Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: Role of PI 3-kinase and ERK  Richard J. Dixon, Nigel J. Brunskill  Kidney International  Volume 56, Issue 6, Pages 2064-2075 (December 1999) DOI: 10.1046/j.1523-1755.1999.00797.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 The p85/p110 phosphatidylinositol 3-kinase (PI 3-kinase) activity in opossom kidney (OK) cell antiphosphotyrosine immunoprecipitates. Serum starved, wild-type, and δp85 OK cells were immunoprecipitated with antiphosphotyrosine antibodies under control conditions (no agonist stimulation) or after stimulation with 10 μ M lysophosphatidic acid (LPA) in the presence or absence of PD98059. The immunoprecipitate was used for in vitro assay of PI 3-kinase using PtdIns as the substrate. Deacylated [32P]-lipid products were separated by high-performance liquid chromatography, and representative elution profiles are displayed. (A) In immunoprecipitates from wild-type cells under control conditions, no appreciable PI 3-kinase activity is observed. (B) When wild-type cells are stimulated with 10 μ M LPA, PI 3-kinase activity becomes associated with tyrosine-phosphorylated proteins and catalyzes the formation of PtdIns(3) as indicated. (C) Preincubation of wild-type cells with 5 μ M PD98059 for 20 minutes has no effect on PI 3-kinase activity stimulated by 10 μ M LPA. (D) In noninduced δp85 OK cells, LPA stimulates a similar response in PI 3-kinase activity. (E) The induction of δp85 expression by IPTG completely abolishes PI 3-kinase activity in response to 10 μ M LPA, the PtdIns(3)P peak being completely diminished. The elution profiles shown are representative of at least three experiments for each condition. Kidney International 1999 56, 2064-2075DOI: (10.1046/j.1523-1755.1999.00797.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Inhibition of PI 3-kinase blocks LPA-induced proliferation in OK cells. (A) Serum-starved cells (approximately 70% confluent) were pretreated (■) or not (▪) with 100 n M wortmannin for 20 minutes. Cells then washed three times with serum-free media and were stimulated with appropriate agents in serum-free media for 24 hours. (B) This shows the effect of LPA on δp85-transfected OK cells. δp85 expression was induced by pre-exposure of the cells to 5 m M IPTG for 18 hours. Control cells were not treated with IPTG. Serum-starved cells were then treated with appropriate agents in serum-free media for 24 hours. The results are presented as percentages ± SEM (derived from at least three experiments with four replicates for each condition) of the control cells, which were incubated with serum-free media. *P < 0.05; **P < 0.01; ***P < 0.001. Kidney International 1999 56, 2064-2075DOI: (10.1046/j.1523-1755.1999.00797.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 LPA causes an increase in phosphorylation of ERK proteins in OK cells. Serum-starved cells were treated with 10 μ M LPA for various times and were then lyzed. Cell lysates were resolved by SDS-polyacrylamide electrophoresis and blotted with antibodies against active (phosphorylated) ERK (see Methods section). This is a representative immunoblot from three independent experiments. Kidney International 1999 56, 2064-2075DOI: (10.1046/j.1523-1755.1999.00797.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 LPA causes an increase in ERK activity in OK cells. (A) Serum-starved OK cells were incubated with 10 μ M LPA for varying times. Cells were then lyzed, and ERK activity was measured by in vitro kinase assay. (B) Serum-starved OK cells were incubated with various concentrations of LPA for five minutes. Cells were then lyzed, and ERK activity was measured by in vitro kinase assay. Results are presented as percentages (± SEM, N = 3) of the control cells that were incubated with serum-free media. Kidney International 1999 56, 2064-2075DOI: (10.1046/j.1523-1755.1999.00797.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 PD98059 inhibits LPA-induced ERK activity and proliferation in OK cells. (A) Serum-starved cells were treated with the appropriate concentration of PD98059 for 20 minutes and were then incubated with 10 μ M LPA, 10% FCS, or serum-free media for five minutes. Cells were then lyzed, and ERK was activity measured by in vitro kinase assay. Results are presented as percentages (± SEM, N = 3) of the control cells that were incubated with serum-free media. Symbols are: (▪) control; (■) 50 μ M PD; (□) 5 μ M PD; (□) 500 n M PD. (B) Serum-starved cells were pretreated with the indicated concentration of PD98059 for 20 minutes and were then stimulated with 10 μ M LPA, 10% FCS, or serum-free media for 24 hours. PD98059 was present throughout the incubation period. Results are presented as percentages (± SEM, N = 3) of the control cells that (■) 5 μ M PD; (■) 500 n M PD; (□) 50 n M PD. *P < 0.05; **P < 0.01; ***P < 0.001. Kidney International 1999 56, 2064-2075DOI: (10.1046/j.1523-1755.1999.00797.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 6 LPA-induced ERK activation is not affected by inhibition of PI 3-kinase. (A) Serum-starved cells were pretreated (□) or not (▪) with 100 n M wortmannin for 20 minutes and were then stimulated with appropriate agent for five minutes. Cells were then lyzed and ERK activity was measured by in vitro kinase assay. (B) The effect of LPA on ERK activity in δp85-transfected OK cells. Symbols are: (▪) control; (□) IPTG pretreated. δp85 expression was induced by pre-exposure of the cells to 5 m M IPTG for 18 hours. Serum-starved cells were then treated with appropriate agents in serum-free media for five minutes. Cells were then lyzed, and ERK activity was measured by in vitro kinase assay. Results are presented as percentages (± SEM, N = 3) of the control cells that were incubated with serum-free media. *P < 0.05. (C) δp85-transfected OK cells were pretreated with IPTG and were serum starved. Cells were treated with 10 μ M LPA for various times and were then lyzed. Cell lysates were resolved by SDS-PAGE and blotted with antibodies against active (phosphorylated) ERK. This is a representative immunoblot from three independent experiments. Kidney International 1999 56, 2064-2075DOI: (10.1046/j.1523-1755.1999.00797.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 7 Pertussis toxin (PTX) pretreatment inhibits LPA-induced ERK activity in OK cells. OK cells were pretreated for 18 hours with 50 ng/ml PTX and then stimulated with agonists for five minutes. Cells then lysed and ERK activity was measured by in vitro kinase assay. Results are presented as percentages (± SEM, N = 3) of the control cells, which were incubated with serum-free media. Symbols are: (▪) no PTX; (□) PTX pretreatment; *P < 0.05; **P < 0.01. Kidney International 1999 56, 2064-2075DOI: (10.1046/j.1523-1755.1999.00797.x) Copyright © 1999 International Society of Nephrology Terms and Conditions