buffer and electrophoresed in 4-20% Novex SDS/PAGE (Invitrogen). Gels were electro-blotted onto polyvinylidene fluoride membranes, blocked with 5% Blotto (Santa Cruz Biotech, Santa Cruz, CA) and probed with antibodies that are specific for STAT1, STAT3, β-Actin (Santa Cruz), Cre (Covance, Berkeley, CA), phosphorylated STAT1 (pSTAT1), or pSTAT3 (Cell Signaling Technology, Beverly, MA). Signals were detected using the ECL system (Amersham, Arlington Heights, IL) or the SuperSignal Chemiluminescent System (Pierce).
Electrophoretic mobility shift assay (EMSA)
Cells were washed once with ice-cold PBS and cytoplasmic protein extracts were prepared from cultured lens epithelial cells using buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM Na3VO4, 25 mM NaF, 10 mM Na-pyrophosphate, and 25 mM p-nitrophenyl guanidinobenzoate), containing 0.05% Nonidet P-40 (Sigma, St. Louis, MO). Lysate was placed on ice for 15 min and centrifuged at 4,000x g at 4 °C for 10 min to remove cytoplasmic proteins. Nuclear proteins were extracted from the pellet using high salt buffer (410 mM KCl, 25% glycerol, and 0.2 mM EDTA in buffer A). Insoluble material was removed by centrifugation at 15,000x g for 10 min. Protein concentrations were determined by the BCA method and extracts were stored at -80 °C until use. Nuclear extract (5 μg) in binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, and 0.1 mM EDTA) containing 0.14 μg/μl poly (dI-dC) were incubated on ice for 20 min. 32P-labeled double stranded DNA probe was then added and incubated for an additional 20 min on ice. Samples were electrophoresed in 5% polyacrylamide gel in 0.25% Tris-borate-EDTA buffer. The m67SIE probe with the following sequence: 5'AGC TTG TCG ACA TTT CCC GTA AAT CGT CGG-3 and 5'-GAT CCT CGA CGA TTT ACG GGA AAT GTC GAC A-3' was used. For super-shift assays, the indicated antibody was added to the binding buffer containing the nuclear extract and pre-incubated on ice for 10 min. The 32P-labeled probe was then added and the entire mixture incubated for an additional 20 min on ice before electrophoresis. Gel-shift grade anti-mouse STAT1 and STAT3 (Santa Cruz) polyclonal antibodies were used.
Generation of mice with disrupted STAT3 gene expression in lens
Generation of STAT3flox/flox mice have previously been described . In these mice, exons 16 to 21 (encoding the SH2 binding domain) in the STAT3 gene is flanked by loxP sites (floxed) and these sites serve as substrate for Cre-mediated recombination. The MLR10 mice are a transgenic strain exhibiting Cre expression in both the lens epithelium and lens fibers under the transcriptional control of an αA-crystallin promoter modified by the insertion of a Pax6 consensus binding element (unpublished). The floxed STAT3 DNA fragment is deleted in offspring of the cross between STAT3flox/flox and MLR10-Cre transgenic mice. The mice with a specific deletion of the STAT3 gene in the lens were identified by genotype analysis using the following allele-specific STAT3 PCR primers: primer1: 5'-GAA GGC AGG TCT CTC TGG TGC TTC-3'; primer 2, 5'-GCT GCC AAC AGC CAC TGC CCC AG-3'; and primer 3: 5'-CAG AAC CAG GCG GCT CGT GTG CG-3' and Cre-specific primers (5'-GCA TTC CAG CTG CTG ACG GT-3' and 5'-CAG CCC GGA CCG ACG ATG AAG-3'). All animal studies were in accordance with NIH institutional guidelines and provisions of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Eyes from 3 month old C57BL/6 wild type (WT), STAT1-/- mice or C56BL/6 mice with a targeted deletion of STAT3 in the lens were fixed for embedding in paraffin wax. Eye sections were deparaffinized and stained with hematoxylin and eosin as previously described .
