Role of connective tissue growth factor (CTGF) module 4 in regulating epithelial mesenchymal transition (EMT) in HK-2 cells.
BACKGROUND
Recent studies have suggested that connective tissue growth factor (CTGF) plays a key role in tissue fibrosis including renal scarring. While studies showed several forms of CTGF with 10-38 kDa in the body fluids, little is known about these small molecule species. We investigated the effect of a 10 kDa CTGF molecule consisting of module 4, on the epithelial mesenchymal transition (EMT) in human proximal tubular cell line (HK-2).


METHODS
HK2 cells were cultured in DMEM medium. The response of cytokeratin (CK) and vimentin (VIM) mRNA and protein expression to the stimulation of rhCTGF(C) were observed by real-time PCR and immunocytochemistry. At the same time, the morphologic changes were observed by microscopy, and expression of alpha-smooth muscle actin (alpha-SMA) and fibronectin (FN) was detected by laser confocal microscope. These effects were compared with CTGF N-terminal [rhCTGF(N)], consisting of module 1-3, and observed in a condition with the addition of anti-CTGF antibody.


RESULTS
RhCTGF(C) induced striking changes in epithelial cells, including changes in cellular morphology, loss of CK, gain VIM and alpha-SMA, and increased levels of fibronectin. Cocultured with anti-CTGF antibody could abrogate most of these effects, while cells treated with rhCTGF(N) showed no significant phenotypic changes comparing to control group.


CONCLUSIONS
Our results suggest that module 4 could induce HK-2 cells EMT, whereas the residual fragment has no similar effect in spite of consisting of 3 modules of CTGF molecule.
Liu
Bi-Cheng
BC
Institute of Nephrology, Zhong Da Hospital, Southeast University, No. 87 Ding Jia Qiao Road, Nanjing 210009, China. liubc64@yahoo.com.cn
Zhang
Jian-Dong
JD
Zhang
Xiao-Liang
XL
Wu
Guo-Qiu
GQ
Li
Min-Xia
MX
D000199:Actins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D017874:Immediate-Early Proteins / Q000494:pharmacology*; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007633:Keratins / Q000096:biosynthesis / Q000187:drug effects; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018613:Microscopy, Confocal; D009130:Muscle, Smooth / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014746:Vimentin / Q000096:biosynthesis / Q000187:drug effects
D000199:Actins; C526317:CCN2 protein, human; D005353:Fibronectins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014746:Vimentin; D055513:Connective Tissue Growth Factor; D007633:Keratins