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Overexpression of megsin induces mesangial cell proliferation and excretion of type IV collagen in vitro
Cellular Immunology 271 (2011) 413–417
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Overexpression of megsin induces mesangial cell proliferation and excretion of type IV collagen in vitro Yunfeng Xia a, Yimin Zhang b, Wei Shi a,⇑, Shuangxin Liu a, Yuanhan Chen a, Xinling Liang a, Zhiming Ye a a b
Department of Nephrology, Guangdong Academy of Medical Sciences, Guangdong General Hospital, Guangzhou, PR China Department of Nephrology, The Second Affiliated Hospital, Sun Yat-sen University, GuangZhou, PR China
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Article history: Received 20 February 2011 Accepted 15 August 2011 Available online 5 September 2011 Keywords: Megsin Transfection Mesangial cell Proliferation Cytokine
a b s t r a c t Over-expression of megsin is associated with mesangial cell (MC) proliferation and extracellular matrix (ECM) accumulation. The underlying pathogenesis is unknown. This study demonstrate that overexpression of megsin induced incorporation of [3H]thymidine in MCs and PDGF-BB, TGF-b1 upregulation. Concentrations of PDGF-BB, TGF-b1 and type IV collagen in the culture medium of MCs transfected with megsin were higher than controls. Anti-PDGF-BB suppressed incorporation of [3H]thymidine in MCs transfected with megsin and mRNA expression of TGF-b1 in stable transformant MCs, suggesting that over-expression of megsin induces cell proliferation and ECM accumulation in MCs, upregulation of PDGF-BB and TGF-b1 is probably the main route involved in pathogenesis. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Megsin is a mesangial cell-predominant gene that encodes a predicted peptide of 380 amino acids [1]. An amino acid homology search revealed that megsin is highly homologous to members of the serine proteinase inhibitor (serpin) superfamily [2], which is a group of structurally related proteins that generally serve as extracellular, irreversible serpins. Megsin is upregulated in IgA nephropathy and diabetic nephropathy, which are both characterized by proliferation of mesangial cells (MCs) and accumulation of extracellular mesangial matrix [2,3]. Immunohistochemistry and in situ hybridization studies have shown that megsin is upregulated in the mesangial area of IgA nephropathy patients compared to normal individuals and patients with other glomerulonephritis [3,4]. Over-expression of megsin in transgenic mice leads to progressive mesangial matrix expansion and an increase in the number of MCs [5]. To date, it is uncertain whether upregulation of megsin induces proliferation of MCs and accumulation of extracellular matrix (ECM) or is only a passive phenomenon of cell proliferation. MCs play a key role in maintaining the structure and function of the glomerulus. In glomerular diseases, MCs that synthesize and secrete cytokines have important roles in the development and progress of these diseases. These cytokines include platelet⇑ Corresponding author. Address: Department of Nephrology, Guangdong Academy of Medical Sciences, Guangdong General Hospital, 106# Zhongshan Road II, Guangzhou 510080, PR China. Fax: +86 020 83827812x62027. E-mail address: [email protected] (W. Shi). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.08.009
derived growth factor-BB (PDGF-BB), transforming growth factor-b1 (TGF-b1), interleukin-10 (IL-10), interleukin-6 (IL-6), interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a), which might be associated with MC proliferation and ECM accumulation. In this study, we asked whether upregulation of megsin in MCs induces secretion of some cytokines that contribute to cell proliferation or ECM accumulation. 2. Materials and methods 2.1. Cell culture A well-characterized, cloned mesangial cell line (1097) isolated from Sprague–Dawley rats [6], was used in this study. MCs were cultured in RPMI 1640 medium (GIBCOÒ, USA) supplemented with 20% (v/v) fetal calf serum (GIBCOÒ, USA) in a 5% (v/v) CO2 atmosphere at 37 °C. 2.2. Construction of megsin gene expression vector Rat megsin cDNA was synthesized and cloned into the pMD18-T vector by the Yingjun Bioengineering Company (Shanghai, China). Hind III and BamH I sites were added to the two termini of megsin cDNA by PCR. The primers used in this step were (restriction sites underlined): sense: 50 -GTC TAA AGC TTA TGG CCT CCC TTG CTG CAG C-30 ; antisense: 50 -ACG CTG GAT CCC GAG GAC ACG AGA CTT TGC CAG-30 . The new rat megsin cDNA was cloned into the Hind III and BamH I sites of pEGFP-N1 to obtain GFP-megsin-wt, and the megsin gene expression vector pEGFP-N1-megsin was
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constructed and identified by electrophoresis and direct sequencing. The plasmid pEGFP-N1 used in this study was a generous gift from the Research Center of Stem Cells of Sun Yat-sen University (GuangZhou, China).
2.3. Gene transfection and cell proliferation assays MCs were cultured in flat-bottomed 24-well plates at 5 103 cells/well. When cells attained 70–80% confluence, transfection of pEGFP-N1-megsin was done with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. To determine the proliferation of MCs at 12, 24 and 48 h post-transfection, [3H]thymidine (2 lCi/ml) was added to each well 6 h before the assay. The incorporation of [3H]thymidine into MCs was used as a measure of DNA synthesis as described [7]. The assay was done three times and in triplicate each time. The stable transformant cells were screened and amplified using G418 (1 mg/ml).
