[Role of CD2-associated protein in podocyte differentiation.].
To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.
Jiang
Hua-Jun
HJ
Department of Nephrology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Chang
Ying
Y
Zhu
Zhong-Hua
ZH
Liu
Jian-She
JS
Deng
An-Guo
AG
Zhang
Chun
C
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology*; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D050199:Podocytes / Q000166:cytology*; D034741:RNA, Small Interfering; D014162:Transfection; D025721:WT1 Proteins / Q000378:metabolism
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; C503968:Synpo protein, mouse; D025721:WT1 Proteins; C113267:nephrin