Abstract
Abstract: :
Purpose: Rod photoreceptors are an excellent system to study intrinsic and extrinsic signal pathways that regulate cell differentiation during mouse retina development. The CNTF–STAT3 signals inhibit rod differentiation in the neonatal mouse retina. Protein Kinase C (PKC) is an intrinsic factor that triggers several signal pathways with multiple functions included cell differentiation. We carried out this study to understand whether PKC is involved in the development of rod photoreceptors. Methods: All mouse protocols were in accordance with ARVO guidelines and were approved by the Animal Care and Use Committee of Yale University School of Medicine. Sixty CD1 mice were used in this study. Most litters were born on E19.5, which was considered equivalent to postnatal day 0 (PN0). E18.5 embryos were dissected in cooled phosphate–buffered saline (PBS). Retinas were isolated and cultured in serum–free medium supplied with L–glutamine and antibiotics. The phorbol ester 12–O–tetradecanoylphorbol 13–acetate (TPA) and/or CNTF were added in the culture medium 5 hours after isolation and kept for 1 day or 5 days. Treated and control retina samples were collected after 5 day or 8 days (equivalent to PN4 or PN7). Immunohistochemistry and western blots were performed to detect the changes of rod photoreceptor marker expression and the protein level of STAT3 and phospho–STAT3. Results: The rod specific marker opsin is expressed earlier and at higher levels in retina explants after treatment with a specific PKC activator TPA, as compared with control cultures. Pre–activation of PKC by TPA abolishes CNTF–induced rod inhibition, but inhibition was not blocked when the TPA was applied at the same time as CNTF. In addition, activation of PKC specifically reduces STAT3 protein levels and decreases STAT3 phosphorylation by CNTF. Activation of PKC also abolished CNTF mediated induction of STAT3 protein in retina explants. Conclusions: PKC–mediated signals may promote rod differentiation through modulation of the STAT3 pathway. These results suggest that cross talk among multiple signal transduction pathways such as CNTF–STAT and PKC contributes to rod development.
Keywords: signal transduction • photoreceptors • retinal development