- Email: [email protected]
SMADS Pathway in High Glucose Circumstances
Nuclear Translocation of SMAD3 May Enhance the TGF-/SMADS Pathway in High Glucose Circumstances Q. Li, F. Ye, Y. Shi, L. Zhang, W. Wang, Z. Tu, J. Qiu, J. Wang, S. Li, H. Bu, and Y. Li ABSTRACT Objectives. Posttransplant diabetes mellitus is one of the most frequent complications after kidney transplantation. It is considered to be one cause of chronic allograft nephropathy. This study sought to investigate the effects of high glucose on the expression and nuclear translocation of Smad3, which is an important signal mediator involved in the fibrotic signal pathway. Methods. The established rat renal mesangial cell line HBZY-1 was cultured in medium with various concentrations of glucose (4.5 mg/mL, 9.0 mg/mL, or 13.5 mg/mL), which was collected at 7, 14, or 21 days. The total expression of Smad3, including both inner and outer nucleus proteins was examined by Western blot analysis. The nuclear translocated Smad3, representing only the inner nucleus protein, was detected by immunofluorescence staining observed under a laser confocal scanning microscope. Results. No significant difference in the total Smad3 expression was demonstrated by Western blot analysis among the three groups of HBZY-1 cells at various concentration of glucose after 7, 14, or 21 days. There was no fluorescence detected in the nucleus at day 7 by immunofluorescence staining; however, robust positive expression of Smad3 was detected at days 14 and 21. Conclusion. As a restricted Smads member, Smad3 protein might not be upregulated in the presence of high glucose. However, with prolonged culture time, Smad3 translocates from cytoplasm to nucleus, which may be a pivotal step in the fibrotic signal pathway.
P
OSTTRANSPLANT DIABETES MELLITUS is a frequent, serious complication after kidney transplantation. The elevated blood glucose concentration may alter the microenvironment of cells in the transplanted kidney. Many studies suggest that hyperglycemia is important in the activation of transforming growth factor- (TGF-) pathway.1–5 TGF- has been confirmed to be the most important molecule in fibrotic lesions, playing an important role in chronic allograft nephropathy. Many molecules participate to mediate the extracellular TGF- signal translocation to the nucleus. Among them, the Smads family is considered to be the most important one. However, effects of TGF- on Smads intracellular signaling driven by higher glucose effects are unclear. MATERIALS AND METHODS Cell Culture The rat mesangial (HBZY-1) cell line was obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum in saturated humidity and 5% CO2. They were passaged about every 2 days. The cultured cells, divided into three groups, were cultured in DMEM with glucose at 4.5 mg/mL (normal level), 9.0 mg/mL, or 13.5 mg/mL for 7, 14, or 21 days.
From the Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, P. R. China. Supported in part by a grant from the National Basic Research Program of China (No. 2003CB515504) and Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education, and in part by the National Natural Scientific Foundation (No. 30571761), and by the Foundation of China Medical Board of New York Inc. (CMB, Inc). Address reprint requests to Hong Bu, Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041 P. R. China. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.092
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2158
Transplantation Proceedings, 38, 2158 –2160 (2006)
NUCLEAR TRANSLOCATION OF SMAD3
2159
Antibodies and Reagents Smad3 goat polyclonal IgG, -actin mouse monoclonal IgG, rabbit anti-goat IgG-HRP secondary antibody, and goat anti-mouse IgGHRP secondary antibody were products of Santa Cruz. Biotinylated protein ladder and HRP-conjugated anti-biotin antibody were from Cell Signaling Technology.
Western Blotting HBZY-1 cells were lysed in SDS-PAGE loading buffer. After separation on 12% SDS-PAGE, the proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked for 30 minutes in TBS containing 0.1% Tween 20 (TBS-T) and 8% (w/v) dry skim milk powder. The PVDF membranes were incubated overnight with specific Smad3 goat polyclonal IgG (1:200). The membrane was then washed with TBS-T and incubated for 2 hours with rabbit anti-goat IgG-HRP secondary antibody (1:4000). -actin mouse monoclonal IgG (1:1000) and goat anti-mouse IgG-HRP secondary antibody (1:4000) were used to ascertain the expression of -actin as a loading control. Biotinylated protein ladder (10 L) and HRPconjugated anti-biotin antibody (1:1000) were used to quantitate the molecular weight of the protein on the PVDF membrane.