STAT and JAK proteins are constitutively expressed in mouse lens cells
IGF1 and PDGF have been shown to activate STAT1 and STAT3 in hematopoietic cells [5,19,20] and expression of these STAT proteins has been detected in the embryonic chick lens . To extend these findings, we prepared whole cell extracts and/or RNA from 6-week old mouse lens or cultured lens cells, performed RT-PCR and/or western blot analyses and examined whether STAT1 and STAT3 are also expressed in the mammalian lens. We show here that STAT1 and STAT3 mRNA (Figure 1A) and proteins (Figure 1B,C) are expressed in Balb/c and FVB/N mouse lenses as well as two lens epithelial cell lines examined. The constitutive expression of STAT1 and STAT3 in the mouse  and chicken  lenses thus provides suggestive evidence that growth factor and cytokine responses in lens may be mediated in part, through activation of STAT1 and STAT3 signaling pathways.
STAT1 and STAT3 signaling pathways are activated in mouse lens
STAT proteins are latent cytoplasmic proteins that require activation by tyrosine phosphorylation to function as signal transducers. It is therefore necessary to demonstrate that the STAT proteins expressed by lens cells are tyrosine-phosphorylated and interact with STAT-binding DNA motifs in the promoter of STAT-inducible genes. Quiescent 1AMLE6 lens epithelial cells were therefore stimulated with IGF-1, PDGFaa or IFNγ and nuclear extracts prepared from the treated or control cells were analyzed by EMSA using the c-fos SIE (c-sis element) GAS probe that binds most activated STATs with high affinity [22,23]. Previous work indicated induction of three DNA binding complexes after IFN stimulation of hematopoietic cells; an upper complex composed of STAT3 homodimers (A), a lower complex comprised of STAT1 homodimers (C) and a middle complex of STAT1-STAT3 heterodimers (B) . Formation of three protein:DNA complexes following treatment of the lens cells with IFNγ (Figure 2A, arrows 1-3) confirms the fidelity of the probe 23 and indicates that lens cells activate JAK/STAT signal transduction pathways in response to cytokine signals. In contrast to IFNγ, IGF-1 or PDGF treatment results in formation of a complex comprising predominantly of STAT3 homodimers (Figure 2A, arrow 1). STAT1 binding activity is not detectable, suggesting that IGF-1 and PDGF preferentially activate STAT3 homodimers in lens cells. In supershift analysis, the addition of STAT3 antibody to the binding reactions supershifted the upper (STAT3/STAT3) complex while STAT1 antibody, or normal mouse serum, could not supershift the complex (Figure 2B), providing direct evidence that IGF-1 and PDGF-induced STAT signaling is preferentially mediated by pSTAT3 homodimers. In some cell types, STAT activation is not detectable by EMSA due to low affinity binding to the probe or because binding to the promoter is indirect, through interaction with other STATs or transcription factors already bound to the promoter [24,25]. The phosphorylation state of STAT1 and STAT3 was therefore examined directly in 1AMLE6 lens epithelial cells stimulated with IGF-1 or PDGFaa. By western blot analyses we show an increase in STAT3 phosphorylation following 15-min treatments with IGF-1 or PDGFaa (Figure 2C) consistent with the induction of STAT3-DNA binding activity (see Figure 2A,B). Interestingly, we also detected activation of STAT1 (Figure 2C). However, in contrast to pSTAT3 that is readily detected by the standard ECL chemilumnescence method, detection of pSTAT1 required the highly sensitive SuperSignal chemilumnescence substrate (see Material section), reflecting the relatively lower level of activated STAT1 (Figure 2C). The blots were stripped and reblotted with β-Actin antibody to indicate that the increase in tyrosine phosphorylation detected following growth factor stimulation was not due to loading unequal amounts of protein extracts.