2.6. Effects of PDGF-BB on proliferation of MCs and mRNA expression of TGF-b1 MCs were cultured in three groups: group I, MCs transfected with megsin gene and incubated with the monoclonal antibody of PDGF-BB (anti-PDGF-BB) (R&D Systems, USA) (0.05 lg/mL) for 24 h; group T, MCs transfected with megsin gene but not incubated with anti-PDGF-BB; group C, MCs transfected with the expression vector and not incubated with anti-PDGF-BB. The proliferation of MCs was measured with a [3H]thymidine incorporation assay and compared within the three groups. Measurements in each group were done three times and in triplicate each time. The stable transformant MCs were divided into similar groups: group I, MCs incubated with anti-PDGF-BB (0.05 lg/mL) for 7 days; group S, MCs not incubated with anti-PDGF-BB; group N, MCs not transfected with megsin gene and not incubated with anti-PDGFBB. The mRNA expression of TGF-b1 in MCs was detected by RTPCR and compared within the three groups. Each assay was done three times and in duplicate each time.
2.4. Gene expression analysis
2.7. Statistical analysis
The mRNA expression of PDGF-BB, TGF-b1, IL-10, IL-6, IL-1b and TNF-a in MCs transfected with megsin gene and in MCs transfected with only the expression vector (pEGFP-N1) (controls) were detected by RT-PCR. Total cellular RNA was extracted using TRIZOLÒ reagent (Life Technologies, USA). The quality of the extracted RNA was monitored by formaldehyde/agarose gel electrophoresis. RNA was quantified by measuring the absorbance (A) at 260 nm and 1.0 lg of total RNA reverse transcribed to cDNA with Moloney murine leukemia virus reverse transcriptase (MBI Fermentas, USA) in a 20 lL reaction mixture containing 50 ng of random hexamer, 0.5 mmol/L dNTPs and 20 U of RNase inhibitor. The cDNA was stored at 20 °C. The primers used for amplifying cytokines are outlined in Table 1. The mRNA expression of cytokines at 12, 24, 48 and 72 h after being transfected with megsin gene were analyzed by the IP Lab gel software from Signal Analytics (Vienna, VA, USA), and the results are expressed as a ratio of the target amplicon to b-actin amplicon. The assay was done three times and in duplicate each time.
SPSS 13.0 software was used for statistical analysis. Analysis of variance (ANOVA) and Student’s t-test were used to assess differences among multiple groups and between two groups, respectively. Statistical significance was set at P < 0.05.
2.5. ELISA Concentrations of PDGF-BB, TGF-b1 and type IV collagen in the culture medium of MCs transfected with megsin gene (samples) or in control MCs were detected by sandwich ELISA using a commercially available kit (R&D Systems) according to the manufacturer’s instructions.
3. Results 3.1. The identification of pEGFP-N1-megsin and assay of MC proliferation Electrophoresis and direct sequencing confirmed that pEGFPN1-megsin is a megsin gene expressing vector, as designed. As observed by fluorescence microscopy, about 90% of MCs were successfully transfected with pEGFP-N1-megsin and expressed green fluorescent protein. The mRNA expression of megsin in MCs transfected with megsin gene upregulated when compared with the controls (Fig. 1). The incorporation of [3H]thymidine into MCs at 24 and 48 h post-transfection was increased significantly when compared to the controls (P < 0.01 and P < 0.05, respectively; Fig. 2), and the peak [3H]thymidine incorporation was at 24 h. 3.2. Cytokine expression in MCs transfected with megsin gene We found that the mRNA expression of PDGF-BB, IL-10, IL-1b and TNF-a in MCs in the control groups was undetectable by
Table 1 Primer sequences and size of PCR products. Cytokines PDGF-BB IL-1b IL-10 IL-6 TNF-a TGF-b1 b-Actin
Primers 0
PCR products (bp) 0
5 -AATGCCAACTTCCTGGTGTG-3 50 -AACTTTCGGTGCTTCCCTTT-30 50 -GTCACTCATTGTGGCTGTGG-30 50 -GGGATTTTGTCGTTGCTTGT-30 50 -CACTGCTATGTTGCCTGCTC-30 50 -GCTCCACTGCCTTGCTTTTA-30 50 -CCACTGCCTTCCCTACTTCA-30 50 -TGGTCCTTAGCCACTCCTTC-30 50 -GGTCCCAACAAGGAGGAGA-30 50 -TGGTATGAAGTGGCAAATCG-30 50 -CGCAACAACGCAATCTATGA-30 50 -GTATCAGTGGGGGTCAGCAG-30 50 -TGGGTATGGGTCAGAAGGAC-30 50 -TAATGTCACGCACGATTTCC-30
335 327 408 498 329 230 504
Fig. 1. Mesangial cells transfected with megsin gene and comparison of the mRNA expression of megsin between samples and controls. (A) MCs transfected with megsin gene seen under the fluorescence microscope (magnification 200). Cells were fixed by cooling acetone and stained with 40 ,6-diamidino-2-phenylindole (DAPI). MCs transfected with pEGFP-N1-megsin expressed green fluorescent protein (green). (B) Comparison of the mRNA expression of megsin between samples and controls. Samples, MCs transfected with megsin gene. Controls, MCs transfected with the expression vector. ⁄P < 0.05 versus controls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The incorporation of [3H]thymidine into MCs transfected with megsin gene. MCs were cultured in flat-bottomed 24-well plates and transfected with megsin gene. Proliferation of MCs at 12, 24 and 48 h post transfection were measured by a [3H]thymidine incorporation assay. Each assay was done three times and in triplicate each time. Samples, MCs transfected with megsin gene. Controls, MCs transfected with the expression vector. ⁄P < 0.05 versus controls; ⁄⁄P < 0.01 versus controls.
