cAMP-responsive element binding protein mediates a cGMP/protein kinase G-dependent anti-apoptotic signal induced by nitric oxide in retinal neuro-glial progenitor cells.
Nitric oxide (NO) is cytoprotective to certain types of neuronal cells. The neuroprotective ability of NO in the retina was reportedly mediated by the cyclic GMP (cGMP) to protein kinase G (PKG) pathway. Cyclic AMP-responsive element binding protein (CREB) plays an essential role in the NO/cGMP/PKG-mediated survival of rat cerebellar granule cells. We tested whether CREB transduces the NO/cGMP/PKG anti-apoptotic cascade in R28 neuro-glial progenitor cells. Apoptosis was induced in R28 cells by serum deprivation for 24 h. Varying concentrations of two NO donors, sodium nitroprusside (SNP) and nipradilol, were added to medium with or without an NO scavenger, a soluble guanylyl cyclase inhibitor, or a PKG inhibitor. The cells were immunostained against activated caspase-3 and counterstained with Hoechst 33258. Apoptosis was quantified by counting activated caspase-3 positive or pyknotic cells. SNP and nipradilol rescued R28 cells from apoptosis in a dose-dependent manner, at an optimal concentration of 1.0 microM and 10 microM, respectively. Higher concentrations were cytotoxic. The NO scavenger and the inhibitors decreased the anti-apoptotic effect of the NO donors. Intracellular cGMP levels were increased after exposure to SNP and nipradilol. Western blotting showed that both NO donors increased CREB phosphorylation, which was blocked when pre-exposed to the inhibitors. Transfection with a dominant negative CREB construct defective of phosphorylation at Ser-133 interfered with the anti-apoptotic activity of SNP. These results indicate that CREB at least in part mediates the cGMP/PKG-dependent anti-apoptotic signal induced by NO in R28 cells.
Nagai-Kusuhara
Azusa
A
Department of Organs Therapeutics, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Nakamura
Makoto
M
Mukuno
Hirokazu
H
Kanamori
Akiyasu
A
Negi
Akira
A
Seigel
Gail M
GM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D019610:Cytoprotection / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011412:Propanolamines / Q000494:pharmacology; D051381:Rats; D012160:Retina / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; D020030:Nitric Oxide Donors; D011412:Propanolamines; D009599:Nitroprusside; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; C036089:nipradilol; D006152:Cyclic GMP