Growth factor-induced lens proliferation and gene transcription are mediated through activation of STAT1 signaling pathways
To examine whether the effects of IGF-1 or PDGF on lens proliferation is mediated in part through activation of STAT pathways, we stimulated lens epithelial cell explants with IGF-1 or PDGF and examined whether lens proliferation, phosphorylation of STAT1 protein, or gene transcription in lens cells is activated by tyrosine kinase or STAT inhibitors. The proliferation assays in Figure 3A show that IGF-1 or PDGF induces significant increases in cell proliferation. We further show that addition of the tyrosine kinase inhibitor, Genistein, a broad spectrum inhibitor of tyrosine-phosphorylated receptors and non-receptor tyrosine kinases, inhibits the proliferative response (Figure 3A). Similarly, AG-490, a specific inhibitor of JAK kinases and STAT pathways [26,27], inhibits lens proliferation induced by IGF-1, as well as IGF-1-induced activation of STAT1 (Figure 3B) and STAT3 (data not shown) in lens cells. We then examined whether IGF-1 or PDGF is able to activate two genes whose expression is dependent on STAT activation. The IFN regulatory factor (IRF) family of transcription factors are immediate targets of STAT proteins whose expression requires STAT activation but not de novo protein synthesis. Two members of the IRF family, IRF-1 and ICSBP (IRF-8), are expressed in the lens. ICSBP is detected primarily in the lens epithelia and bow region while IRF-1 is expressed in the epithelia and lens fibers [14,28]. We show in Figure 3C that both IGF-1 and PDGF upregulates expression of ICSBP. However, these growth factors appear to have marginal effects on IRF-1 gene transcription in the 1AMLE6 lens epithelial cells, suggesting that STAT-dependent transcriptional activities of IGF-1 and PDGF targets the IRF member whose expression is spatially restricted to the lens epithelia. It is of note that although β-actin transcripts were readily detected after 25 cycles of amplification, 35 cycles was optimal for detection of IRF-1 transcripts in mouse lens explants. However, under these RT-PCR conditions we are able to detect IRF-1 transcripts in αTN4-1 cells after 25 cycles of amplification (data not shown) and this is consistent with a recent report by Awasthi and Wagner . Although the latter results suggest that the steady state level of IRF-1 is higher in SV40 T-Antigen-transformed αTN4-1 lens cells, they underscore the fact that IRF-1 is constitutively expressed in lens cells and are consistent with a previous report of IRF-1 expression in the mouse lens . Thus, activation of STAT-dependent gene transcription by IGF-1 and PDGF, taken together with inhibition of IGF-1/PDGF-induced lens proliferation by AG-490, suggest that regulation of cellular processes of the lens by growth factors is mediated in part through JAK/STAT pathways.
Targeted deletion of STAT3 in the lens
Among the STATs, STAT3 appears to be the predominant one used by IGF-1 and PDGF in the lens. We therefore sought to elucidate its physiologic role in lens development. However, STAT3 deletion leads to embryonic lethality in the mouse . We therefore employed the Cre-lox recombination system to generate mice with lens-specific deletion of STAT3. The targeting construct and the experimental strategy is shown in Figure 4A,B. We first generated the MLR10 mice expressing Cre recombinase under the control of a modified αA-crystallin promoter which leads to expression of Cre in both the lens epithelium and lens fiber cells (unpublished). The MLR10 mice were then mated with transgenic mice expressing STAT3 gene flanked by loxP sites. The resulting double heterozygous mice were inter-crossed to obtain mice with a targeted deletion of STAT3 in the lens. These mice are referred to as STAT3Δ/δ. Genomic DNA from the lens and tail were analyzed using PCR primers described in Methods and the expected sizes of the PCR products correlate with the genotypes shown in Figure 4C. PCR analysis of lens from STAT3+/+ mice produces a 140 bp band that characterizes the WT genotype while the heterozygous mice (STAT3Δ/f) contain a 260 bp from the floxed allele as well as the 140 bp band from the wild type allele. A 370 bp band is detected in lens DNA from the homozygous (STAT3Δ/Δ) or heterozygous transgenic mice. The PCR results are confirmed by western blot analysis showing no STAT3 expression in STAT3Δ/Δ mouse lens (Figure 4D).