RT-PCR. However, when transfected with megsin gene, a timedependent mRNA expression of PDGF-BB in MCs was detected (Fig. 3A) but the expression of IL-10, IL-1b and TNF-a remained undetectable. The mRNA expression of IL-6 and TGF-b1 was detected in MCs whether transfected with megsin gene or not. Transfection of megsin gene resulted in time-dependent upregulation of the mRNA expression of TGF-b1 (Fig. 3B), but no change was found in the mRNA expression of IL-6 (Fig. 3C). 3.3. Influence of over-expression of megsin on excretion of PDGF-BB, TGF-b1 and type IV collagen in MCs Transfection of megsin gene resulted in significantly increased concentrations of PDGF-BB and TGF-b1 in the culture medium of MCs at 24, 48 and 72 h post-transfection when compared to the controls; and the peak excretion was at 48 h (Fig. 4A and B). The excretion of type IV collagen was also increased significantly at 48 and 72 h post-transfection with megsin gene (Fig. 4C). 3.4. Effects of anti-PDGF-BB in cell proliferation and TGF-b1 expression When MCs were transfected with megsin gene and incubated with anti-PDGF-BB for 24 h, the incorporation of [3H]thymidine into MCs decreased significantly when compared with MCs transfected with megsin gene but not incubated with anti-PDGF-
Fig. 4. Concentrations of PDGF-BB, TGF-b1 and type IV collagen in the culture medium of MCs transfected with megsin gene. (A) Concentrations of PDGF-BB in the culture medium of MCs at different time post-transfection. (B) Concentrations of TGF-b1 in the culture medium of MCs at different time post-transfection. (C) Concentrations of type IV collagen in the culture medium of MCs at different time post-transfection. Concentrations of the three cytokines were measured by ELISA and compared between samples and controls. Each assay was done three times and in duplicate each time. ⁄P < 0.05 versus controls; ⁄⁄P < 0.01 versus controls.
BB (P < 0.05; Fig. 5). Incubation of the stable transformant MCs with anti-PDGF-BB for 7 days resulted in downregulation of mRNA expression of TGF-b1 (Fig. 6).
Fig. 3. The mRNA expression of PDGF-BB, TGF-b1 and IL-6 in MCs transfected with megsin gene. (A) The mRNA expression of PDGF-BB in MCs at different times posttransfection with megsin gene. (B) The mRNA expression of TGF-b1 in MCs at different times post-transfection. (C) The mRNA expression of IL-6 in MCs at different times post-transfection. The mRNA expression of PDGF-BB, TGF-b1 and IL-6 in MCs were measured by RT-PCR and compared to that of controls. Each assay was done three times and in duplicate each time. M, DNA marker; C, controls; S, stable group. ⁄P < 0.05 versus controls.
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Fig. 5. Effects of anti-PDGF-BB on proliferation of MCs transfected with megsin gene Cell proliferation was measured by a [3H]thymidine incorporation assay three times and in triplicate each time. Group I, MCs transfected with megsin gene and incubated with anti-PDGF-BB for 24 h; group T, MCs transfected with megsin gene but not incubated with anti-PDGF-BB; group C, MCs transfected with the expression vector and not incubated with anti-PDGF-BB. ⁄⁄P < 0.01 versus group C; NP < 0.05 versus group T.
Fig. 6. Effects of anti-PDGF-BB in the mRNA expression of TGF-b1 in stable transformant MCs. The mRNA expression of TGF-b1 in three groups of MCs was measured by RT-PCR and compared with each other. Each assay was done three times and in duplicate each time. Group I, stable transformant MCs incubated with anti-PDGF-BB for 7 days; group S, stable transformant MCs not incubated with antiPDGF-BB; group N, normal MCs not transfected with megsin gene and not incubated with anti-PDGF-BB. M, DNA marker. ⁄P < 0.05 versus group S.
