[The relationship between MERTK gene and protein kinase C in the phagocytic process of human retinal pigment epithelial cells].
OBJECTIVE
To investigate relationship of the expression of MERTK gene and the activity of protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (hRPE) cells.


METHODS
Cultured hRPE cells were incubated with rod outer segments (ROS) suspension (containing ROS 1x10(10)/L) at 37 degrees C, then cells were rinsed at different times to terminate the phagocytosis. The kinetic of phagocytosis was measured by double-fluorescent labeling. The activity of PKC and the expression level of MERTK gene were measured by counting gamma-32P radio-activity with liquid scintillation and RT-PCR respectively. Change of MERTK gene expression was measured after hRPE was treated cells with stimulator or inhibitor of PKC. Statistical analysis was performed by SPSS 13.0 software.


RESULTS
The phagocytic assay showed that the quality of bound and ingested ROS by hRPE cells increased. The quality of ingested ROS by hRPE cells at 24 hours was (2.85+/-0.11)x10(6), which reached maximum contrast with control (0.00+/-0.00)x10(6) (t=47.64, P<0.05). The activity of PKC (both in cytoplasm and on membrane) decreased during all the incubation periods compared with control [cytoplasm: (329.63+/-14.26) nmolxg(-1)xmin(-1)and on membrane: (467.67+/-68.87) nmolxg(-1)xmin(-1)], and reached the minimum at 24 h[cytoplasm: (151.13+/-17.67) nmolxg(-1)xmin(-1) and on membrane: (152.45+/-64.83) nmolxg(-1)xmin(-1); cytoplasm t=89.66 and membrane t=10.31, P<0.05]. The level of MERTK mRNA increased in pulse-chase and long-time incubation test. The gray level for 90 min was 1.8853+/-0.0077, contrasted with control 0.7246+/-0.0062, F=16,060.2167 and P<0.05. The gray level for 24 h was 0.5946+/-0.0082, contrasted with control 0.3343+/-0.0064, F=919.8421 and P<0.05. When up-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA was decreased in the proceeding incubating with ROS contrasted with control (pulse-chase group F=17,142.2331, long time group F=1886.4614; P<0.05). After down-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA waved between 4.4670+/-0.0092 and 5.7034+/-0.0095 in the first 30 min of incubating with ROS, which lower than control 0.9117+/-0.0021 (F=199,012.9138, P<0.05).


CONCLUSIONS
The lower activity of PKC and the higher expression MERTK gene are very important for sustaining phagocytic process of ROS by hRPE cells. MERTK gene and PKC both as up-stream regulators are negative-correlated in the phagocytic process of hRPE.
Sun
Yu-zhao
YZ
Department of Ophthalmology, the First Clinical College of China Medical University, Shenyang 110001, China.
Hong
Jing
J
D000328:Adult; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D010587:Phagocytosis; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D000076202:c-Mer Tyrosine Kinase
D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C097072:MERTK protein, human; D020794:Receptor Protein-Tyrosine Kinases; D000076202:c-Mer Tyrosine Kinase; D011493:Protein Kinase C