Lens-specific deletion of STAT3 did not alter the lens development program
To elucidate the role of STAT signaling pathways in lens development, lenses from wild type, STAT1-/- and STAT3Δ/Δ mice were analyzed by histology. Hematoxylin and eosin staining of eye sections revealed a normal eye morphology with no apparent effects on the lens, cornea, iris, choroids, and the retina (Figure 5). No developmental defects are observed in mice that are 1 year old.
Growth factors transduce signals that induce transcription of genes which allow cells to adapt to subtle changes in their environment. Although the JAK/STAT signal transduction pathway activates transcription of a wide array of genes that regulate proliferation, differentiation and survival of several cell types, the role of this evolutionarily conserved pathway in the mammalian lens is not well characterized. In this study we have investigated whether activation of STAT signaling pathway in the lens by growth factors play a role in regulating the growth of the mammalian lens. We show that IGF-1 and PDGF, two growth factors implicated in inducing proliferation of lens cells [9-11], mediate their effects in 1AMLE6 lens epithelial cells in part through activation of STAT3 and to a lesser extent STAT1. We further show that activation of STAT signaling and STAT-dependent gene transcription by IGF-1 and PDGF are inhibited by inhibitors of JAK kinases and STAT3, providing a direct link between STAT signaling pathways and regulation of essential cellular processes in the vertebrate lens.
A number of in vitro studies have shown that IGF-1 or PDGF stimulates proliferation of lens explants [11,31]. However, the underlying mechanism and extent to which these factors participate in stimulating lens proliferation in vivo is largely unknown. Receptors for IGF-1 and PDGF are ligand-activated tyrosine kinases and they associate with STAT proteins either in the presence or absence of the cognate agonist . In contrast to cytokines that require obligatory phosphorylation of STATs by JAKs to initiate the signaling cascade, STATs are directly phosphorylated by IGF-1 or PDGF receptors following ligand binding . Several factors suggest that IGF-1 and PDGF mediate their effects in the lens in part through STAT pathways. These include; (i) inhibition of IGF-1/PDGF-induced proliferation by AG-490 (Figure 3A), (ii) activation of gene transcription in the lens by IGF-1 and PDGF (Figure 3C), and (iii) expression of STAT3/STAT1 proteins in chick  and mouse [14-16] lenses and inhibition of IGF-1-induced STAT1 activation by AG-490 (Figure 3B). Thus, growth factor-induced activation of STAT pathways may play important physiologic roles in the lens.
Embryonic lethality of STAT3-deficient mice suggest that STAT3 regulates critical pathways in development and analyses of tissue-specific STAT3-deficient mice have revealed that STAT3 plays a crucial role in a variety of biological functions including cell growth, anti-apoptosis, apoptosis, and cell motility [6,33]. To investigate the role of STATs in lens development, we utilized a Cre-loxP recombination strategy to generate a transgenic mouse line with targeted deletion of STAT3 in the lens (Figure 4). Analysis of the STAT3Δ/Δ lens and lenses from STAT1 null mice allowed assessment of STAT3 and STAT1 in the development of the lens. Interestingly, and in contrast to developmental defects resulting from targeted deletion of STAT3  or STAT1  in hematopoietic cells, histological analysis of the STAT3 or STAT1 null lenses revealed an apparently normal mouse lens phenotype in both cases. However, this result should be interpreted with caution in view of the fact that the promoter use to direct lens-specific cre expression and functional deletion of STAT3 in the lens is activated only after day 10.5 of embryonic development. Because expression of STAT3 is detected as early as E4.5 during early post-implantation stages of murine development , we cannot rule out the possibility that STAT3 may be essential at very early stages of lens development and that deletion of STAT3 at E10.5 may be inconsequential to normal lens development. It is also of note that tissue-specific deletion of STATs does not always produce abnormalities in every cell type or developmental pathway of a non-hematopoietic tissue. For example, the requirement of STAT3 pathway is selective in mutant mice with keratinocyte-specific ablation of STAT3 . While the STAT3 pathway is required for keratinocyte differentiation  and regulation of keratinocyte growth factor signaling  it is not required for skin morphogenesis. Thus, similar to the skin, STAT signaling pathways may differentially regulate cellular processes in the lens but are not necessarily indispensable.