4. Discussion As a new member of the serpin superfamily, megsin gene was cloned in 1998 and several studies suggested that over-expression of megsin is likely to be a cause of MC proliferation and ECM expansion [4,5,8], but the underlying pathogenesis is still unknown. In the related studies, many cytokines were reported to be implicated in MC proliferation and ECM expansion, among them, PDGF-BB and TGF-b1 may be the most attractive ones [9–11]. The results of our study indicate that over-expression of megsin in MCs in vitro results in cell proliferation and upregulation of mRNA expression of PDGF-BB and TGF-b1, as well as the increased secretion of PDGF-BB, TGF-b1 and type I collagen. However, no change was found in the expression of IL-10, IL-6, IL-1b or TNF-a in the MCs. Mesangial cells and their matrix form the central stalk of the glomerulus and are part of a functional unit interacting with endothelial cells and podocytes. Mesangial matrix components per se, such as type IV collagen, laminin, and fibronectin, influence mesangial cell growth and proliferation both directly and indirectly, the latter by their ability to bind growth factors, influencing their activation and release [12]. Intriguingly, it was found in this study that the proliferation of MCs and the expression of TGF-b1 in MCs transfected with megsin gene were suppressed by anti-PDGF-BB. As we know, PDGF-BB is a potent mitogen for MCs that can also stimulate ECM production in vitro [13,14]. The glomerular PDGF-B chain and its receptor
are expressed in experimental glomerulonephritis and in human IgA nephropathy [15,16]. Several studies in vivo, including the effects of recombinant PDGF-BB infusion in rats [17], the effects of intrarenal PDGF-BB gene transfer with HVJ-liposome in rats [18] and the effects of PDGF receptor tyrosine kinase inhibitor on experimental glomerulonephritis [19], all confirmed that PDGF-BB plays a key role in MC proliferation and ECM accumulation. PDGF-BB also induces the expression of TGF-b1 [9,20,21], and the latter is another important cytokine that regulates cell proliferation and ECM accumulation by stimulating their synthesis and/or decreasing the activity of some extracellular proteases [22,23]. Activation of the TGF-b1 loop leads to cell cycle arrest and ECM synthesis [24,25]. In MCs, TGF-b1 inhibits growth and stimulates the synthesis of collagens, fibronectin, laminin and proteoglycans [26–29]. In an earlier study, Miyata analyzed the abnormal glomeruli in megsin transgenic mice and observed accumulation of type IV collagen and laminin, but no accumulation of type I collagen or fibronectin was observed [5]. IL-10, IL-6, IL-1b and TNF-a are all cytokines that can be synthesized by MCs and contribute to cell proliferation or ECM accumulation by paracrine secretion [30–36]. However, no significant difference of the mRNA expression of the four cytokines in MCs transfected with megsin gene was detected in this study. To date, we know still little about megsin. This study is a preliminary exploration of the effects that megsin might have in some glomerular diseases. Our results suggest two possibilities: (1) Over-expression of megsin promotes MC proliferation and ECM accumulation in some glomerular diseases; (2) PDGF-BB and TGF-b1 play an important role in the megsin-induced cell proliferation and ECM accumulation. The upregulation of PDGF-BB probably plays a key role in promoting cell proliferation and TGF-b1 expression. Owing to the shortage of some commercial reagents regarding megsin and the paucity of our experience with megsin, there are many limitations in this study and there is a clear need for further studies. For example, as the effects of cytokines in vitro is quite different with that of cytokines in vivo, related studies in vivo should be carried out in the future. In conclusion, the results of this study suggest that overexpression of megsin causes MC proliferation and ECM accumulation. The upregulation of PDGF-BB and TGF-b1 is probably the main route involved in the pathogenesis. The upregulation of PDGF-BB probably plays a key role in stimulating cell proliferation and the excretion of TGF-b1 in MCs. Acknowledgments This study was supported by the Science and Technology Scheme Project Foundation of Guangdong Science and Technology Department No. (2007B030701003). We are grateful to Prof. Peng Xiang for the generous gift of plasmid pEGFP-N1 and helpful suggestions. References [1] R. Inagi, T. Miyata, M. Nangaku, H. Ueyama, K. Takeyama, S. Kato, K. Kurokawa, Transcriptional regulation of a mesangium predominant gene, megsin, J. Am. Soc. Nephrol. 13 (2002) 2715–2722. [2] T. Miyata, M. Nangaku, D. Suzuki, R. Inagi, K. Uragami, H. Sakai, K. Okubo, K. Kurokawa, A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy, J. Clin. Invest. 102 (1998) 828–836. [3] D. Suzuki, T. Miyata, M. Nangaku, H. Takano, N. Saotome, M. Toyoda, Y. Mori, S.Y. Zhang, R. Inagi, M. Endoh, K. Kurokawa, H. Sakai, Expression of megsin mRNA, a novel mesangium-predominant gene, in the renal tissues of various glomerular diseases, J. Am. Soc. Nephrol. 10 (1999) 2606–2613. [4] R. Inagi, T. Miyata, D. Suzuki, M. Toyoda, T. Wada, Y. Ueda, Y. Izuhara, H. Sakai, M. Nangaku, K. Kurokawa, Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy, Biochem. Biophys. Res. Commun. 286 (2001) 1098–1106.
