Decorin gene transfer inhibited the expression of TGFbeta1 and ECM in rat mesangial cells.
OBJECTIVE
To explore the regulative role of decorin on the ECM gene-expression in diabetic nephropathy, recombinant adenovirus expressing rat decorin (Ad-decorin) was constructed to further investigate the effects of decorin overproduction on the expression of TGFbeta1 and ECM in rat mesangial cells (RMCs) in high glucose condition.


METHODS
The recombinant decorin adenovirus and lacz adenovirus(Ad-lacz), as a control, were constructed. RT-PCR, restriction enzyme digestion, western blot and gene sequence were used for validating correctness of Ad-decorin. MTT was used to examine the biological function of decorin (decorin expressed by Ad-decorin transduced CHO cells was used to interact with TGFbeta1 which can inhibit the proliferation of Mv1Lu cells). Then Ad-decorin was transferred into rat mesangial cells cultured in high-glucose (450 mg/dL) media and Ad-lacz was as the control transducer. TGFbeta1, decorin, collagen IV, fibronectin, laminin and tenascin mRNA in RMCs at 24, 48 and 72 hours after Ad-decorin infection were determined with RT-PCR. The distribution and expression of TGFbeta1 protein was detected in RMCs at 96 hours after Ad-decorin infection by immunoperoxidase cell staining.


RESULTS
RT-PCR, restriction enzyme digestion, western blot and gene sequence all confirmed that Ad-decorin could express correct decorin mRNA and protein. MTT showed that decorin protein expressed by Ad-decorin-transfected CHO cells abrogated the inhibitive effect of TGFbeta1 on the proliferation of Mv1Lu cells. Decorin mRNA significantly increased in Ad-decorin transduced RMCs at all the observed time points, reached the peak at 24 hours(2.2-fold, P<0.05) and the overexpression lasted to the end of the observation at 72 hours(1.7-fold, P<0.05) compared to that in Ad-lacz transduced RMCs. Meanwhile, TGFbeta1 mRNA level began to fall at 48 hours (-20%, P<0.05) in Ad-decorin transduced RMCs and went to the valley at 72 hours (-46, P<0.05). ECM components, such as tenascin, laminin, fibronectin and collagen IV, were reduced notably in the Ad-decorin transduced RMCs from the 48 hours to the end of study versus those in the Ad-lacz transduced RMCs. Cellular immunohistochemistry further confirmed that the Ad-decorin transduced RMCs produced much less TGFbeta1 compared with the Ad-lacz transduced RMCs.


CONCLUSIONS
The constructed recombinant decorin adenovirus can highly efficiently express biologically active decorin. Overexpression of decorin down-regulates the expression of TGFbeta1 and ECM components from RMCs. These results suggest that overexpression of decorin may be one of the therapeutic approaches to diabetic nephropathy.
Wu
F
F
Department of Endocrinology, the Affiliated Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 3 East Qingchun Road, Hangzhou 310003, China.
Yao
H
H
Bader
A
A
Dong
F
F
Zhu
F
F
Wu
N
N
Wang
B
B
Li
H
H
Brockmeyer
N H
NH
Altmeyer
P
P
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000096:biosynthesis; D058575:Decorin; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism*; D005784:Gene Amplification; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007124:Immunoenzyme Techniques; D050527:Mesangial Cells / Q000378:metabolism*; D011509:Proteoglycans / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014161:Transduction, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D000099222:betaIG-H3 Protein
D018076:DNA, Complementary; D058575:Decorin; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C546880:Dcn protein, rat