Expression and activation of STAT proteins during mouse retina development

doi:10.1016/S0014-4835(03)00002-2

Experimental Eye Research

Volume 76, Issue 4, 1 April 2003, Pages 421-431

https://doi.org/10.1016/S0014-4835(03)00002-2 Get rights and content

Abstract

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis.

Introduction

Mammalian eye development and function are influenced by a wide array of peptide growth factors and cytokines. The ways in which the signals from these peptide factors are transduced into altered cell metabolism and gene expression remain poorly understood. It has been shown that signal transducer and activator of transcription proteins (STAT proteins) play a key role in integrating extrinsic signals and nuclear responses. Originally identified and isolated from cell culture systems responding to cytokines such as interferon, seven STAT proteins have so far been identified in mouse and human (Fu, 1995, Darnell, 1997). Each contains an SH2 domain and can be activated through phosphorylation by a number of receptor tyrosine kinases (Fu, 1992, Schindler et al., 1992). After phosphorylation, in a number of cases the activated STAT proteins dimerize in the cytoplasm and are then translocated to the nucleus where they can modulate transcription.

The importance of STAT proteins in the immune system has been shown by targeted disruption of STAT proteins in mouse that result in severe malfunctions of both cellular and humoral immune responses (Leonard and O'Shea, 1998, Takeda and Akira, 2000). These knockouts also show that each STAT protein appears to have specific functions. For example, IL-12 induced development of Th1 cells was not observed in Stat4 knockout mice and IL-4 induced development of Th2 cells was impaired in Stat6 knockout mice (Kaplan et al., 1996a, Kaplan et al., 1996b, Shimoda et al., 1996, Thierfelder et al., 1996).

Since targeted disruption of the Stat3 gene resulted in embryonic lethality, and there is some evidence for expression of STAT proteins during embryonic development, it seems likely that these proteins play a wider developmental role than initially thought (Duncan et al., 1997) (Chai and Fu, unpublished results). Within the nervous system Stat3 has been shown to play a role in specification of glial cell fate in a dissociated cell culture system and in the responses of mature retinal cells to stress (Bonni et al., 1997, Rajan and McKay, 1998, Peterson et al., 2000).

As a first step in determining the role of STAT proteins in retina development we have measured their expression and activation at various developmental stages. The distinct and changing patterns of expression through out retina development, suggest that many members of the STAT protein family play key roles in the formation of retina structures.

Section snippets

Reagents

Antibodies against STAT proteins and phosphorylated proteins are listed in Table 1. Anti-STAT polyclonal antibodies, and immunizing peptides, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphorylated Stat1 (P-Tyr701) and Stat3 (P-Tyr705) polyclonal antibodies were purchased from New England Biolabs (Boston, MA, USA). Reactivity of all STAT antibodies was removed by pre-incubation with the appropriate peptide. Anti-phosphorylated-STAT antibodies selectively recognize

Specificity of antibody labelling

Our conclusions in this study depend upon both the sensitivity of the labelling method and the specificity of the antibodies used. The results presented were obtained using sections of paraffin-embedded fixed material, as recommended by the antibody supplier. We have confirmed the labelling patterns in selected cases using cryostat sections of fixed material (data not shown). Thus, within the normal limits of immunocytochemical detection we feel that we have detected all significant STAT

Discussion

It is well established that STAT proteins play a vital role in the development of innate and adaptive immunity in many organisms (Darnell, 1997). Recent findings suggest that these molecules also function during development of many tissues, but these roles remain poorly understood. Using cell lysates from different regions of the nervous system it was found that Stat6 is expressed and possibly regulated during development (Cattaneo et al., 1999). Stat3 has been implicated in specification of

Acknowledgements

The authors would like to thank Dr Thomas Welte for critical discussion and Ms Lan Ji for excellent technical assistance. XYF is a recipient of Career Development Award (KO4AE01356) from the National Institutes of Health and CJB is a Senior Scientific Investigator of Research to Prevent Blindness, Inc. Supported in part by grants from the National Institutes of Health and the Kemper Foundation.

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Toll-like receptors (TLRs) are essential receptors of the innate immune system and are first responders for protection against bacterial and viral pathogens. Recently, several TLRs have also been implicated in regulating cell death and survival in non-pathogen injuries such as stroke and oxidative stress. Investigating the role of TLRs during central nervous system damage is an important focus of research that may reveal new mechanisms underlying the cellular response to injury and survival. Retinal pigmented epithelium (RPE) cells form an epithelial layer underneath the neural retina that maintains the function of photoreceptors and are the primary cell type affected in the retinal disease age-related macular degeneration (AMD). Predicted loss of function polymorphisms in the TLR3 gene are associated with protection from AMD but the role of TLR3 in regulating RPE survival during AMD-like injury, such as high oxidative stress, is not known. Therefore the purpose of this study is to evaluate the effect of TLR3 signaling on RPE viability during oxidative stress. We demonstrated that TLR3 activation in the presence of oxidative stress injury significantly increased RPE cell viability, in contrast to TLR3 reducing cell viability in the absence of cellular injury. Furthermore, we show signal transducer and activator of transcription 3 (STAT3) signaling as an essential mediator of TLR3-regulated protection of RPE cells. STAT3 signaling was increased by TLR3 activation and knockdown of STAT3 transcripts using siRNA abolished the protective effect of TLR3 during oxidative stress. Together, these results demonstrate a novel pro-survival role for TLR3 signaling within the RPE during injury. These findings support the concept that dysregulation of TLR3 activity may contribute to the development of AMD, suggesting that precise regulation of the TLR3 pathway during AMD-associated injury could be of therapeutic interest.
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In our previous report (35) we observed strong activation of STAT3 in the RPE after intravitreal injection of LIF, which prompted us to identify RPE functions that are modulated by STAT3. Members of the STAT family of transcription factors, such as STAT1, STAT2, STAT5b, STAT6, and STAT3, were found in the adult RPE (57). In particular, phosphorylated STAT3 (pSTAT3) was localized to RPE nuclei and along with STAT5a was suggested as the most important modulator of RPE function.
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Expression and activation of STAT proteins during mouse retina development

Abstract

Introduction

Section snippets

Reagents

Specificity of antibody labelling

Discussion

Acknowledgements

J. Biol. Chem.

Trends Neurosci.

J. Biol. Chem.

Cell

Trends Genet.

Immunity

Blood

J. Biol. Chem.

Brain Res. Mol. Brain Res.

Brain Res.

Cytokine Growth Factor Rev.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Brain Res.

Int. Rev. Cytol.

Brain Res.

J. Biol. Chem.

Transforming growth factor-beta-3 is mitogenic for rat retinal progenitor cells in vitro

J. Neurobiol.

Production of interleukin-6 by human retinal pigment epithelium in vitro and its regulation by other cytokines

Curr. Eye Res.

Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway

Science

Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells

J. Cell Sci.

STATs and gene regulation

Science

STAT signaling is active during early mammalian development

Dev. Dyn.

Postmitotic cells fated to become rod photoreceptors can be respecified by CNTF treatment of the retina

Development

Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor

Nature

A direct signaling pathway through tyrosine kinase activation of SH2 domain-containing transcription factors

J. Leukoc. Biol.

Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity

EMBO J.