Immunofluorescence Staining Cells were grown on glass slides in DMEM in the presence of various concentration of glucose for different times as described above. Then the cells were fixed with 4% paraformaldehyde for 15 minutes at 4°C and permeabilized in 0.1% Triton X-100 for 10 minutes at 4°C. The cells were then blocked in 10% rabbit blocking serum at 37°C for 25 minutes followed by incubation with Smad3 goat polyclonal IgG (1:100) for 1 hour. After washes with PBS three times, cells were incubated with FITC-conjugated rabbit anti-goat IgG (1:20) at 37°C for 1 hour. The results were evaluated under a confocal laser scanning microscope.
RESULTS Total Smad3 Expression
The total expression of Smad3 in all groups with longer culturing times was detected by Western blot analysis. As showed in Fig 1, the quantity of Smad3 did not increase compared with the control group in all nine samples. The Nuclear Translocation of Smad3
To track the location of Smad3 in the cell, Smad3 was detected with FITC-conjugated antibody and observed under a laser confocal scanning microscope (LCSM). The value of integrated optical density (IOD) of fluorescence was used as an indicator to represent the quantity of Smad3. The value of the nucleus in the high glucose groups did not vary compared with the control group at 7 days. This value increased in the high glucose groups at 14 and particularly 21 days compared with the control group. DISCUSSION
Posttransplant diabetes mellitus is one factor that disturbs the normal microenvironment of the newly transplanted kidney. Several studies have revealed that hyperglycemia
Fig 1. The expression of Smad3 in different glucose concentrations with accumulated time. 1 ⫽ 4.5 mg/mL glucose in DMEM in 7 days; 2 ⫽ 9.0 mg/mL glucose in DMEM in 7 days; 3 ⫽ 13.5 mg/mL glucose in DMEM in 7 days; 4 ⫽ 4.5 mg/mL glucose in DMEM in 14 days; 5 ⫽ 9.0 mg/mL glucose in DMEM in 14 days; 6 ⫽ 13.5 mg/mL glucose in DMEM in 14 days; 7 ⫽ 4.5 mg/mL glucose in DMEM in 21 days; 8 ⫽ 9.0 mg/mL glucose in DMEM in 21 days; 9 ⫽ 13.5 mg/mL glucose in DMEM in 21 days.
might activate the TGF- signal pathway, leading to fibrosis.6 –11 Our experiments were focused on the intracellular signaling events of the TGF- pathway in high concentrations of glucose. As shown in Fig 1, high concentrations of glucose did not affect the total expression of Smad3 in HBZY-1 cells. The immunofluorescence staining showed that at an early stage of hyperglucose in the cultures, and the expression of Smad3 was restricted to the plasma. However, robust expression of Smad3 could be seen in the nucleus under LSCM with extended culture times in high glucose media. Several members of the Smads family are involved in downstream TGF- signal pathway, which mediates the extracellular signals to the nucleus to stimulate transcription of target genes. The intracellular signaling cascade is activated after TGF- binds to its membrane receptor complex. The TGF--receptor complex relays the extracellular signals into the cell by phosphorylating its intracellular substrates Smad2/Smad3.12,13 Then the phosphorylated Smad2/Smad3 combines with Smad4 to form a complex that translocates to the nucleus and functionally collaborates with distinct transcription factors to turn off or turn on TGF-regulated gene expression.14 The nuclear translocation of Smad3 is a restriction step in the TGF- signal pathway. We have observed a stability of the total quantity of Smad3 expressed in the HBZY-1 cell line in the presence of high glucose. This result is similar to the report about the stability of total Smad3 in human peritoneal mesothelial cells.1 Our results suggested that high glucose seemed to stimulate Smad3 translocation from cytoplasm to nucleus to activate the TGF- signal pathway, thus leading to fibrosis and chronic allograft nephropathy. REFERENCES 1. Yao Q, Qian JQ, Lin XH, et al: Int J Artif Organs 27:828, 2004 2. Hayashida T, Schnaper HW, et al: J Am Soc Nephrol 15:2032, 2004