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Overexpression of megsin induces mesangial cell proliferation and excretion of type IV collagen in vitro Yunfeng Xia a, Yimin Zhang b, Wei Shi a,⇑, Shuangxin Liu a, Yuanhan Chen a, Xinling Liang a, Zhiming Ye a a b
Department of Nephrology, Guangdong Academy of Medical Sciences, Guangdong General Hospital, Guangzhou, PR China Department of Nephrology, The Second Affiliated Hospital, Sun Yat-sen University, GuangZhou, PR China
a r t i c l e
i n f o
Article history: Received 20 February 2011 Accepted 15 August 2011 Available online 5 September 2011 Keywords: Megsin Transfection Mesangial cell Proliferation Cytokine
a b s t r a c t Over-expression of megsin is associated with mesangial cell (MC) proliferation and extracellular matrix (ECM) accumulation. The underlying pathogenesis is unknown. This study demonstrate that overexpression of megsin induced incorporation of [3H]thymidine in MCs and PDGF-BB, TGF-b1 upregulation. Concentrations of PDGF-BB, TGF-b1 and type IV collagen in the culture medium of MCs transfected with megsin were higher than controls. Anti-PDGF-BB suppressed incorporation of [3H]thymidine in MCs transfected with megsin and mRNA expression of TGF-b1 in stable transformant MCs, suggesting that over-expression of megsin induces cell proliferation and ECM accumulation in MCs, upregulation of PDGF-BB and TGF-b1 is probably the main route involved in pathogenesis. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Megsin is a mesangial cell-predominant gene that encodes a predicted peptide of 380 amino acids [1]. An amino acid homology search revealed that megsin is highly homologous to members of the serine proteinase inhibitor (serpin) superfamily [2], which is a group of structurally related proteins that generally serve as extracellular, irreversible serpins. Megsin is upregulated in IgA nephropathy and diabetic nephropathy, which are both characterized by proliferation of mesangial cells (MCs) and accumulation of extracellular mesangial matrix [2,3]. Immunohistochemistry and in situ hybridization studies have shown that megsin is upregulated in the mesangial area of IgA nephropathy patients compared to normal individuals and patients with other glomerulonephritis [3,4]. Over-expression of megsin in transgenic mice leads to progressive mesangial matrix expansion and an increase in the number of MCs [5]. To date, it is uncertain whether upregulation of megsin induces proliferation of MCs and accumulation of extracellular matrix (ECM) or is only a passive phenomenon of cell proliferation. MCs play a key role in maintaining the structure and function of the glomerulus. In glomerular diseases, MCs that synthesize and secrete cytokines have important roles in the development and progress of these diseases. These cytokines include platelet⇑ Corresponding author. Address: Department of Nephrology, Guangdong Academy of Medical Sciences, Guangdong General Hospital, 106# Zhongshan Road II, Guangzhou 510080, PR China. Fax: +86 020 83827812x62027. E-mail address: [email protected] (W. Shi). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.08.009
derived growth factor-BB (PDGF-BB), transforming growth factor-b1 (TGF-b1), interleukin-10 (IL-10), interleukin-6 (IL-6), interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a), which might be associated with MC proliferation and ECM accumulation. In this study, we asked whether upregulation of megsin in MCs induces secretion of some cytokines that contribute to cell proliferation or ECM accumulation. 2. Materials and methods 2.1. Cell culture A well-characterized, cloned mesangial cell line (1097) isolated from Sprague–Dawley rats [6], was used in this study. MCs were cultured in RPMI 1640 medium (GIBCOÒ, USA) supplemented with 20% (v/v) fetal calf serum (GIBCOÒ, USA) in a 5% (v/v) CO2 atmosphere at 37 °C. 2.2. Construction of megsin gene expression vector Rat megsin cDNA was synthesized and cloned into the pMD18-T vector by the Yingjun Bioengineering Company (Shanghai, China). Hind III and BamH I sites were added to the two termini of megsin cDNA by PCR. The primers used in this step were (restriction sites underlined): sense: 50 -GTC TAA AGC TTA TGG CCT CCC TTG CTG CAG C-30 ; antisense: 50 -ACG CTG GAT CCC GAG GAC ACG AGA CTT TGC CAG-30 . The new rat megsin cDNA was cloned into the Hind III and BamH I sites of pEGFP-N1 to obtain GFP-megsin-wt, and the megsin gene expression vector pEGFP-N1-megsin was
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constructed and identified by electrophoresis and direct sequencing. The plasmid pEGFP-N1 used in this study was a generous gift from the Research Center of Stem Cells of Sun Yat-sen University (GuangZhou, China).
2.3. Gene transfection and cell proliferation assays MCs were cultured in flat-bottomed 24-well plates at 5 103 cells/well. When cells attained 70–80% confluence, transfection of pEGFP-N1-megsin was done with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. To determine the proliferation of MCs at 12, 24 and 48 h post-transfection, [3H]thymidine (2 lCi/ml) was added to each well 6 h before the assay. The incorporation of [3H]thymidine into MCs was used as a measure of DNA synthesis as described [7]. The assay was done three times and in triplicate each time. The stable transformant cells were screened and amplified using G418 (1 mg/ml).
