Abstract
Purpose: :
Both intrinsic and extrinsic signals regulate rod photoreceptor development. Activation of STAT3 blocks differentiation of rod photoreceptors from dividing progenitors. Here we test the hypothesis that PKC isoforms promote rod differentiation by inhibiting phosphorylation of STAT3. In addition we investigate whether insulin growth factor 1 (IGF-1) activates PKC during retinal development.
Methods: :
Animal use was in accordance with ARVO/IACUC guidelines. Retinal explant cultures were established from embryos of wild type and PKC Gamma knockout mice (Jackson laboratories) in serum free medium (Ultraculture, Lonza) with L-glutamine and antibiotics and kept for 1 to 8 days. PMA (100nM), LIF (20ng/mL), and a specific PKC Beta 1 inhibitor LY 333531 (calbiochem) (30nM) were added to the culture medium 2 hours after explant isolation. Treated and control retina samples were collected for immunohistochemistry and western blots. In vitro PKC/STAT3 interactions were investigated in NIH3T3 cells. 100nM of phorbol 12 myristate 13 acetate (PMA, LC Laboratories) was used to activate PKC and 20ng/mL of LIF (Millipore) to activate STAT3. 100nM of Go7874 (calbiochem) was used to inhibit PKC alpha and beta isoforms. STAT3 activation was monitored by western blot.
Results: :
We previously showed that there is an intense expression of both PKC-beta1 and Gamma between E17.5 and PN5 in the outer nuclear layer. PMA induced opsin expression in WT retinas in the presence of LY33351 and in PKC gamma KO retinas, but not in LY33351 treated PKC gamma KO retinas. In these opsin negative explants there was an increase in glial marker expression. IGF1 also stimulated rod photoreceptor differentiation in a PKC-dependent manner. In cultured 3T3 cells activation of PKC with either PMA or IGF1 reduced STAT3 phosphorylation, and inhibition of PKC increased STAT3 phosphorylation.
Conclusions: :
Activation of PKC gamma or PKC beta1 can remove the block to rod photoreceptor differentiation by STAT3 activation. Extrinsic factors such as IGF-1 that regulate rod production may do so by through a PKC-STAT3 pathway.Key words: Retina development, Rod photoreceptor, STAT3, PKC
Keywords: retinal development • photoreceptors • retinal culture