Abstract
Purpose: :
It is known that the ciliary neurotrophic factor (CNTF) activates the JAK-STAT pathway to induce protein expression, and that this activation subsequently levels off, due to negative control of the JAK-STAT pathway. The suppressors of cytokine signaling (SOCS) protein family constitutes one class of negative regulators of cytokine activity. In the present study, we have examined some temporal aspects of CNTF action on SOCS3 gene expression as well as its relationship to the JAK-STAT pathway in the retinal Müller cell line, rMC-1.
Methods: :
The retinal Muller cell line, rMC-1, was treated with CNTF (50ng/ml) for different time periods, ranging from 15 min to 90min. In a second set of experiments, the Müller cell line was transfected with a dominant-negative STAT3 mutant using the lipofectin method 24-48 hrs prior to the treatment of rMC-1 cells with CNTF. Total RNA was prepared using TRIZOL Reagent from both sets of experiments. cDNA was then generated using M-MLV reverse transcriptase for use in quantitative real time PCR (qPCR). qPCR was carried out using short oligonucleotide primers (<150bp) against either suppressors of cytokine signaling 3 (SOCS3) or Glial fibrillary acidic protein (GFAP). As an internal control, cDNA samples were also amplified with primers for glyceraldehyde phosphate dehydrogenase (GAPDH). All qPCR reactions were carried out in a Bio-Rad myiQ Real Time PCR Detection System using the DNA-binding dye SYBR Green I. For Real-Time qPCR Data Analysis we used relative quantification normalized to the reference gene GAPDH using the 2-Ct (Livak) Method.
Results: :
In Müller cell cultures treated with CNTF, there was a rapid increase of SOCS3 mRNA by 15 min with a 2.5 fold increase relative to GAPDH. By 30 min the level of SOCS3 is more than 3.5 fold higher than in untreated Muller cells. But by 60min of CNTF treatment of rMC-1 cells the level of SOCS3 has declined to under 2 fold higher and by 90min the levels of SOCS3 have returned to baseline. By comparison the peak level of GFAP mRNA expression in rMC-1 cells stimulated with CNTF is seen at 60min with very high levels still detected at 90min.
Conclusions: :
The retinal Müller cell line, rMC-1, expresses SOCS3. CNTF treatment results in an immediate up-regulation of SOCS3 levels. This upregulation of SOCS3 is transient and returns to baseline levels within 90min. SOCS3 may be the main negative feedback regulator of CNTF activation of the JAK-STAT pathway in retinal Müller cells.
Keywords: Muller cells • cytokines/chemokines • retina