Volume 56, Issue 4, Pages 1378-1390 (October 1999) TGF-βbgr-activating kinase-1 inhibits cell cycle and expression of cyclin D1 and A in LLC-PK1 cells  Yoshio Terada, Osamu Nakashima, Seiji Inoshita, Michio Kuwahara, Sei Sasaki, Fumiaki Marumo  Kidney International  Volume 56, Issue 4, Pages 1378-1390 (October 1999) DOI: 10.1046/j.1523-1755.1999.00665.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Effect of the TAK1 pathway and MKK1 pathway on [3H]-thymidine incorporation in LLC-PK1 cells. (A) Cells were transfected with plasmids containing TAK1, TAK1dN, TAK1K63W, or pNK11 (vector) and incubated with DMEM medium containing 10% FCS in the presence or absence of TGF-βbgr (20 ng/ml) for 24 hours. [3H]-thymidine incorporation was measured during the last 4 hours. SB203580 (10 μM; a p38K inhibitor) was added just after the transfection of TAK1dN plasmid. Results are means ± SEM of five or six independent experiments. Symbols are: (□) TGF-βbgr; () control; *P < 0.05; #P < 0.01. (B) The cells were transfected with wild type MKK1, MKK1(S222A), MKK1(S222E), or pCDNA3 (vector) and incubated with DMEM medium without FCS. PD98059 (100 μM; MKK1 inhibitor) was added just after the transfection of MKK1 (S222E). Results are the means ± SEM of five or six independent experiments (*P < 0.05; #P < 0.01; NS is not significant). Kidney International 1999 56, 1378-1390DOI: (10.1046/j.1523-1755.1999.00665.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Effects of TAK1 activation on protein expression of cyclins D1, D2, D3, E, and A. Immunoblots of cyclin D1 (A), cyclin D2 (B), cyclin D3 (C), cyclin E (D), and cyclin A (E) were performed using whole cell lysates of LLC-PK1 cells. Exponentially growing cells were transfected with TAK1dN or empty vector (pNK11) and incubated with DMEM medium containing 10% FCS. Whole cell lysates were separated by SDS-PAGE gels. Cyclin D1, D2, D3, E, and A protein levels were detected by Western blot analysis. For the detection of cyclin D1 and cyclin E, the primary antibodies (diluted 1/2500) and a second antibody, horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (diluted 1/2500), were used. The bands were visualized by the Amersham ECL system with 5 to 10 minutes of exposure. For the detection of cyclins D2, D3, and A, the primary antibodies (diluted 1/1000) and a second antibody, HRP-conjugated goat antirabbit IgG (diluted 1/2500), were used. The bands were visualized by the Amersham ECL system with 10 to 30 minutes of exposure after extensive washing of the membranes. Immunoblots of cyclins D1, D2, D3, E, and A were performed at least five times. Values were obtained by densitometry of immunoblots of cyclin D1 (A), cyclin D2 (B), cyclin D3 (C), cyclin E (D), and cyclin A (E), and expression is presented as fraction of expression in control (pNK11). Results are the means ± SEM of five independent experiments (*P < 0.05). Kidney International 1999 56, 1378-1390DOI: (10.1046/j.1523-1755.1999.00665.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Involvement of the MKK3/6-p38K in TAK1 pathway in LLC-PK1 cells. (A) Cells were transfected with plasmids containing wild TAK1, TAK1dN, TAK1K63W, or pNK11 (vector) and incubated with DMEM medium containing 10% FCS in the absence of TGF-βbgr for 24 hours. SB203580 (10 μM; p38K inhibitor) was added just after the transfection of TAK1dN. Immunoblots of phospho-specific MKK3/6 (A) and cyclin D1 (D) were performed using whole cell lysates of LLC-PK1 cells. As described in the methods section, phosphorylation of p38K (B) and JNK (C) was detected by immunoprecipitation using p38K and JNK antibodies followed by immunoblots with phospho-specific p38K and JNK antibodies. For the detection of cyclin D1, phospho-specific MKK3/6, p38K, and JNK, primary antibodies (diluted 1/2500) and a second antibody, horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (diluted 