2.6. Effects of PDGF-BB on proliferation of MCs and mRNA expression of TGF-b1 MCs were cultured in three groups: group I, MCs transfected with megsin gene and incubated with the monoclonal antibody of PDGF-BB (anti-PDGF-BB) (R&D Systems, USA) (0.05 lg/mL) for 24 h; group T, MCs transfected with megsin gene but not incubated with anti-PDGF-BB; group C, MCs transfected with the expression vector and not incubated with anti-PDGF-BB. The proliferation of MCs was measured with a [3H]thymidine incorporation assay and compared within the three groups. Measurements in each group were done three times and in triplicate each time. The stable transformant MCs were divided into similar groups: group I, MCs incubated with anti-PDGF-BB (0.05 lg/mL) for 7 days; group S, MCs not incubated with anti-PDGF-BB; group N, MCs not transfected with megsin gene and not incubated with anti-PDGFBB. The mRNA expression of TGF-b1 in MCs was detected by RTPCR and compared within the three groups. Each assay was done three times and in duplicate each time.
2.4. Gene expression analysis
2.7. Statistical analysis
The mRNA expression of PDGF-BB, TGF-b1, IL-10, IL-6, IL-1b and TNF-a in MCs transfected with megsin gene and in MCs transfected with only the expression vector (pEGFP-N1) (controls) were detected by RT-PCR. Total cellular RNA was extracted using TRIZOLÒ reagent (Life Technologies, USA). The quality of the extracted RNA was monitored by formaldehyde/agarose gel electrophoresis. RNA was quantified by measuring the absorbance (A) at 260 nm and 1.0 lg of total RNA reverse transcribed to cDNA with Moloney murine leukemia virus reverse transcriptase (MBI Fermentas, USA) in a 20 lL reaction mixture containing 50 ng of random hexamer, 0.5 mmol/L dNTPs and 20 U of RNase inhibitor. The cDNA was stored at 20 °C. The primers used for amplifying cytokines are outlined in Table 1. The mRNA expression of cytokines at 12, 24, 48 and 72 h after being transfected with megsin gene were analyzed by the IP Lab gel software from Signal Analytics (Vienna, VA, USA), and the results are expressed as a ratio of the target amplicon to b-actin amplicon. The assay was done three times and in duplicate each time.
SPSS 13.0 software was used for statistical analysis. Analysis of variance (ANOVA) and Student’s t-test were used to assess differences among multiple groups and between two groups, respectively. Statistical significance was set at P < 0.05.
2.5. ELISA Concentrations of PDGF-BB, TGF-b1 and type IV collagen in the culture medium of MCs transfected with megsin gene (samples) or in control MCs were detected by sandwich ELISA using a commercially available kit (R&D Systems) according to the manufacturer’s instructions.
3. Results 3.1. The identification of pEGFP-N1-megsin and assay of MC proliferation Electrophoresis and direct sequencing confirmed that pEGFPN1-megsin is a megsin gene expressing vector, as designed. As observed by fluorescence microscopy, about 90% of MCs were successfully transfected with pEGFP-N1-megsin and expressed green fluorescent protein. The mRNA expression of megsin in MCs transfected with megsin gene upregulated when compared with the controls (Fig. 1). The incorporation of [3H]thymidine into MCs at 24 and 48 h post-transfection was increased significantly when compared to the controls (P < 0.01 and P < 0.05, respectively; Fig. 2), and the peak [3H]thymidine incorporation was at 24 h. 3.2. Cytokine expression in MCs transfected with megsin gene We found that the mRNA expression of PDGF-BB, IL-10, IL-1b and TNF-a in MCs in the control groups was undetectable by
Table 1 Primer sequences and size of PCR products. Cytokines PDGF-BB IL-1b IL-10 IL-6 TNF-a TGF-b1 b-Actin
Primers 0
PCR products (bp) 0
5 -AATGCCAACTTCCTGGTGTG-3 50 -AACTTTCGGTGCTTCCCTTT-30 50 -GTCACTCATTGTGGCTGTGG-30 50 -GGGATTTTGTCGTTGCTTGT-30 50 -CACTGCTATGTTGCCTGCTC-30 50 -GCTCCACTGCCTTGCTTTTA-30 50 -CCACTGCCTTCCCTACTTCA-30 50 -TGGTCCTTAGCCACTCCTTC-30 50 -GGTCCCAACAAGGAGGAGA-30 50 -TGGTATGAAGTGGCAAATCG-30 50 -CGCAACAACGCAATCTATGA-30 50 -GTATCAGTGGGGGTCAGCAG-30 50 -TGGGTATGGGTCAGAAGGAC-30 50 -TAATGTCACGCACGATTTCC-30
335 327 408 498 329 230 504
Fig. 1. Mesangial cells transfected with megsin gene and comparison of the mRNA expression of megsin between samples and controls. (A) MCs transfected with megsin gene seen under the fluorescence microscope (magnification 200). Cells were fixed by cooling acetone and stained with 40 ,6-diamidino-2-phenylindole (DAPI). MCs transfected with pEGFP-N1-megsin expressed green fluorescent protein (green). (B) Comparison of the mRNA expression of megsin between samples and controls. Samples, MCs transfected with megsin gene. Controls, MCs transfected with the expression vector. ⁄P < 0.05 versus controls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The incorporation of [3H]thymidine into MCs transfected with megsin gene. MCs were cultured in flat-bottomed 24-well plates and transfected with megsin gene. Proliferation of MCs at 12, 24 and 48 h post transfection were measured by a [3H]thymidine incorporation assay. Each assay was done three times and in triplicate each time. Samples, MCs transfected with megsin gene. Controls, MCs transfected with the expression vector. ⁄P < 0.05 versus controls; ⁄⁄P < 0.01 versus controls.
