Abstract:AIM:To investigate relation between the phagocytic fuction of retinal pigment epithelial(RPE)cells and the signal transduction pathway of MERTK-Ras-extracellular signal regulated kinase kinase(MEK)-myosin light chain kinase(MLCK)-myosin.
METHODS: Cultured 3～5 passage RPE cells of C57BL/6 mouse were incubated with rod outer segments(ROS)suspension(containing ROS 1×107 /ml)at 37℃, then cells were rinsed at different times(30,60,120,180,240min)to terminate the phagocytosis. The kinetics of phagocytosis was measured by double-fluorescent labeling. The activity levels of MERTK, Ras, MEK and MLCK at different incubation times were measured by Western Blot with antibodies of MERTK, Ras and phospho-antibodies of MEK and MLCK, respectively. To repeat the measurement of the phagpcytic kinetics and activity levels of MERTK, Ras, MEK and MLCK at different incubation times after interference to Ras and Mertk gene in RPE cells by plasmid transfection.
RESULTS: The phagocytic kinetics showed that the ingestion occurred at 30min of incubation. Ingested ROS by RPE cells increased until saturated at 180min. The protein levels of MERTK, Ras, MEK and MLCK in RPE cells increased during all the incubation periods compared with control group(P<0.05). After interference to Ras and MERTk gene in RPE cells by plasmid transfection, the protein levels of MERTK, Ras, MEK and MLCK and the quality of ingested ROS sustained at the lower levels during all the incubation periods, only a few ingested ROS were seen at 180 min. Compared with untransfected RPE cells, the protein levels of MERTK, Ras, MEK and MLCK and the quality of ingested ROS in siRas-RPE cells and siMERTK-RPE cells decreased distinctly at 120 min and 180 min during incubation(P<0.05).
CONCLUSION: Ras-MEK-MLCK-myosin signal pathway is the downstream of MERTK receptor in the phagocytic process of RPE cells from mice.
