Effect of knocking down the insulin receptor on mouse rod responses.

Previous experiments have shown that the insulin receptor (IR) is expressed in mammalian rods and contributes to the protection of photoreceptors during bright-light exposure. The role of the insulin receptor in the production of the light response is however unknown. We have used suction-electrode recording to examine the responses of rods after conditionally knocking down the insulin receptor. Our results show that these IR knock-down rods have an accelerated decay of the light response and a small decrease in sensitivity by comparison to littermate WT rods. Our results indicate that the insulin receptor may have some role in controlling the rate of rod response decay, but they exclude a major role of the insulin receptor pathway in phototransduction

Protein tyrosine phosphatase-1B regulates the tyrosine phosphorylation of the adapter Grb2-associated binder 1 (Gab1) in the retina

Abstract Background Gab1 (Grb2-associated binder 1) is a key coordinator that belongs to the insulin receptor substrate-1 like family of adaptor molecules and is tyrosine phosphorylated in response to various growth factors, cytokines, and numerous other molecules. Tyrosine phosphorylated Gab1 is able to recruit a number of signaling effectors including PI3K, SHP2 and PLC-γ. In this study, we characterized the localization and regulation of tyrosine phosphorylation of Gab1 in the retina. Results Our immuno localization studies suggest that Gab1 is expressed in rod photoreceptor inner segments. We found that hydrogen peroxide activates the tyrosine phosphorylation of Gab1 ex vivo and hydrogen peroxide has been shown to inhibit the protein tyrosine phosphatase PTP1B activity. We found a stable association between the D181A substrate trap mutant of PTP1B and Gab1. Our studies suggest that PTP1B interacts with Gab1 through Tyrosine 83 and this residue may be the major PTP1B target residue on Gab1. We also found that Gab1 undergoes a light-dependent tyrosine phosphorylation and PTP1B regulates the phosphorylation state of Gab1. Consistent with these observations, we found an enhanced Gab1 tyrosine phosphorylation in PTP1B deficient mice and also in retinas treated ex vivo with a PTP1B specific allosteric inhibitor. Conclusions Our laboratory has previously reported that retinas deficient of PTP1B are resistant to light damage compared to wild type mice. Since Gab1 is negatively regulated by PTP1B, a part of the retinal neuroprotective effect we have observed previously in PTP1B deficient mice could be contributed by Gab1 as well. In summary, our data suggest that PTP1B regulates the phosphorylation state of retinal Gab1 in vivo

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression

<p>Abstract</p> <p>Background</p> <p>Human adenovirus type 19 (HAdV-19) is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of p38 mitogen-activated protein kinase (MAPK) in HAdV-19 infection, with particular attention to the role of p38 MAPK in the transcriptional control of interleukin-8 (IL-8), a chemokine previously shown to be central to the initiation of adenovirus keratitis.</p> <p>Results</p> <p>We found that infection of corneal cells with HAdV-19 led to activation of p38 MAPK and its downstream targets, HSP-27 and ATF-2, within 15 to 30 minutes post-infection. Infection also induced phosphorylation of IκB and NFκB in a p38 MAPK-dependent fashion. Furthermore, HAdV-19 induced an interaction between p38 MAPK and NFκB-p65, followed by nuclear translocation of activated NFκB-p65 and its binding to the IL-8 promoter. The interaction between p38 MAPK and NFκB-p65 was inhibited in concentration-dependent fashion by SB203580, a chemical inhibitor of p38 MAPK, but not by SP600125, an inhibitor of JNK – another MAPK implicated in chemokine expression by HAdV-19 infected cells. IL-8 gene expression in HAdV-19 infection was significantly reduced in the presence of sequence-specific p38 MAPK siRNA but not control siRNA.</p> <p>Conclusion</p> <p>These results provide the first direct evidence for transcriptional regulation of IL-8 in HAdV-19 infected cells through the activation of the p38 MAPK signaling pathway. The p38 MAPK pathway may play a biologically important role in regulation of IL-8 gene expression in the adenovirus-infected cornea.</p

