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Jae-Chang Jung, Man-Ill Huh, Sharada Paudelkattel, Yeon-Hee Kim; Modulation of Staurosporine Induced Neuronal Cell Differentiation and Neurite Outgrowth of Chick Retinal Neuroblasts by Inhibition of MMPs Activities. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2251.
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PKC dependent signaling pathway is well known regulator of Matrix metalloproteinases (MMPs) which play an important role during development. The goal of this study is to investigate whether injection of staurosporine (STS) into the early developing chick embryos in ovo affects the differentiation of retinal neuroblasts during retinal development, and is to compare whether inhibition of MMP activities differentially regulates the STS-induced neuronal cell differentiation and cell outgrowth in both coating and non-coating culture conditions.
Neuroblasts isolated from an early stage of developing chick retinas at embryonic day 6 days (E6) were primary cultured in coating culture dishes with poly DL-ornithine and also non-coating dishes for 3 days. Neuroblasts were treated with STS in the presence of GM6001 (broad MMPs inhibitor), GI254023X (ADAM10 specific inhibitor), and amiloride (uPA inhibitor). Furthermore, to investigate the differentiation pattern of neuroblasts in ovo, STS and/or amiloride were directly injected into developing eggs at E6. Two, four, and six days after injections, eye balls were harvested and embedded in OCT. Indirect immunofluorescence staining for Tuj-1, visinin, neurofilament, CD44v6, and N-cadherin in cultured cells and frozen sectioned retinas was performed, and their expression levels were also examined by Western blot analysis.
In both culture conditions, STS induced morphological changes differentially and also neurite outgrowth. Cell cycle analysis showed that STS treated cells were growth arrested at the G2-M phage. High levels of visinin and neurite outgrowth as well as cleavage forms of both CD44v6 and N-cadherin induced by STS were strongly inhibited by GM6001 and amiloride. Furthermore, expression level of visinin induced by STS in photoreceptor cell layer was also dramatically decreased by amiloride in ovo.
This study suggests that STS may regulate MMPs activation, cell-cell and cell-ECM interactions, and subsequently induces neuronal cell differentiation in early developing chick retina.
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