In context to the physiologic role of STATs in the lens, our data showing activation of STAT1 and STAT3 in 1AMLE6 lens epithelial cells by IGF-1 and PDGF, caused us to examine the possibility that these polypeptide growth factors may function to control expression of genes whose expression is spatially restricted to the lens epithelia. In fact, a recent report has suggested that a major function of IGFs in the lens is to provide spatial cues that restrict proliferation of lens epithelial cells to germinative and transition zones of the lens . Members of the IRF (interferon regulatory factor) family of transcription factors are important cell cycle regulators  and their activation in the lens by STAT1  makes them potential targets of IGF-1 or PDGF regulation in the lens. The most abundant IRFs in the lens are IRF-1 and IRF-2.14 IRF-2 is a potent inducer of cell proliferation and it is oncogenic in many cell types while IRF-1 is a potent growth inhibitor and a tumor suppressor that negatively regulates the transcriptional activities of IRF-2 . In most cell types, the ratio of IRF-1:IRF-2 is fixed and whether the cell divides or differentiates depends in part on a net increase or decrease of IRF-1 relative to IRF-2 . Interestingly, in the mouse lens expression of IRF-1 and IRF-2 is spatially regulated, with IRF-1 expression restricted to the lens fiber while IRF-2 is present in both the anterior epithelia and fibers . Another IRF member whose expression is spatially regulated in the lens is ICSBP (IRF-8) . In contrast to IRF-1 and IRF-2 that are ubiquitously expressed, ICSBP expression has been detected only in lymphocytes and the mouse lens [28,42]. In the lens its expression is restricted to the anterior epithelia and bow region of the lens [14,28]. In this study, we show that expression of ICSBP is upregulated following stimulation of lens epithelial explants by either IGF-1 or PDGF. However, unlike ICSBP, neither IGF-1 nor PDGF is able to upregulate expression of IRF-1 (Figure 3C). The selective activation of the transcription of ICSBP but not the IRF-1 gene suggests that the effect of by IGF-1 or PDGF on transcription of IRF family genes is restricted to the proliferating epithelia of the mammalian lens. This observation is also in concert with the notion that IGF-1 functions to regulate the spatial pattern of gene expression in the lens through its effects on expression of critical regulatory proteins requested in the lens epithelia and bow region.
In summary, the data communicated in this report show that PDGF and IGF1 mediate their effects on cellular proliferation and activation of gene transcription in lens, in part, through activation of STAT3 and to a lesser extent STAT1 signal transduction pathways. However, deleting STAT3 in the lens after day 10.5 in embryonic development does not result in overt developmental lens defects. Similarly, STAT1 deletion does not affect lens development suggesting that although STAT pathways may regulate cellular processes in the lens, they are not essential to the normal lens developmental program. These results are particularly surprising in view of the fact that activation of STAT proteins has been detected in the chick lens  and the developing mouse eye  and constitutive activation of STAT1 in the mouse lens by ectopic lens expression of IFNγ results in the inhibition of lens differentiation and cataract in transgenic mice [14,15]. It may well be that because several growth factors and cytokines present in the lens activate STATs, this evolutionarily conserved signaling pathway may be under stringent control. Whereas a deficiency in any particular STAT pathway can be compensated by functional redundancy of the many mammalian STAT proteins induced by many agonists, chronic or prolonged activation of a particular STAT protein may perturb the homeostatic balance in STAT-dependent growth factor signaling, culminating in pathologic changes of lens.
