Abstract
Purpose: :
Intrinsic and extrinsic signal pathways that regulate cell differentiation during mouse retinal development can be studied using rod photoreceptors as a model. Intrinsic factors including STAT3 and PKC play important roles in cell differentiation. Activation of STAT3 by CNTF specifically blocks differentiation of rod photoreceptors. Here we test the hypothesis that PKC activation promotes development of rod photoreceptors by inhibiting STAT3.
Methods: :
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. 20ng/mL of LIF (Millipore) was used to activate STAT3. 100nM of Go7478 (calbiochem) was used to inhibit PKC isoforms. Western blots were used to monitor STAT3 activation after PKC activation/inhibition. Immunocytochemistry was utilized to study PKC-alpha, -beta1, -beta2 and -gamma expression during retinal development. Animal protocols were in accordance with ARVO / IACUC guidelines. Explant cultures of mouse retina were used to study the effects of PKC activation and PKC-STAT3 interactions on rod development. Embryos from various ages were dissected in cooled phosphate-buffered saline (PBS). Retinas were isolated and cultured in serum free medium (Ultraculture, Lonza). PMA and/or CNTF were added in the culture medium 2 hours after isolation and kept for 1 to 5 days. Treated and control retina samples were collected to perform immunohistochemistry staining and western blots to detect the changes of rhodopsin marker expression and the protein level of STAT3 and phospho-STAT3.
Results: :
Our histological experiments show that PKC-beta2 is absent from adult and developing retina. PKC-alpha has a late expression appearing at postnatal day 5 (PN5) in bipolar and amacrine layers. In the outer nuclear layer, there is an intense expression of PKC-beta1 between E17.5 and PN5. PKC-beta1 staining in bipolar cells appears after PN3. The expression of rhodopsin, a rod specific marker, is induced at an early stage in retina explants cultured in the presence of PMA. Addition of PMA to retinas cultured in the presence of LIF abolished STAT3 induced inhibition of photoreceptor development. Our in vitro data shows that activation of PKC results in reduction of STAT3 phosphorylation. In addition, inhibition of PKC results in increase STAT3 phosphorylation. The expression pattern of PKC-beta1 suggests an involvement in modulation of rod development.
Keywords: retinal development • photoreceptors • gene/expression