RT-PCR. However, when transfected with megsin gene, a timedependent mRNA expression of PDGF-BB in MCs was detected (Fig. 3A) but the expression of IL-10, IL-1b and TNF-a remained undetectable. The mRNA expression of IL-6 and TGF-b1 was detected in MCs whether transfected with megsin gene or not. Transfection of megsin gene resulted in time-dependent upregulation of the mRNA expression of TGF-b1 (Fig. 3B), but no change was found in the mRNA expression of IL-6 (Fig. 3C). 3.3. Influence of over-expression of megsin on excretion of PDGF-BB, TGF-b1 and type IV collagen in MCs Transfection of megsin gene resulted in significantly increased concentrations of PDGF-BB and TGF-b1 in the culture medium of MCs at 24, 48 and 72 h post-transfection when compared to the controls; and the peak excretion was at 48 h (Fig. 4A and B). The excretion of type IV collagen was also increased significantly at 48 and 72 h post-transfection with megsin gene (Fig. 4C). 3.4. Effects of anti-PDGF-BB in cell proliferation and TGF-b1 expression When MCs were transfected with megsin gene and incubated with anti-PDGF-BB for 24 h, the incorporation of [3H]thymidine into MCs decreased significantly when compared with MCs transfected with megsin gene but not incubated with anti-PDGF-
Fig. 4. Concentrations of PDGF-BB, TGF-b1 and type IV collagen in the culture medium of MCs transfected with megsin gene. (A) Concentrations of PDGF-BB in the culture medium of MCs at different time post-transfection. (B) Concentrations of TGF-b1 in the culture medium of MCs at different time post-transfection. (C) Concentrations of type IV collagen in the culture medium of MCs at different time post-transfection. Concentrations of the three cytokines were measured by ELISA and compared between samples and controls. Each assay was done three times and in duplicate each time. ⁄P < 0.05 versus controls; ⁄⁄P < 0.01 versus controls.
BB (P < 0.05; Fig. 5). Incubation of the stable transformant MCs with anti-PDGF-BB for 7 days resulted in downregulation of mRNA expression of TGF-b1 (Fig. 6).
Fig. 3. The mRNA expression of PDGF-BB, TGF-b1 and IL-6 in MCs transfected with megsin gene. (A) The mRNA expression of PDGF-BB in MCs at different times posttransfection with megsin gene. (B) The mRNA expression of TGF-b1 in MCs at different times post-transfection. (C) The mRNA expression of IL-6 in MCs at different times post-transfection. The mRNA expression of PDGF-BB, TGF-b1 and IL-6 in MCs were measured by RT-PCR and compared to that of controls. Each assay was done three times and in duplicate each time. M, DNA marker; C, controls; S, stable group. ⁄P < 0.05 versus controls.
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Fig. 5. Effects of anti-PDGF-BB on proliferation of MCs transfected with megsin gene Cell proliferation was measured by a [3H]thymidine incorporation assay three times and in triplicate each time. Group I, MCs transfected with megsin gene and incubated with anti-PDGF-BB for 24 h; group T, MCs transfected with megsin gene but not incubated with anti-PDGF-BB; group C, MCs transfected with the expression vector and not incubated with anti-PDGF-BB. ⁄⁄P < 0.01 versus group C; NP < 0.05 versus group T.
Fig. 6. Effects of anti-PDGF-BB in the mRNA expression of TGF-b1 in stable transformant MCs. The mRNA expression of TGF-b1 in three groups of MCs was measured by RT-PCR and compared with each other. Each assay was done three times and in duplicate each time. Group I, stable transformant MCs incubated with anti-PDGF-BB for 7 days; group S, stable transformant MCs not incubated with antiPDGF-BB; group N, normal MCs not transfected with megsin gene and not incubated with anti-PDGF-BB. M, DNA marker. ⁄P < 0.05 versus group S.
