Nitric oxide regulates AKT phosphorylation and nuclear translocation in cultured retinal cells
Graphical abstract
Introduction
Nitric oxide (NO) is synthesized from l-arginine (L-Arg) which is converted to l-citrulline and NO in the presence of O2, NADPH and tetrahydrobiopterin in a nitric oxide synthase (NOS)-catalyzed reaction [1], [2]. Many brain regions are able to synthesize NO [3], although it is most actively formed in the cerebellum [4], [5]. NO is a potent bioactive molecule acting as a diffusible messenger in intercellular communication and intracellular signaling. In postsynaptic neurons, NO is formed following activation of glutamate receptors, mainly the NMDA subtype [6], although recent evidence also indicates the involvement of AMPA receptors [7], [8]. After activation of these receptors, calcium is transiently increased in the cytosol and forms a complex with calmodulin, which binds to and activates neuronal nitric oxide synthase (nNOS) [2]. Previous work has shown the presence of NOS in the vertebrate retina [9], [10]. We have also shown a NADPH diaphorase activity in retinal cells, which could be inhibited by L-Arg analogs and stimulated by calcium during early stages of chick retina development. Moreover, this diaphorase activity was localized in distinct cell types within the retina, including photoreceptors and amacrine cells [11]. Glutamate is able to stimulate NOS activity in the chick retina and this effect is mediated by the activation of NMDA receptors [12], [13], [14]. On the other hand, we have demonstrated the presence of a high affinity system for L-Arg uptake in cultured retinal neurons and glial cells. Furthermore, L-Arg could be released from glial cells and taken up by neurons in these cultures [15]. However, immunocytochemistry for nNOS or l-citruline in mixed neuron/glia cultures as well as measuring nitrite formation in purified neuronal cultures indicated the presence of NOS and NO production in neurons, but not in glial cells [7], [15], [16]. Significantly, we have also shown that the activation of NMDA receptors promoted the inhibition of protein synthesis and, as a consequence, increased the intracellular pool of L-Arg available to be used for the synthesis of NO in cultured retinal cells [17]. NO is also closely involved in the control of glial cell proliferation [18], vitamin C uptake [19] and the modulation of Src, ERK and CREB activation in the retina [7], [8]. Hence, these results indicate the importance of NO in neuronal–glial communication and glutamate/Src/ERK/CREB linkage during retinal development [7], [8], [16], [18].
AKT plays a crucial function in intracellular signaling pathways associated with cell survival and proliferation. AKT activation is usually achieved by a tyrosine kinase receptor-mediated phosphoinositide 3′ kinase (PI3K) activation, which is known to be wortmannin-sensitive [20]. During its activation, AKT is recruited to the plasma membrane where it binds to the PI3K-catalyzed products, PI(3,4,5)P3 and PI(3,4)P2, exposing both Threonine-308 (Thr308) and Serine 473 (Ser473) residues, which in turn can be phosphorylated by 3-phosphoinositide-dependent kinase (PDK1) and mammalian target of rapamycin complex-2 (mTORC2) [21], respectively. This double phosphorylation results in a full activation of AKT [22], [23]. Once activated, AKT induces the phosphorylation of a number of nuclear and cytosolic proteins that regulate cell metabolism, growth and survival. In line with this, recent data showed that genetic depletion of PTEN, a phosphatase that counter-balances PI3K activity and thereby diminishes the downstream activation of AKT, promotes a robust disturbance in retinal neurogenesis [24] through a mechanism related to Notch signaling [25].
Previous studies from our laboratory showed the participation of PI3K in promotion of cell survival by NO in retinal neuronal cultures [16]. Accordingly, herein we present evidence that delineate a novel signaling pathway in which NO, as well as its upstream activating system represented by NMDA receptors, activates AKT. Furthermore, we show that exogenous or endogenous NO elicits AKT translocation to the nucleus, an effect dependent on a PI3K-regulated AKT phosphorylation. NO is also able to protect retinal cells from hydrogen peroxide-induced cell death through a PI3K-dependent pathway. Therefore, the present data highlight the importance of NO in the regulation of survival pathways related to AKT activation in retinal neurons and indicate that NO plays a pivotal role during CNS development.
Section snippets
Reagents
LY294002 and NMDA were from Biomol (Plymouth Meeting, PA, USA). Wortmannin, LY83583, 1-oxo-9.12-epoxy-1H-diidolo[1,2,3-fg:3′,2′,1′kl]pyrrolo[3], [4][1], [6] benzodiazocine-10carboxylic acid methyl ester (KT5823), HEPES, NG-nitro-l-arginine methyl ester (L-NAME), 3-(5′hydroxymethyl-2′-furyl)-1-1benzyl indazole (YC-1), 1H[1], [2], [4]-oxadiazole [4,3-a]quinoxalin-1-one (ODQ), dimethyl sulfoxide (DMSO), diamidino-2-phenylindole, 4′,6-diamidino-2-phenylindole (DAPI), 7-Nitroindazole (7-NI),
Starvation of retinal cultures is essential for lowering basal AKT phosphorylation level
It is well known that serine-threonine kinases can be activated by serum added to the culture medium. The strategy of serum withdrawal was used in different cell cultures and does not cause any cellular alteration or damage but to reduce the basal phosphorylation level of different proteins [28]. Interestingly, starvation of our cultures using serum-free medium for 24 h had no effect on basal AKT phosphorylation when compared with cultures maintained with serum (3% FBS). On the other hand, when
Discussion
NO is able to exert its cellular functions through the activation of the canonical sGC/cGMP/PKG pathway or by protein nitrosylation and nitration [44]. However, very little is known about the signaling pathways triggered by such stimulatory events. Our recent studies revealed that the canonical pathway is involved in the activation of different kinases including ERK as well as the activation of the transcription factor CREB in retinal cells [7], [8]. Here we have studied in cultures of chick
Conclusions
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SNAP, a NO donor, or L-Arg, the NOS substrate, promotes AKT phosphorylation when added to cultured retinal cells in a concentration and time-dependent manner.
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The phosphorylation occurs in the residues Ser473 and Thr308, and the phosphorylation at both residues is completely prevented by the PI3K inhibitors LY294002 or wortmannin, or the mTor inhibitor KU0063794.
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The effect of NO on AKT phosphorylation is mediated by the canonical signaling pathway involving activation of sGC, cGMP production and
Acknowledgments
We thank Luzeli R. de Assis and Sarah A. Rodrigues for the technical assistance. We greatly acknowledge Dr. Edward Ziff for his critical review of the manuscript. This work was supported by grants from CNPq, Capes (Procad 0236055), Faperj and Pronex/Faperj. TAM-G, CCP and TGE were recipients of fellowships from Capes. RPC was a fellow from CNPq and Faperj.
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The first three authors (Telmo A. Mejía-García, Camila C. Portugal, and Thaísa G. Encarnação) have equal contributions to the work.