Modulation of Mouse Rod Photoreceptor Responses by Grb14 Protein*

Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14(-/-) mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τREC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14(-/-) mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τD, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-4

against phospho- and total p38 MAPK reveals phosphorylation in HAdV-19 infected cells, reduced in the presence of the Src inhibitor PP2 (10 μM). Densitometry values for the phosphorylated protein (normalized to the corresponding total protein) are shown above each lane. (B) An p38 MAPK assay performed at 15 and 30 min after infection shows increased phosphorylation of the ATF-2 substrate signifying p38 MAPK activity upon HAdV-19 infection of keratocytes. (C) Densitometric quantification from three experiments of the phosphorylated ATF-2 band in the p38 MAPK assay revealed increased activity in virus infected keratocytes at both 15 and 30 min post-infection (p = 0.0012 and p = 0.0025, respectively). (D & E) Western blot analysis using phospho- and total antibodies against HSP27 and ATF-2 respectively, reveals increases in phosphorylation in HAdV-19 infected cells that was absent in the presence of PP2.<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-3

Otein expression as compared to control siRNA in both mock and virus infected cells. (B) IL-8 production in virus infected keratocytes was reduced to mock infected levels by p38 MAPK-specific siRNA but not by control siRNA (*p = 0.0009).<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-0

against phospho- and total p38 MAPK reveals phosphorylation in HAdV-19 infected cells, reduced in the presence of the Src inhibitor PP2 (10 μM). Densitometry values for the phosphorylated protein (normalized to the corresponding total protein) are shown above each lane. (B) An p38 MAPK assay performed at 15 and 30 min after infection shows increased phosphorylation of the ATF-2 substrate signifying p38 MAPK activity upon HAdV-19 infection of keratocytes. (C) Densitometric quantification from three experiments of the phosphorylated ATF-2 band in the p38 MAPK assay revealed increased activity in virus infected keratocytes at both 15 and 30 min post-infection (p = 0.0012 and p = 0.0025, respectively). (D & E) Western blot analysis using phospho- and total antibodies against HSP27 and ATF-2 respectively, reveals increases in phosphorylation in HAdV-19 infected cells that was absent in the presence of PP2.<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-2

Duced by pre-treatment with SB203580 (SB: 2, 5, 10, or 20 μM). GAPDH mRNA levels are shown as a control. (B) At 4 hr after infection, IL-8 protein was also significantly increased by ELISA, and this increase was reduced in dose-dependent fashion by SB203580 (p = 0.0041). Error bars represent the standard error of the mean. The figure shown represents four independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p

Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression-1

On, and lysates prepared 30 min after infection. (A) NFκB-p65 and IκB phosphorylation increased with virus compared to mock infection, and were reduced in cells pre-treated with SB203580 in dose-dependent fashion. Densitometry values for the phosphorylated protein (normalized to the corresponding actin levels) are shown above each lane. (B) Immunoprecipitation assay reveals association of p38 MAPK with NFκB-p65 in virus infected (V) but not in mock (M) infected cells. This association was blocked by the p38 MAPK inhibitor SB203580 (SB) but not by the JNK inhibitor SP600125 (SP). Isotype control did not immunoprecipitate any NFκB-p65. (C) NFκB-p65 activation in HAdV-19 infection analyzed by confocal microscopy. The left column shows DAPI staining for nuclei (blue), the middle column for p65 (yellow), and the right column a merging of the left 2 rows. (c) Mock infected keratocytes show mostly cytoplasmic localization of NFκB-p65. (f) HAdV-19 infected keratocytes at 20 min post-infection show nuclear localization of NFκB-p65. (i) Nuclear translocation of NFκB-p65 was reduced in the presence of 10 μM, and (l) completely blocked with 20 μM SB203580. Bottom row (m, n, o), represent isotype control. (D) Electromobility shift assay showing NFκB-p65 binding to IL-8 promoter in HAdV-19 infected keratocytes. Extracts from HAdV-19 infected cell nuclei show more binding to NFκB-specific IL-8 probe (lane 2) as compared to nuclear protein from mock infected cells (lane 6) or when pretreated with SB203580 (lane 10). Binding specificity of the probe is shown with 100 molar excess of unlabelled probe (lanes 3 and 7). SB203580 (SB) blocked NFκB binding in virus infected nuclear extracts (lane 10). Dose dependent supershifts using increasing amounts of NFκB-p65 antibody are shown in virus infected nuclear extracts (lanes 4 and 5). No shifts were observed in nuclear extracts from mock infected cells (lanes 8 and 9) or SB203580 treated cells (lane 11).<p><b>Copyright information:</b></p><p>Taken from "Human adenovirus type 19 infection of corneal cells induces p38 MAPK-dependent interleukin-8 expression"</p><p>http://www.virologyj.com/content/5/1/17</p><p>Virology Journal 2008;5():17-17.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2265692.</p><p></p