4. Discussion As a new member of the serpin superfamily, megsin gene was cloned in 1998 and several studies suggested that over-expression of megsin is likely to be a cause of MC proliferation and ECM expansion [4,5,8], but the underlying pathogenesis is still unknown. In the related studies, many cytokines were reported to be implicated in MC proliferation and ECM expansion, among them, PDGF-BB and TGF-b1 may be the most attractive ones [9–11]. The results of our study indicate that over-expression of megsin in MCs in vitro results in cell proliferation and upregulation of mRNA expression of PDGF-BB and TGF-b1, as well as the increased secretion of PDGF-BB, TGF-b1 and type I collagen. However, no change was found in the expression of IL-10, IL-6, IL-1b or TNF-a in the MCs. Mesangial cells and their matrix form the central stalk of the glomerulus and are part of a functional unit interacting with endothelial cells and podocytes. Mesangial matrix components per se, such as type IV collagen, laminin, and fibronectin, influence mesangial cell growth and proliferation both directly and indirectly, the latter by their ability to bind growth factors, influencing their activation and release [12]. Intriguingly, it was found in this study that the proliferation of MCs and the expression of TGF-b1 in MCs transfected with megsin gene were suppressed by anti-PDGF-BB. As we know, PDGF-BB is a potent mitogen for MCs that can also stimulate ECM production in vitro [13,14]. The glomerular PDGF-B chain and its receptor
are expressed in experimental glomerulonephritis and in human IgA nephropathy [15,16]. Several studies in vivo, including the effects of recombinant PDGF-BB infusion in rats [17], the effects of intrarenal PDGF-BB gene transfer with HVJ-liposome in rats [18] and the effects of PDGF receptor tyrosine kinase inhibitor on experimental glomerulonephritis [19], all confirmed that PDGF-BB plays a key role in MC proliferation and ECM accumulation. PDGF-BB also induces the expression of TGF-b1 [9,20,21], and the latter is another important cytokine that regulates cell proliferation and ECM accumulation by stimulating their synthesis and/or decreasing the activity of some extracellular proteases [22,23]. Activation of the TGF-b1 loop leads to cell cycle arrest and ECM synthesis [24,25]. In MCs, TGF-b1 inhibits growth and stimulates the synthesis of collagens, fibronectin, laminin and proteoglycans [26–29]. In an earlier study, Miyata analyzed the abnormal glomeruli in megsin transgenic mice and observed accumulation of type IV collagen and laminin, but no accumulation of type I collagen or fibronectin was observed [5]. IL-10, IL-6, IL-1b and TNF-a are all cytokines that can be synthesized by MCs and contribute to cell proliferation or ECM accumulation by paracrine secretion [30–36]. However, no significant difference of the mRNA expression of the four cytokines in MCs transfected with megsin gene was detected in this study. To date, we know still little about megsin. This study is a preliminary exploration of the effects that megsin might have in some glomerular diseases. Our results suggest two possibilities: (1) Over-expression of megsin promotes MC proliferation and ECM accumulation in some glomerular diseases; (2) PDGF-BB and TGF-b1 play an important role in the megsin-induced cell proliferation and ECM accumulation. The upregulation of PDGF-BB probably plays a key role in promoting cell proliferation and TGF-b1 expression. Owing to the shortage of some commercial reagents regarding megsin and the paucity of our experience with megsin, there are many limitations in this study and there is a clear need for further studies. For example, as the effects of cytokines in vitro is quite different with that of cytokines in vivo, related studies in vivo should be carried out in the future. In conclusion, the results of this study suggest that overexpression of megsin causes MC proliferation and ECM accumulation. The upregulation of PDGF-BB and TGF-b1 is probably the main route involved in the pathogenesis. The upregulation of PDGF-BB probably plays a key role in stimulating cell proliferation and the excretion of TGF-b1 in MCs. Acknowledgments This study was supported by the Science and Technology Scheme Project Foundation of Guangdong Science and Technology Department No. (2007B030701003). We are grateful to Prof. Peng Xiang for the generous gift of plasmid pEGFP-N1 and helpful suggestions. References [1] R. Inagi, T. Miyata, M. Nangaku, H. Ueyama, K. Takeyama, S. Kato, K. Kurokawa, Transcriptional regulation of a mesangium predominant gene, megsin, J. Am. Soc. Nephrol. 13 (2002) 2715–2722. [2] T. Miyata, M. Nangaku, D. Suzuki, R. Inagi, K. Uragami, H. Sakai, K. Okubo, K. Kurokawa, A mesangium-predominant gene, megsin, is a new serpin upregulated in IgA nephropathy, J. Clin. Invest. 102 (1998) 828–836. [3] D. Suzuki, T. Miyata, M. Nangaku, H. Takano, N. Saotome, M. Toyoda, Y. Mori, S.Y. Zhang, R. Inagi, M. Endoh, K. Kurokawa, H. Sakai, Expression of megsin mRNA, a novel mesangium-predominant gene, in the renal tissues of various glomerular diseases, J. Am. Soc. Nephrol. 10 (1999) 2606–2613. [4] R. Inagi, T. Miyata, D. Suzuki, M. Toyoda, T. Wada, Y. Ueda, Y. Izuhara, H. Sakai, M. Nangaku, K. Kurokawa, Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy, Biochem. Biophys. Res. Commun. 286 (2001) 1098–1106.
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