2160 3. Li JH, Huang XR, Zhu HJ, et al: Kidney Int 63:2010, 2003 4. Isono M, Chen S, Hong SW, et al: Biochem Biophys Res Commun 296:1356, 2002 5. Abdel-Wahab N, Wicks SJ, et al: Biochem J 362:643, 2002 6. Rocco M, Chen Y, et al: Kidney Int 41:107, 1992 7. Ziyadeh FN, Sharma K, Ericksen M, et al: J Clin Invest 93:536, 1994 8. Hoffman B, Sharma K, Zhu Y, et al: Kidney Int 54:1107, 1998 9. Han DC, Isono M, Hoffman BB, et al: J Am Soc Nephrol 10:1891, 1999
LI, YE, SHI ET AL 10. Border WA, Yamamoto T, Noble NA, et al: Diabetes Metab Rev 12:309, 1996 11. Reeves WB, Andreoli TE, et al: Proc Natl Acad Sci USA 97:7667, 2000 12. Liu X, Sun Y, Constantinescu SN, et al: Proc Natl Acad Sci USA 94:10669, 1997 13. Nakao A, Imamura T, Souchelnystkyi S, et al: Embo J 16:5353, 1997 14. Xuedong L, Yin S, Robert A, et al: Cytokine Growth F R 12:1, 2001
P
OSTTRANSPLANT DIABETES MELLITUS is a frequent, serious complication after kidney transplantation. The elevated blood glucose concentration may alter the microenvironment of cells in the transplanted kidney. Many studies suggest that hyperglycemia is important in the activation of transforming growth factor- (TGF-) pathway.1–5 TGF- has been confirmed to be the most important molecule in fibrotic lesions, playing an important role in chronic allograft nephropathy. Many molecules participate to mediate the extracellular TGF- signal translocation to the nucleus. Among them, the Smads family is considered to be the most important one. However, effects of TGF- on Smads intracellular signaling driven by higher glucose effects are unclear. MATERIALS AND METHODS Cell Culture The rat mesangial (HBZY-1) cell line was obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum in saturated humidity and 5% CO2. They were passaged about every 2 days. The cultured cells, divided into three groups, were cultured in DMEM with glucose at 4.5 mg/mL (normal level), 9.0 mg/mL, or 13.5 mg/mL for 7, 14, or 21 days.
From the Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, P. R. China. Supported in part by a grant from the National Basic Research Program of China (No. 2003CB515504) and Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education, and in part by the National Natural Scientific Foundation (No. 30571761), and by the Foundation of China Medical Board of New York Inc. (CMB, Inc). Address reprint requests to Hong Bu, Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041 P. R. China. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.092
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2158
Transplantation Proceedings, 38, 2158 –2160 (2006)
NUCLEAR TRANSLOCATION OF SMAD3
2159
Antibodies and Reagents Smad3 goat polyclonal IgG, -actin mouse monoclonal IgG, rabbit anti-goat IgG-HRP secondary antibody, and goat anti-mouse IgGHRP secondary antibody were products of Santa Cruz. Biotinylated protein ladder and HRP-conjugated anti-biotin antibody were from Cell Signaling Technology.
Western Blotting HBZY-1 cells were lysed in SDS-PAGE loading buffer. After separation on 12% SDS-PAGE, the proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked for 30 minutes in TBS containing 0.1% Tween 20 (TBS-T) and 8% (w/v) dry skim milk powder. The PVDF membranes were incubated overnight with specific Smad3 goat polyclonal IgG (1:200). The membrane was then washed with TBS-T and incubated for 2 hours with rabbit anti-goat IgG-HRP secondary antibody (1:4000). -actin mouse monoclonal IgG (1:1000) and goat anti-mouse IgG-HRP secondary antibody (1:4000) were used to ascertain the expression of -actin as a loading control. Biotinylated protein ladder (10 L) and HRPconjugated anti-biotin antibody (1:1000) were used to quantitate the molecular weight of the protein on the PVDF membrane.
Immunofluorescence Staining Cells were grown on glass slides in DMEM in the presence of various concentration of glucose for different times as described above. Then the cells were fixed with 4% paraformaldehyde for 15 minutes at 4°C and permeabilized in 0.1% Triton X-100 for 10 minutes at 4°C. The cells were then blocked in 10% rabbit blocking serum at 37°C for 25 minutes followed by incubation with Smad3 goat polyclonal IgG (1:100) for 1 hour. After washes with PBS three times, cells were incubated with FITC-conjugated rabbit anti-goat IgG (1:20) at 37°C for 1 hour. The results were evaluated under a confocal laser scanning microscope.