1/2500), were used. The bands were visualized by the Amersham ECL system with 5 to 10 minute exposure. For the detection of phospho-specific MKK3/6, p38K, and JNK, the primary antibodies (diluted 1/1500) and a second antibody, HRP-conjugated goat antirabbit IgG (diluted 1/2500), were used. The bands were visualized by the Amersham ECL system with 10 to 30 minutes of exposure after extensive washing of the membranes. Immunoblots of cyclin D1. Values were obtained by densitometry of immunoblots of phospho-specific MKK3/6 (A), phospho-specific p38K (B), phospho-specific JNK (C), and cyclin D1 (D), and expression is presented as fraction of expression in control (pNK11). Results are means ± SEM of five independent experiments (*P < 0.05). Kidney International 1999 56, 1378-1390DOI: (10.1046/j.1523-1755.1999.00665.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 Involvement of the TAK1-MKK6-p38K pathway in cyclin D1 promoter activity and protein expression in LLC-PK1 cells. (A) Cells were transfected with plasmids containing TAK1, TAK1dN, TAK1K63W, or pNK11 (vector) and incubated with DMEM containing 10% FCS in the presence (□) or absence () of TGF-βbgr (20 ng/ml) for 24 hours. LLC-PK1 cells were cotransfected with cyclin D1 promoter-luciferase construct and βbgr-galactosidase reporter construct. SB203580 (10 μM) was added just after the transfection of TAK1dN. Luciferase enzyme units were normalized to βbgr-galactosidase. The N-fold increase in luciferase activity was calculated relative to the basal level of cyclin D1-luciferase transfected with the empty vector. Results are means ± SEM of five or six independent experiments (*P < 0.05; #P < 0.01). (B) After cotransfecting the LLC-PK1 cells with cyclin D1 promoter-luciferase construct, βbgr-galactosidase reporter construct, and expression vectors containing TAK1, MKK6, and p38K constructs, they were incubated with DMEM medium containing 10% FCS in the absence of TGF-βbgr for 24 hours. The N-fold increase in luciferase activity was calculated relative to the level of cyclin D1-luciferase activity transfected with TAK1dN, MKK6(Glu), and p38K. Results are means ± SEM of four or six independent experiments. (#P < 0.01). Kidney International 1999 56, 1378-1390DOI: (10.1046/j.1523-1755.1999.00665.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 Involvement of the TAK1-MKK6-p38K pathway in cyclin A promoter activity in LLC-PK1 cells. (A) Cells were cotransfected with cyclin A promoter-luciferase construct, βbgr-galactosidase reporter construct, and expression vectors containing TAK1 constructs, TAK1, TAK1dN, TAK1K63W, or pNK11 (vector), and they were then incubated with DMEM medium containing 10% FCS in the presence (□) or absence () of TGF-βbgr (20 ng/ml) for 24 hours. LLC-PK1 cells were cotransfected with cyclin A promoter-luciferase construct, βbgr-galactosidase reporter construct, and expression vectors containing TAK1 constructs. SB203580 (10 μM) was added after the transfection of TAK1dN. Luciferase enzyme units were normalized to βbgr-galactosidase. The N-fold increase in luciferase activity was calculated relative to the basal level of cyclin A-luciferase transfected with the empty vector. Results are means ± SEM of five or six independent experiments (*P < 0.05; #P < 0.01). (B) Immunoblots of cyclin A were performed using whole cell lysate of LLC-PK1 cells. For the detection of cyclin A the primary antibodies (diluted 1/1000) and a second antibody, horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (diluted 1/2500), were used. The bands were visualized by the Amersham ECL system with 10 to 30 minutes of exposure after extensive washing of the membranes. Values were obtained by densitometry of immunoblots of cyclin cyclin A, and expression is presented as fraction of expression in control (pNK11). Results are means ± SEM of five independent experiments (*P < 