Temporal aspects of Ciliary Neurotrophic Factor on the Suppressor of Cytokine Signaling 3 Expression and the JAK/STAT Pathway in Rat Retinal Müller cells

Abstract

The cytokine ciliary neurotrophic factor (CNTF) is a neuroprotective agent in the central nervous system. CNTF activates the JAK/STAT signaling pathway to induce protein expression, which subsequently levels off due to negative control. One class of negative regulators is the suppressors of cytokine signaling (SOCS) proteins. The goal of my research was to study the temporal aspects of CNTF on SOCS3 gene expression and its relationship to the JAK/STAT pathway; and examine the effects of CNTF on levels of other proteins involved in the JAK/STAT pathway. Retinal Müller cells were treated with CNTF for different time periods from 0 to 90 minutes. The total RNA collected was converted into cDNA for use in quantitative real-time PCR. At 15 minutes, the CNTF-treated cells showed a 2.5 fold increase in SOCS3 mRNA, which increased to 3.5 fold by 30 minutes. The level of SOCS3 mRNA returned to baseline by 90 minutes. A separate set of cells were transfected with a dominant-negative STAT3 mutant prior to the treatment with CNTF at different time periods from 0 to 90 minutes. The total RNA collected was converted into cDNA for use in quantitative real-time PCR. The dominant-negative STAT3 mutant appears to negate up-regulation of SOCS3 in response to CNTF. These experimental results indicate SOCS3 as a negative feedback regulator of CNTF activation of the JAK/STAT pathway. CNTF-treated cells were analyzed using Western immunoblotting techniques to study the levels of other proteins such as P-AMPK, P-MAPK, and P-AKT that are involved in the JAK/STAT pathway. There were no significant changes in levels of P-AMPK and P-AKT over 90-minutes. A large increase in the level of P-MAPK was observed at 15 minutes and a substantial decrease was observed at 30 minutes. The P-MAPK level returned to baseline by 60 minutes. The role of P-MAPK in the JAK/STAT pathway of retinal cells is under further investigation.