Influence of all-trans retinoic acid on the secretion of TGF-β2 and the intracellular second messengers IP3 and cAMP in guinea pig retinal pigment epithelium cells

Abstract

Objective To evaluate the role of all-trans retinoic acid (ATRA) on proliferation and function in the secretion of TGF-β2 and in the related signal cascades in cultured guinea pig retinal pigment epithelium cells (RPE). Methods RPE cells were taken from 3-week-old guinea pig eyes. The 2-3 passage cells in the logarithmic growth phase were used for the experiment. Cells were verified by keratin immunohistochemistry. When cells almost attained confluence, the medium was changed to a DMEM/F12 medium without FBS (fetal bovine serum) for 24 hours before being used. The cells were then divided into 3 groups. ①The medium was changed to a DMEM-F12 medium without FBS but contained different concentrations 6f the drug ATRA (5×10-6, 10×10-6, 40×10-6 mol/L). After 24 hours, cell proliferation was analyzed using an MTT assay. ②The medium was changed to DMEM/F12 containing 10×10-6 mol/L ATRA. The TGF-β2 secreted by RPE cells was tested using an ELISA kit at 2, 4, 6, 8, and 16 hours. ③ Intracellular 1, 4, 5 -trisphosphate (IP3) and cyclic adenosine monophosphate (cAMP) were extracted at 0 min, 5 min, 30 min, 2 h and 6 h, and their concentrations were measured with the ELISA method and radioimmunoassay. The same dose of DMSO was added to all of the control groups. Results Twenty-four hours after ATRA concentrations of 5×10-6 mol/L, 10×10-6 mol/L, and 40×10-6 mol/L were added to RPE cells, the respective OD values were 0.099±0.008, 0.117±0.008, and 0.087±0.011. Compared to the controls, 0.103 0.017, cell proliferation was inhibited by 40×10-6 mol/L ATRA (P<0.05). After 10×10-6 mol/L ATRA was added, the secretion of TGF-β2 increased in the first 6 hours (P<0.05), and then decreased. And the decrease was time related. Intracellular IP3 was inhibited at each time point. However, the amount of cAMP production increased at 30 min and 2 h. Conclusion A concentration of 40×10-6 mol/L ATRA can inhibit the proliferation of guinea pig RPE cells. But the RPE cells can grow well in lower concentrations. The secretion of TGF-β2 increased in the short term but there was a time-related decrease. And the ATRA influence on RPE cells may correlate with a decreased second messenger IP3. Key words: Retinoic acid; Guinea pig; Retina; Pigment epithelial of eye; Myopia; Transforming growth factor β2; Second messenger systems