Activation of STAT signaling pathways and induction of suppressors of cytokine signaling (SOCS) proteins in mammalian lens by growth factors

PURPOSE. This study was conducted to examine whether the effects of growth factors are mediated in the lens by Janus kinase/signal transducers and activators of transcription (JAK/ STAT) pathways and whether they induce expression of suppressors of cytokine signaling (SOCS), a novel family of feedback regulators of cytokine and growth factor activities. METHODS. STAT activation and SOCS expression were analyzed in transgenic or wild-type mouse lens and lens epithelial cells stimulated with growth factors by immunohistochemistry, RT-PCR, Northern, Western, proliferation, or transient reporter assays. RESULTS. STATs were constitutively expressed at low levels and activated by insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)-aa, and FGF-1 or -2 in the lens. The Intensity of STAT signaling increased at high FGF-2 concentration and FGFs act in synergy with IGF-1 or PDGFaa to enhance STAT signaling and SOCS expression. Growth factor-induced proliferation of lens cells is inhibited by AG-490, a specific inhibitor of JAK2/STAT3. CONCLUSIONS. This is the first report that FGFs activate STAT pathways in the lens and that SOCS proteins are constitutively expressed and upregulated by growth factors in this tissue. Physiological relevance of STAT pathways in the lens is underscored by inhibition of lens cell proliferation by inhibitors of JAK/STAT pathways and by the aberrant proliferation of lens epithelium in the posterior pole of transgenic mice with constitutively activated STAT1 in the lens. Common activation of STAT pathways by FGF-1, FGF-2, IGF-1, or PDGFaa and their synergistic activation of STATs and SOCS in lens cells suggest that activities and crosstalk between these factors are sensitive to the steady state levels of activated STATs in the lens and may be under feedback regulation by SOCS family proteins. (Invest Ophthalmol Vis Sci

A Novel Alternative Spliced Variant of the Transcription Factor AP2α Is Expressed in the Murine Ocular Lens

AbstractThe AP2α gene encodes a transcription factor containing a basic, helix–span–helix DNA-binding/dimerization domain, which is developmentally regulated and retinoic acid inducible. Recent reports about AP2α null mice indicate that AP2α plays an important role in embryogenesis, especially in craniofacial development and midline fusion. Ocular development is also affected in these null mice. As AP2α may be involved in transcriptional regulation in the lens, it was important to examine the expression of the AP2α gene in the lens. Four AP2α mRNA variants have been previously isolated from whole mouse embryos. Variants 1, 3, and 4 are transcriptional activators that are transcribed from different promoters and variant 2 is a repressor lacking the activation domain encoded by exon 2. Usingin situ-PCR, we found that AP2α is expressed in the lens epithelia but not in the lens fibers. RT-PCR analysis of lens mRNA with amplimers specific for each variant revealed that AP2α variants 1, 2, and 3 are expressed in newborn mouse lenses. However, variant 4 is not expressed in the lens. In this report we characterized a novel isoform, which we named variant 5, expressed in the lens and kidney. Variant 5, which is generated by alternative splicing, may function as a repressor due to the partial deletion of the proline-rich transactivation domain encoded by exon 2. This is the first molecular characterization of AP2α gene expression in the lens. Our results indicate that two activator and two repressor AP2α isoforms may play a role in regulating gene expression in the lens

Interferon Regulatory Transcription Factors Are Constitutively Expressed and Spatially Regulated in the Mouse Lens

AbstractInterferon 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 γ 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

PKC inhibitor prevents MIP membrane localization in differentiating lens explants

MIP immunofluorescence (red) shows MIP localization in the cell membrane (; indicated with large arrows) of rat lens epithelial explants cultured in 100 ng/ml FGF-2 and 0.2% DMSO for 72 h (control; as described in Methods; and ). MIP is retained in the cytosolic compartment (; large arrows point to cell with strong MIP signal in the cytoplasmic compartment; numerous MIP-positive vesicles were also seen, small arrow) in rat lens epithelial explants cultured for 72 h (100 ng/ml FGF-2 in the presence of 4 μM Go6976 plus 0.2% DMSO as described in Methods; panels and ). and show the merged images of MIP immunofluorescence ( and , respectively) with the ones for green nuclei staining. Green fluorescence indicates nuclei stained with SYTOX green dye. Scale bars represent 20 μm. and show one of the z stack confocal images of MIP immunofluorescence cell distribution through the thickness of the cultured explant in the control () and Go6976 () experiments, respectively.<p><b>Copyright information:</b></p><p>Taken from "PKC putative phosphorylation site Ser is required for MIP/AQP0 translocation to the plasma membrane"</p><p></p><p>Molecular Vision 2008;14():1006-1014.</p><p>Published online 29 May 2008</p><p>PMCID:PMC2405813.</p><p></p

Mutation of PKC putative phosphorylation site (Ser) prevents MIP translocation from the trans-Golgi network to the plasma membrane

Immunofluorescence of RK13 cells transfected with pCMV- MIP (WT; -) or pCMV-MIP Ala (S235A; -) for 72 h is shown. and as well as and show the merged images of MIP red immunofluorescence with the corresponding images of their DAPI nuclear staining. and as well as and show the trans-Golgi network marker 38K (TGN) green immunofluorescence. and show the merged images of MIP immunofluorescence and DAPI nuclear staining with their respective TGN green immunofluorescence ( and ; and ; and ; and , respectively). Spatial quantification was performed along a path across the plasma membrane, indicated by a white line with prominent end points in the merged images (, , , and ). Red and green fluorescence was quantified separately and plotted as a function of distance along the path (, , , and ). Blue broken lines in the spatial quantification graphs indicate the approximate location of the plasma membrane (except the left line in , which corresponds to the TGN region in ). Note that WT MIP and TGN vesicles colocalize at the plasma membrane (peaks are indicated with blue lines in and right peak in ). MIP Ala mutant (S235A) does not colocalize with TGN vesicles at the plasma membrane (blue lines; and ). and show colocalization (yellow) of WT MIP (red immunofluorescence) and TGN 38K (green immunofluorescence) in the cytoplasmic compartment in addition to the localization of WT MIP in the plasma membrane. and show MIP Ala mutant punctate distribution in the cytosolic compartment (red immunofluorescence) and colocalization (yellow) with trans-Golgi network 38K (green) in and . and as well as and show cell images from either duplicate experiments or in different fields of the same cell culture of WT or MIP Ala mutant (S235A), respectively. Scale bars represent 10 μm. Note that three cells in , , and (one cell at the right side and two cells in the upper part of the panels) that did not uptake the transfected MIP expression plasmid served as negative controls. They show no red immunofluorescence in contrast to two transfected cells showing the red immunofluorescence. All the cells in the panel (transfected and non-transfected) show green immunofluorescence for TGN38.<p><b>Copyright information:</b></p><p>Taken from "PKC putative phosphorylation site Ser is required for MIP/AQP0 translocation to the plasma membrane"</p><p></p><p>Molecular Vision 2008;14():1006-1014.</p><p>Published online 29 May 2008</p><p>PMCID:PMC2405813.</p><p></p