A crosstalk between hSiah2 and Pias E3-ligases modulates Pias-dependent activation.

Protein inhibitor of activated STAT (Pias) and human homologues of seven in absentia (hSiah) proteins both exhibit properties of ubiquitin-family peptides conjugating enzymes. Pias present E3-ligase activity for small ubiquitin-related modifiers (Sumo) covalent attachment to their targets. This post-translational modification is responsible for the activation of different transcription factors such as AP1. HSiah proteins possess ubiquitin-E3-ligase activity that triggers their partners to proteasomal-dependent degradation. The present study identifies Pias as a new hSiah2-interacting protein. We demonstrate that hSiah2 regulates specifically the proteasome-dependent degradation of Pias proteins. On reverse, Pias does not prevent hSiah2 degradation. We provide evidences for hSiah2-dependent degradation of Pias as being a mechanism in the regulation of c-jun N-terminal kinase-activating pathways. This report describes a new interconnection between sumoylation and ubiquitination pathways by regulating the levels of the E3-ligases available for these processes.

Regulation of FGF soluble receptor type 1 (SR1) expression and distribution in developing, degenerating, and FGF2-treated retina.

The spatial and temporal patterns of expression and content of the fibroblast growth factor (FGF) soluble receptor SR1, a specific inhibitor of FGF, were investigated during embryonic and postnatal development of the retina in Fisher rats. As early as at embryonic day 18 (E18), SR1 mRNA and protein were detected in the retina. SR1 protein was strongly associated with the differentiating ganglion cells and its distribution paralleled the radial pattern of retinal development, from center to periphery. From E18 to postnatal day 5, the levels of both SR1 mRNA and SR1 protein remained constant. Thereafter, they decreased rapidly, by a factor of 5 in the adult retina. SR1 was labeled in the inner nuclear layer, but never in the photoreceptor nuclei. In the neural retina of RCS dystrophic rats, the levels SR1 mRNA and SR1 protein were 2 to 3 times higher than those in the normal congenic controls, before and during photoreceptor degeneration. These results provide the first evidence that a natural FGF inhibitor is regulated during retina development and degeneration and suggest that changes in SR1 content may be involved in the regulation of FGF activities in retina. This was confirmed in vivo in RCS rats, in which delayed photoreceptor apoptosis by intravitreal injection of FGF2 was accompanied by a downregulation of SR1 expression. Dev Dyn 2000;217:24-36.

Requirement for nitric oxide in retinal neuronal cell death induced by activated Müller glial cells.

Retinal Müller glial cells express the inducible isoform (-2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Müller glial (RMG) cells from mice disrupted for the gene of NOS-2 [NOS-2 (-/-)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN-gamma-stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been observed in cocultures with RMG cells from NOS-2 (-/-) mice and because inhibition of NOS-2 induction by transforming growth factor-beta or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superoxide dismutase partially prevented neuronal cell death induced by NO, whereas the presence of a poly(ADP-ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large release of NO from RMG cells is responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated.

Role of nitric oxide in photoreceptor survival in embryonic chick retinal cell culture.

The presence of nitric oxide synthase (NOS) in chick retina during development has allowed us to study the role of nitric oxide (NO) during retinal differentiation in dissociated chick retinal cell culture from embryonic day 6. We have demonstrated the presence of nicotinamide adenine dinucleotide phosphate diaphorase staining in these cultures after 3 days in vitro (Div), with a maximal intensity after 8 Div, corresponding to embryonic day 14. Immunohistochemistry studies confirmed the presence of the two isoforms of NOS, NOS-I and -III, in dissociated retinal cell cultures at 8 Div. Addition of NG-monomethyl-L-arginine, a NOS inhibitor, to retinal cell cultures prevented NO production but did not modify the appearance and the survival of ganglion and amacrine cells. However, immunohistochemical analysis with distinct markers for photoreceptor cells (rods and cones) showed that inhibition of endogenous NOS in retinal cell cultures prevented the developmental decrease of rod number between 5 and 8 Div, thus supporting the hypothesis that NO may be involved in the cell death of rods during the development of the retina.

Fibroblast growth factor (FGF) soluble receptor 1 acts as a natural inhibitor of FGF2 neurotrophic activity during retinal degeneration.

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

Paracrine effects of phosphorylated and excreted FGF1 by retinal pigmented epithelial cells.

