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Decorin Gene Transfer Inhibited the Expression of Tgfβ1 and Ecm in Rat Mesangial Cells

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Decorin Gene Transfer Inhibited the Expression of Tgfβ1 and Ecm in Rat Mesangial Cells 360 EU RO PE AN JOUR NAL OF MED I CAL RE SEARCH August 16, 2007 Eur J Med Res (2007) 12: 360-368 © I. Holzapfel Publishers 2007 DECORIN GENE TRANSFER INHIBITED THE EXPRESSION OF TGFβ1 AND ECM IN RAT MESANGIAL CELLS F. Wu1, H. Yao2, A. Bader3, F. Dong2, F. Zhu1, N. Wu2, B. Wang1, H. Li1, N. H. Brockmeyer3, P. Altmeyer3 1Department of Endocrinology, the Affiliated Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China; 2Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China; 3Department of Dermatology and Allergy, Ruhr-University, Bochum, Germany Abstract chemistry further comfirmed that the Ad-decorin Objective: To explore the regulative role of decorin on transduced RMCs produced much less TGFβ1 com- the ECM gene-expression in diabetic nephropathy, re- pared with the Ad-lacz transduced RMCs. combinant adenovirus expressing rat decorin (Ad- Conclusion: The constructed recombinant decorin ade- decorin) was constructed to further investigat the ef- novirus can highly efficiantly express biologically ac- fects of decorin overproduction on the expression of tive decorin. Overexpression of decorin down-regu- TGFβ1 and ECM in rat mesangial cells (RMCs) in lates the expression of TGFβ1 and ECM components high glucose condition. from RMCs. These results suggest that overexpression Methods: The recombinant decorin adenovirus and of decorin may be one of the theraputic approaches lacz adenovirus(Ad-lacz), as a control, were construct- to diabetic nephropathy. ed. RT-PCR, restriction enzyme digestion, western blot and gene sequence were used for validating cor- INTRODUCTION rectness of Ad-decorin. MTT was used to examine the biological function of decorin (decorin expressed by Diabetic nephropathy (DN) is associated with the ac- Ad-decorin transduced CHO cells was used to interact cumulation of extracellular matrix (ECM) proteins in with TGFβ1 which can inhibit the proliferation of the glomerulus and is represented morphologically by Mv1Lu cells). Then Ad-decorin was transferred into thickening and expansion of the glomerular basement rat mesangial cells cultured in high-glucose membrane and the mesangium. Hyperglycemia is the (450mg/dL) media and Ad-lacz was as the control primary etiologic factor in the metabolic abnormalities transducer. TGFβ1, decorin, collagen IV, fibronectin, and vascular complications of diabetes. Prolonged ex- lamnin and tenascin mRNA in RMCs at 24, 48 and 72 posure to high glucose is an important contributor to hours after Ad-decorin infection were determined the development of diabetic nephropathy both in with RT-PCR. The distribution and expression of types 1 and 2 diabetes [1, 2, 3]. Although the mecha- TGFβ1 protein was detected in RMCs at 96 hours af- nisms underlying the effects of chronic hyperglycemia ter Ad-decorin infection by immunoperoxidase cell on the kidney are not fully understood, TGFβ1 is now staining. considered to be a key molecule that aggravates dia- Results: RT-PCR, restriction enzyme digestion, west- betic nephropathy [4, 5, 6]. ern blot and gene sequence all confirmed that Ad- Several lines of evidence revealed critical roles of decorin could express correct decorin mRNA and TGFβ1 during the progression of glomerular lesions protein. MTT showed that decorin protein expressed in diabetic nephropathy: 1) TGFβ1 expression is up- by Ad-decorin-transfected CHO cells abrogated the regulated by glucose and enhances extracellular matrix inhibitive effect of TGFβ1 on the proliferation of (ECM) accumulation in mesangial cells [7] ; 2) TGFβ1 Mv1Lu cells. Decorin mRNA significantly increased in expression levels are markedly increased in mesangial Ad-decorin transduced RMCs at all the observed time areas in animals or in patients after the onset of dia- points, reached the peak at 24 hours(2.2-fold, P <0.05) betic nephropathy [8]; and 3) importantly, neutraliza- and the overexpression lasted to the end of the obser- tion of TGFβ1 actions with a specific antibody sup- vation at 72hours(1.7-fold, P <0.05) compared to that presses glomerular hypertrophy as well as sclerosis in in Ad-lacz transduced RMCs. Meanwhile, TGFβ1 vivo [9, 10]. A distortion of the balance between ECM mRNA level began to fall at 48 hours (-20%, P <0.05) synthesis and turnover may result in an abnormal in Ad-decorin transduced RMCs and went to the val- ECM accumulation in the mesangium in diabetic ley at 72 hours (-46, P <0.05). ECM components, such nephropathy. TGFβ stimulates the synthesis of key as teascin, laminin, fibronectin and collagen IV, were extracellular matrix molecules including type I colla- reduced notably in the Ad-decorin transduced RMCs gen, type IV collagen, fibronectin, and laminin. And from the 48 hours to the end of study versus those in TGFβ also decreases matrix degradation by inhibiting the Ad-lacz transduced RMCs. Cellular immunohisto- proteases as well as activating protease inhibitors [9, 11, 12, 13]. Although the pathogenesis of glomeru- losclerosis is uncertain, it is likely that all three major cells of the glomerulus participate in the fibrotic The first two authors contributed equally to this study. process. Among the resident cells of the glomerulus, August 16, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 361 mesangial cells are primarily responsible for excessive CONSTRUCTION OF ADENOVIRUS-DECORIN ECM deposition [10] and ECM accumulation often (AD-DECORIN) PLASMID appears to begin in the mesangium [14]. There is in- creasing evidence indicating that mesangial autocrine Decorin gene fragment digested by BglII and HindIII activation of TGFβ mediates the effects of high glu- was inserted into the pShuttle-CMV vector(Stratagene cose concentrations [15]. Company, USA) digested with same enzymes. The in- All above provide one strategy to inhibit the pro- sert fragment was confirmed by restriction digestion gression of diabetic nephropathy. One such approach and sequence analysis. Purified shuttle vector plus is the use of the endogenous proteoglycan decorin, gene of decorin in sufficient quantity was linearized by which is refered to as the naturally occurring inhibitor PmeI restriction enzyme. For the recombination, the of TGFβ [16]. Decorin is a small leucine-rich Proteo- BJ5183-AD-1 cells (Stratagene Company, USA) were glycan, and consists of a 40 kD core protein and one transformed with the linearized shuttle vector (con- glycosaminoglycan chain. It is known for its ability to taining the gene of decorin). A recombination event interact with several matrix molecules, including vari- that took place in the bacterial cells resulted in the ous types of collagen, fibronectin, thrombospondin production of recombinant AdEasy plasmid DNA in- and growth factors. Most of these interactions are me- cluding decorin gene (Ad-decorin plasmid). And the diated by the core protein [17]. More importantly, transformed cell suspension was inoculated on LB there appears to be a role for decorin in the regulation agar plates with kanamycin (50µg/ml). After 18h, of TGFβ activity [16, 18]. clonies were picked on the plates and identified by In the present study, we constructed a recombinant PCR with vector-primers firstly. The olignucleotide adenovirus of decorin gene to further investigate the primers (Sangong, China) were used as follows: functional ability of overexpression of decorin to 5’-GAAGTGAAATCTGAATAATTTTGT (upstream), block the selected effects of TGFβ1 in rat mesangial 5’-GTGGGGGTCTTATGTAGTTTTG, (downstream). cells. The size of amplified PCR product is 1181bp. The clonies containing exact decorin gene were confirmed METHODS with digestion of PacI, BglII and HindIII restricion enzyme and PCR. Recombinant Ad-decorin plasmid EXTRACTION OF TOTAL RNA was digested with PacI enzyme and purified. The Ad-decorin plasmid or Ad-lacz (β-galactosi- Total RNA in the kidney of a 8weeks SD rat (Animal dase gene) plasmid was transferred with calcium phos- study center of Zhejiang University) was abstracted in phate into Ad293 cells (Human adenovirus trans- accordance with the manufacturer’s introductions. The formed kidney cells, Stratagene company, USA.), re- RNA was washed with 75% ethanol and briefly vacu- spectively. After incubation at 37 °C for 10 days, the lum-dried. In the end, the RNA was dissolved in Ad293 cells, which appeared cytopathic effects, were DEPC-H2O. The integrity of the total RNA was ana- harvested to freeze-thaw and vortex for 3 times to lyzed by 1% agarose gel electrophoresis alongside produce virus. The resulting adenovirus was amplified RNA marker, and the purity of the total RNA was and purified and plaque tittered on Ad293 cells. Final- checked by the ration of OD260/280. ly, the titer of adenovirus was adapt to 1×1010 pfu/ml with phosphate-buffer saline (PBS). Another adeno- CDNA SYNTHESIS AND AMPLIFICATION OF virus vector containing β-galactosidase gene (Ad-lacz) DECORIN GENE alone was constructed by the same way as a control. First-strand cDNA was synthesized by reverse tran- IDENTIFYING THE EXPRESSION OF DECORIN IN CHO scription(RT) of 2 µg total RNA using oligo dT18 and CELLS 200u superscript II reverse transcriptase(Invitrogen) at 42 °C for 70 min according to the protocal. PCR were Confluent CHO cells (Chinese Hamster Overy cells, carried out in a final volume of 50µl with 2µl of dena- the American Type Cell Culture Collection), 1.0 × tured cDNA and 2.5u Taq DNA polymerase High Fi- 10 6/60 mm dish, were treated with 1.0×10 8 pfu Ad- delity platinum(Invitrogen), 1µM of both primers and decorin or Ad-lacz, in 2ml/60 mm-dish of serum free PCR buffer containing 1.5mm Mgcl2 and 200µm of media. After 24h of incubation, the supernatant was each dNTP. The olignucleotide primers(Sangong, Chi- discarded and cells were washed 2 times with PBS. 5ml na) were used as follows: 5’-GCAGATCTATGAAG of standard medium per 60 mm-dish was added. Un- GCAACTCTCGTCTT-3’ (upstream) and 5’-CGAAG infected cells, cultured in the same conditions, served CTTGCTTACTTGTAGTTCCCAAG-3’(down- an additional negative control.
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