The authors thank Drs. David Levy and Regina Raz of NYU School of Medicine (NY) for kindly providing the STAT1-/- and STAT3 floxed mice.
1. Darnell JE Jr. STATs and gene regulation. Science 1997; 277:1630-5.
2. Chang TL, Peng X, Fu XY. Interleukin-4 mediates cell growth inhibition through activation of Stat1. J Biol Chem 2000; 275:10212-7.
3. Kaplan MH, Wurster AL, Smiley ST, Grusby MJ. Stat6-dependent and -independent pathways for IL-4 production. J Immunol 1999; 163:6536-40.
4. Jacobson NG, Szabo SJ, Weber-Nordt RM, Zhong Z, Schreiber RD, Darnell JE Jr, Murphy KM. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J Exp Med 1995; 181:1755-62.
5. Bromberg JF. Activation of STAT proteins and growth control. Bioessays 2001; 23:161-9.
6. Akira S. Functional roles of STAT family proteins: lessons from knockout mice. Stem Cells 1999; 17:138-46.
7. Raz R, Lee CK, Cannizzaro LA, d'Eustachio P, Levy DE. Essential role of STAT3 for embryonic stem cell pluripotency. Proc Natl Acad Sci U S A 1999; 96:2846-51.
8. Kira M, Sano S, Takagi S, Yoshikawa K, Takeda J, Itami S. STAT3 deficiency in keratinocytes leads to compromised cell migration through hyperphosphorylation of p130(cas). J Biol Chem 2002; 277:12931-6.
9. Lang RA. Which factors stimulate lens fiber cell differentiation in vivo?. Invest Ophthalmol Vis Sci 1999; 40:3075-8.
10. Klok E, Lubsen NH, Chamberlain CG, McAvoy JW. Induction and maintenance of differentiation of rat lens epithelium by FGF-2, insulin and IGF-1. Exp Eye Res 1998; 67:425-31.
11. Kok A, Lovicu FJ, Chamberlain CG, McAvoy JW. Influence of platelet-derived growth factor on lens epithelial cell proliferation and differentiation. Growth Factors 2002; 20:27-34.
12. Lovicu FJ, McAvoy JW. FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling. Development 2001; 128:5075-84.
13. Potts JD, Kornacker S, Beebe DC. Activation of the Jak-STAT-signaling pathway in embryonic lens cells. Dev Biol 1998; 204:277-92.
14. Li W, Nagineni CN, Efiok B, Chepelinsky AB, Egwuagu CE. Interferon regulatory transcription factors are constitutively expressed and spatially regulated in the mouse lens. Dev Biol 1999; 210:44-55.
15. Egwuagu CE, Sztein J, Chan CC, Mahdi R, Nussenblatt RB, Chepelinsky AB. gamma Interferon expression disrupts lens and retinal differentiation in transgenic mice. Dev Biol 1994; 166:557-68.
16. Egwuagu CE, Sztein J, Chan CC, Reid W, Mahdi R, Nussenblatt RB, Chepelinsky AB. Ectopic expression of gamma interferon in the eyes of transgenic mice induces ocular pathology and MHC class II gene expression. Invest Ophthalmol Vis Sci 1994; 35:332-41.
17. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994; 264:1415-21.
18. Sun JK, Iwata T, Zigler JS Jr, Carper DA. Differential gene expression in male and female rat lenses undergoing cataract induction by transforming growth factor-beta (TGF-beta). Exp Eye Res 2000; 70:169-81.
19. Levy DE, Darnell JE Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 2002; 3:651-62.
20. Bromberg J, Darnell JE Jr. The role of STATs in transcriptional control and their impact on cellular function. Oncogene 2000; 19:2468-73.
21. Zhang SS, Wei JY, Li C, Barnstable CJ, Fu XY. Expression and activation of STAT proteins during mouse retina development. Exp Eye Res 2003; 76:421-31.
22. Vignais ML, Sadowski HB, Watling D, Rogers NC, Gilman M. Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins. Mol Cell Biol 1996; 16:1759-69.