RESULTS Total Smad3 Expression
The total expression of Smad3 in all groups with longer culturing times was detected by Western blot analysis. As showed in Fig 1, the quantity of Smad3 did not increase compared with the control group in all nine samples. The Nuclear Translocation of Smad3
To track the location of Smad3 in the cell, Smad3 was detected with FITC-conjugated antibody and observed under a laser confocal scanning microscope (LCSM). The value of integrated optical density (IOD) of fluorescence was used as an indicator to represent the quantity of Smad3. The value of the nucleus in the high glucose groups did not vary compared with the control group at 7 days. This value increased in the high glucose groups at 14 and particularly 21 days compared with the control group. DISCUSSION
Posttransplant diabetes mellitus is one factor that disturbs the normal microenvironment of the newly transplanted kidney. Several studies have revealed that hyperglycemia
Fig 1. The expression of Smad3 in different glucose concentrations with accumulated time. 1 ⫽ 4.5 mg/mL glucose in DMEM in 7 days; 2 ⫽ 9.0 mg/mL glucose in DMEM in 7 days; 3 ⫽ 13.5 mg/mL glucose in DMEM in 7 days; 4 ⫽ 4.5 mg/mL glucose in DMEM in 14 days; 5 ⫽ 9.0 mg/mL glucose in DMEM in 14 days; 6 ⫽ 13.5 mg/mL glucose in DMEM in 14 days; 7 ⫽ 4.5 mg/mL glucose in DMEM in 21 days; 8 ⫽ 9.0 mg/mL glucose in DMEM in 21 days; 9 ⫽ 13.5 mg/mL glucose in DMEM in 21 days.
might activate the TGF- signal pathway, leading to fibrosis.6 –11 Our experiments were focused on the intracellular signaling events of the TGF- pathway in high concentrations of glucose. As shown in Fig 1, high concentrations of glucose did not affect the total expression of Smad3 in HBZY-1 cells. The immunofluorescence staining showed that at an early stage of hyperglucose in the cultures, and the expression of Smad3 was restricted to the plasma. However, robust expression of Smad3 could be seen in the nucleus under LSCM with extended culture times in high glucose media. Several members of the Smads family are involved in downstream TGF- signal pathway, which mediates the extracellular signals to the nucleus to stimulate transcription of target genes. The intracellular signaling cascade is activated after TGF- binds to its membrane receptor complex. The TGF--receptor complex relays the extracellular signals into the cell by phosphorylating its intracellular substrates Smad2/Smad3.12,13 Then the phosphorylated Smad2/Smad3 combines with Smad4 to form a complex that translocates to the nucleus and functionally collaborates with distinct transcription factors to turn off or turn on TGF-regulated gene expression.14 The nuclear translocation of Smad3 is a restriction step in the TGF- signal pathway. We have observed a stability of the total quantity of Smad3 expressed in the HBZY-1 cell line in the presence of high glucose. This result is similar to the report about the stability of total Smad3 in human peritoneal mesothelial cells.1 Our results suggested that high glucose seemed to stimulate Smad3 translocation from cytoplasm to nucleus to activate the TGF- signal pathway, thus leading to fibrosis and chronic allograft nephropathy. REFERENCES 1. Yao Q, Qian JQ, Lin XH, et al: Int J Artif Organs 27:828, 2004 2. Hayashida T, Schnaper HW, et al: J Am Soc Nephrol 15:2032, 2004
2160 3. Li JH, Huang XR, Zhu HJ, et al: Kidney Int 63:2010, 2003 4. Isono M, Chen S, Hong SW, et al: Biochem Biophys Res Commun 296:1356, 2002 5. Abdel-Wahab N, Wicks SJ, et al: Biochem J 362:643, 2002 6. Rocco M, Chen Y, et al: Kidney Int 41:107, 1992 7. Ziyadeh FN, Sharma K, Ericksen M, et al: J Clin Invest 93:536, 1994 8. Hoffman B, Sharma K, Zhu Y, et al: Kidney Int 54:1107, 1998 9. Han DC, Isono M, Hoffman BB, et al: J Am Soc Nephrol 10:1891, 1999
LI, YE, SHI ET AL 10. Border WA, Yamamoto T, Noble NA, et al: Diabetes Metab Rev 12:309, 1996 11. Reeves WB, Andreoli TE, et al: Proc Natl Acad Sci USA 97:7667, 2000 12. Liu X, Sun Y, Constantinescu SN, et al: Proc Natl Acad Sci USA 94:10669, 1997 13. Nakao A, Imamura T, Souchelnystkyi S, et al: Embo J 16:5353, 1997 14. Xuedong L, Yin S, Robert A, et al: Cytokine Growth F R 12:1, 2001