We have recently shown that both inhibition of endogenous Fibroblast growth factor (FGF) synthesis in non dividing lens epithelial cells (Renaud et al. J. Biol. Chem 1996, 271: 2801-2811) and inhibition of secreted FGF1 in confluent quiescent retinal pigmented epithelial (RPE) cells (Guillonneau et al., Exp. Cell. Res. 1997, in press) induce rapid cell apoptosis. In addition, FGF2-stimulated release of endogenous FGF1 is associated with reduced apoptosis in RPE cells. We now show that a single addition of exogenous FGF2 to RPE cells induces after 4 days of culture, a great accumulation of FGF1 within the cells. Concomitantly we observe that FGF1 was released into the extracellular medium. Secreted FGF1 from RPE cells, purified from culture medium and added to either Go-arrested RPE or RMG cells at low plating density induced cell proliferation, whereas when it is added once to serum-depleted confluent RPE and RMG cells it prevented apoptosis. Both endogenous and secreted FGF1 are phosphorylated. In addition, FGF2 stimulated the production and release of phosphorylated FGF1 by RPE cells. We show that this secreted form of phosphorylated FGF1 binds to the high affinity tyrosine kinase receptors of RPE and RMG cells on retinal sections and to heparan sulfate proteoglycan in RPE cell extracellular matrix. In contrast to non-phosphorylated FGF1, phosphorylated secreted FGF1 was not degraded after internalization but accumulated within RPE and RMG cells, and is rapidly translocated to the nucleus suggesting a role in signal transduction and gene expression pathways. These results show that exogenous FGF2 activities might be mediated indirectly by phosphorylation and that secretion of FGF1 may function as a paracrine trophic factor for retinal cells.

Developmental expression of nitric oxide synthase isoform I and III in chick retina.

During our studies on the multiple possible functions of nitric oxide (NO) in chick retinal development and physiology, we have demonstrated the presence and the activity of NO synthase (NOS-I and III) in certain neuronal populations (photoreceptors, amacrine cells in the inner nuclear and ganglion cells) and also in synaptic-rich regions in the developing chick retina. Both enzymes, detected by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, immunohistochemistry and Western blotting, appeared between embryonic days 6 and 12, and followed a spatial and temporal pattern of expression which correlated with the differentiation of the neuronal layers. Evaluation of the conversion of [3H]-labeled arginine to [3H]-citrulline, confirmed the presence of a calcium-dependent NOS activity in the cytosolic and particulate retinal extracts during the development. This pattern of NOS expression suggests that the regulated release of NO during key phases of development might be one mechanism involved in the regulation of retinal differentiation.

FGF2-stimulated release of endogenous FGF1 is associated with reduced apoptosis in retinal pigmented epithelial cells.

Both inhibition of endogenous fibroblast growth factor (FGF) synthesis on nondividing lens epithelial cells and inhibition of secreted FGF1 in confluent quiescent retinal pigmented epithelial (RPE) cells induce rapid cell apoptosis (Renaud et al., 1996, J. Biol. Chem., 271, 2801-2811). In addition several studies demonstrate that exogenous FGF2 can promote retinal cell survival in vitro and in vivo. To determine the possible relationship between exogenous FGF2, endogenous FGF1, and cell survival, we examined the protective effect of a single dose of exogenous FGF2 on long-term culture of quiescent RPE cells after serum withdrawal. After 4 days of culture, a dramatic and sustained upregulation of FGF1 protein expression occurs specifically in response to exogenous FGF2. After addition of FGF2 (20 ng/ml), RPE cells express fourfold more FGF1 after Day 7 than after Day 1 of culture. This phenomenon is FGF2 dose-dependent. In contrast, neither serum nor FGF2 have an effect on total endogenous FGF2 expression. In addition, in response to exogenous FGF2, FGF1 is secreted in significant amounts into the extracellular medium at a rate comparable to FGF1 accumulation within the cell. Furthermore, in the absence of serum, significant increase in cell death occurs on Day 6 of culture, whereas addition of exogenous FGF2 induces a twofold decrease of RPE cell apoptosis. In the presence of exogenous FGF2, addition of a specific anti-FGF1 neutralizing antibody induces a rapid apoptosis of RPE cell cultures. Thus, we speculate that exogenous FGF2 may indirectly prolong cell survival by increasing synthesis and secretion of endogenous FGF1 and that endogenous FGF1, directly in response to exogenous FGF2, may function as an autocrine trophic factor in RPE cells.