23. Yu CR, Lin JX, Fink DW, Akira S, Bloom ET, Yamauchi A. Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-alpha. J Immunol 1996; 157:126-37.
24. Kanno Y, Kozak CA, Schindler C, Driggers PH, Ennist DL, Gleason SL, Darnell JE Jr, Ozato K. The genomic structure of the murine ICSBP gene reveals the presence of the gamma interferon-responsive element, to which an ISGF3 alpha subunit (or similar) molecule binds. Mol Cell Biol 1993; 13:3951-63.
25. Driggers PH, Ennist DL, Gleason SL, Mak WH, Marks MS, Levi BZ, Flanagan JR, Appella E, Ozato K. An interferon gamma-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes. Proc Natl Acad Sci U S A 1990; 87:3743-7.
26. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, Roifman CM. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 1996; 379:645-8.
27. Levitzki A, Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 1995; 267:1782-8.
28. Li W, Nagineni CN, Ge H, Efiok B, Chepelinsky AB, Egwuagu CE. Interferon consensus sequence-binding protein is constitutively expressed and differentially regulated in the ocular lens. J Biol Chem 1999; 274:9686-91.
29. Awasthi N, Wagner BJ. Interferon-gamma induces apoptosis of lens alphaTN4-1 cells and proteasome inhibition has an antiapoptotic effect. Invest Ophthalmol Vis Sci 2004; 45:222-9.
30. Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, Kishimoto T, Akira S. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci U S A 1997; 94:3801-4.
31. Liu J, Chamberlain CG, McAvoy JW. IGF enhancement of FGF-induced fibre differentiation and DNA synthesis in lens explants. Exp Eye Res 1996; 63:621-9.
32. Wang YZ, Wharton W, Garcia R, Kraker A, Jove R, Pledger WJ. Activation of Stat3 preassembled with platelet-derived growth factor beta receptors requires Src kinase activity. Oncogene 2000; 19:2075-85.
33. Akira S. Roles of STAT3 defined by tissue-specific gene targeting. Oncogene 2000; 19:2607-11.
34. Duncan SA, Zhong Z, Wen Z, Darnell JE Jr. STAT signaling is active during early mammalian development. Dev Dyn 1997; 208:190-8.
35. Sano S, Itami S, Takeda K, Tarutani M, Yamaguchi Y, Miura H, Yoshikawa K, Akira S, Takeda J. Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis. EMBO J 1999; 18:4657-68.
36. Hauser PJ, Agrawal D, Hackney J, Pledger WJ. STAT3 activation accompanies keratinocyte differentiation. Cell Growth Differ 1998; 9:847-55.
37. Liang Q, Mohan RR, Chen L, Wilson SE. Signaling by HGF and KGF in corneal epithelial cells: Ras/MAP kinase and Jak-STAT pathways. Invest Ophthalmol Vis Sci 1998; 39:1329-38.
38. Shirke S, Faber SC, Hallem E, Makarenkova HP, Robinson ML, Overbeek PA, Lang RA. Misexpression of IGF-I in the mouse lens expands the transitional zone and perturbs lens polarization. Mech Dev 2001; 101:167-74.
39. Taniguchi T, Harada H, Lamphier M. Regulation of the interferon system and cell growth by the IRF transcription factors. J Cancer Res Clin Oncol 1995; 121:516-20.
40. Harada H, Kitagawa M, Tanaka N, Yamamoto H, Harada K, Ishihara M, Taniguchi T. Anti-oncogenic and oncogenic potentials of interferon regulatory factors-1 and -2. Science 1993; 259:971-4.
41. Vaughan PS, van Wijnen AJ, Stein JL, Stein GS. Interferon regulatory factors: growth control and histone gene regulation--it's not just interferon anymore. J Mol Med 1997; 75:348-59.
42. Tamura T, Ozato K. ICSBP/IRF-8: its regulatory roles in the development of myeloid cells. J Interferon Cytokine Res 2002; 22:145-52.