0.05). (C) LLC-PK1 cells were cotransfected with cyclin A promoter-luciferase construct, βbgr-galactosidase reporter construct, and expression vectors containing TAK1, MKK6, p38K constructs, and they were then incubated with DMEM containing 10% FCS in the absence of TGF-βbgr for 24 h. The N-fold increase in luciferase activity was calculated relative to the level of cyclin A-luciferase transfected with TAK1dN, MKK6(Glu), and p38K(TGY). Results are means ± SEM of four or six independent experiments (#P < 0.01). Kidney International 1999 56, 1378-1390DOI: (10.1046/j.1523-1755.1999.00665.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 6 Stimulation of cyclin A and cyclin D1 promoter activity by the MKK1-p44/p42 MAP kinase pathway. (A) After cotransfecting the cells with cyclin A promoter-luciferase construct, βbgr-galactosidase reporter construct, and expression vectors containing MKK1 constructs [wild type MKK1, MKK1(S222E), MKK1(S222A), or pCDNA3 (vector)], they were incubated with DMEM medium without FCS. PD98059 (100 μM; MKK1 inhibitor) was added just after the transfection of MKK1(S222E). Results are means ± SEM of four or five independent experiments (*P < 0.05; #P < 0.01). (B) Whole cell lysates were separated by SDS-PAGE gels. The cyclin A protein levels were detected by Western blot analysis. For the detection of cyclin A, the primary antibodies (diluted 1/1000) and a second antibody, horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (diluted 1/2500), were used. The bands were visualized by the Amersham ECL system with a 10 to 30 minute exposure after extensive washing of the membranes. Values were obtained by densitometry of immunoblots of cyclin cyclin A, and expression is presented as fraction of expression in control (pNK11). Results are means ± SEM of four independent experiments (*P < 0.05). (C) After cotransfecting the cells with cyclin D1 promoter-luciferase construct, βbgr-galactosidase reporter construct, and expression vectors containing MKK1 constructs [wild type MKK1, MKK1(S222E), MKK1(S222A), or pCDNA3 (vector)], and incubated with DMEM medium without FCS. PD98059 (100 μM; MKK1 inhibitor) was added just after the transfection of MKK1(S222E).Whole cell lysates were separated by SDS-PAGE gels. Cyclin D1 protein levels were detected by Western blot analysis. Luciferase enzyme units were normalized to βbgr-galactosidase. The N-fold increase in luciferase activity was calculated relative to the basal level of luciferase activity transfected with the empty vector. Results are means ± SEM of four or five independent experiments (*P < 0.05; #P < 0.01). (D) Whole cell lysates were separated by SDS-PAGE gels. The cyclin D1 protein levels were detected by Western blot analysis. For the detection of cyclin D1 the primary antibodies (diluted 1/2500) and a second antibody, HRP-conjugated goat antirabbit IgG (diluted 1/2500), were used. The bands were visualized by the Amersham ECL system with 5 to 10 minutes of exposure after extensive washing of the membranes. Values were obtained by densitometry of immunoblots of cyclin cyclin D1, and expression is presented as fraction of expression in control (pNK11). Results are means ± SEM of four independent experiments (*P < 0.05). Kidney International 1999 56, 1378-1390DOI: (10.1046/j.1523-1755.1999.00665.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 7 Effects of TAK1 pathway on cell cycle progression in LLC-PK1 cells. LLC-PK1 cells expressing pNK11 (empty vector) or TAK1dN were incubated in DMEM medium with or without 10% FCS for 24 hours. The percentages of S phase and G2/M phase were analyzed by flow cytometry using a FACS flow cytometer. Cell cycle analyses by flow cytometry were performed for four times. Kidney International 1999 56, 1378-1390DOI: (10.1046/j.1523-1755.1999.00665.x) Copyright © 1999 International Society of Nephrology Terms and Conditions