Differential Effects of Inhibitor Combinations on Lysophosphatidic Acid-Mediated Chemokine Secretion in Unprimed and Tumor Necrosis Factor-α-Primed Synovial Fibroblasts

Abstract

Lysophosphatidic acid (LPA) is a pleiotropic bioactive lysophospholipid involved in inflammatory mediator synthesis. Signaling through p38MAPK, ERK, Rho kinase, and MSK-CREB contributes to LPA-mediated IL-8 production in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. The study was undertaken to investigate how LPA activates MSKs and how signaling crosstalk between TNFα and LPA contributes to the super-production of cytokines/chemokines. RAFLS pretreated or not with TNFα were stimulated with LPA. Immunoblotting with phospho-antibodies monitored MSK activation. Cytokine/chemokine production was measured using ELISA and multiplex immunoassays. LPA induced MSK activation by signaling through ERK whereas p38MAPK, Rho kinase, NF-κB or PI3K contribute to IL-8 synthesis mainly via MSK-independent pathways. Priming with TNFα enhanced LPA-mediated MSK phosphorylation and cytokine/chemokine production. After priming with TNFα, inhibition of ERK or MSK failed to attenuate LPA-mediated IL-8 synthesis even if the MSK-CREB signaling axis was completely or partially inhibited. In TNFα-primed cells, inhibition of LPA-mediated cytokine/chemokine synthesis required a specific combination of inhibitors such as p38MAPK and ERK for IL-8 and IL-6, and Rho kinase and NF-κB for MCP-1. The ability of the signaling inhibitors to block LPA induced cytokine/chemokine synthesis is dependent on the inflammatory cytokinic environment. In TNFα-primed RAFLS the super-production of IL-8 and IL-6 induced by LPA occurs mainly via MSK-independent pathways, and simultaneous inhibition of at least two MAPK signaling pathways was required to block their synthesis. Since simultaneous inhibition of both the p38MAPK and ERK-MSK-CREB pathways are required to significantly reduce LPA-mediated IL-8 and IL-6 production in TNFα-preconditioned RAFLS, drug combinations targeting these two pathways are potential new strategies to treat rheumatoid arthritis.

Keywords: CREB Fibroblast-like synoviocytes; ERK; MSK; lysophosphatidic acid; p38MAPK; tumor necrosis factor-α.

FIGURE 1

Priming of RAFLS with TNFα transiently enhances MSK phosphorylation and CREB phosphorylation, but not MSKs or CREB protein expression. RAFLS were treated with TNFα (80 ng/ml) for indicated time points. Cell lysates were subjected to Western blot analyses with the indicated antibodies. (A) Kinetics of MSK1/MSK2 and CREB phosphorylation in response to stimulation with TNFα. The upper panel is a Western blot representative of three independent experiments and lower panels are the densitometry quantification analysis. Only the upper band recognized by the p-MSK1/2 antibody was scanned and normalized to actin. (B) Priming of RAFLS with TNFα for 8 h did not affect the expression of MSKs or CREB. Upper panels are Western blots representative of at least three independent experiments and lower panels are the densitometry quantification analysis. Depending on the protein molecular weight data were normalized with respect to actin or GAPDH as a loading control. The basal phospho-protein levels in unstimulated RAFLS were set to 100%. Data are the mean value ± SEM. The values in A and B were subjected to a one-way ANOVA, Dunnett’s multiple comparison test and T-test, respectively. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 2

Priming of RAFLS with TNFα enhances LPA-mediated MSK phosphorylation and CREB phosphorylation. RAFLS were treated with or without TNFα (80 ng/ml) for 8 h before stimulation with 5 μM LPA for 5 min (A–F), 10 min (G,H), and 30 min (I,J). Cell lysates were subjected to Western blot analyses with the indicated antibodies. The left (A,C,E,G,I) are Western blots representative of independent experiments with similar results. The right (B; n = 22), (D; n = 17), (F; n = 10), (H; n = 3), and (J; n = 4) are the densitometry quantification analysis. In panel A only the upper band recognized by the p-MSK1/2 antibody was scanned. Data were normalized with respect to actin or GAPDH as a loading control. The non-treated sample (NT) was set to 100% for comparison between experiments. Data are the mean value ± SEM. The values were subjected to a two-way ANOVA, Sidak’s multiple comparison test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

FIGURE 3

Inhibition of ERK1/2 blocks LPA-mediated MSK1 Ser-376/MSK2 Ser-360 phosphorylation in TNFα-primed RAFLS. RAFLS were treated with or without TNFα (80 ng/ml) for 8 h before stimulation with 5 μM LPA for 5 min. Where indicated the cells were pre-treated for 30 min with the inhibitors prior to stimulation with LPA. The levels of p-MSK1 Ser-376/MSK2 Ser-360 were monitored as described in “Materials and Methods.” Upper panels are Western blots representative of 4 (A,C) or 6 (B,D) independent experiments with similar results. Lower panels are the densitometry quantification of p-MSK1 Ser376/MSK2 Ser-360. Only the upper band recognized by the p-MSK1/2 antibody was scanned and normalized with respect to actin as a loading control. The non-treated sample (NT) was set to 100% for comparison between experiments. Data are the mean value ± SEM. The resulting values were subjected to a two-way ANOVA, Sidak’s multiple comparison test. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 4

Inhibition of ERK1/2 blocks LPA-mediated MSK1 Ser-212 phosphorylation in TNFα-primed RAFLS. The cells were pre-treated with the inhibitor of p38MAPK (A), Rho kinase (B), ERK (C), and PI3K (D) for 30 min prior to stimulation with LPA. The samples were analyzed exactly as described in Figure 3, except that p-MSK1 Ser-212 was monitored. Data are the mean value ± SEM. The values were subjected to a two-way ANOVA, Sidak’s multiple comparison test. ^∗∗p < 0.01.

FIGURE 5

Combinations of signaling inhibitors attenuate LPA-mediated MSK phosphorylation. RAFLS were treated with or without TNFα (80 ng/ml) for 8 h before stimulation with 5 μM LPA for 5 min. Where indicated, cell samples were pre-treated for 30 min with the indicated combinations of inhibitors prior to stimulation with LPA. Inhibitor concentrations were 10 μM for PD98059, SB203580, and Y27632, and 200 nM for wortmannin. The levels of p-MSK1/MSK2 were monitored as described in Materials and Methods. Upper (A–D) are Western blots representative of at least 3 independent experiments with similar results. The lower panels are the densitometry quantification of p-MSK1 Ser376/MSK2 Ser-360 (A,B) and p-MSK1 Ser-212 (C,D) from 3 (A–C) or 6 (D) independent experiments. Protein bands were scanned and data were normalized with respect to actin as a loading control. The non-treated sample was set to 100% for comparison between experiments. Data are the mean value ± SEM. The resulting values were subjected to a one-way ANOVA, Dunnett’s multiple comparison test for selected groups. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

FIGURE 6

Lysophosphatidic acid-mediated CREB phosphorylation in TNFα-primed RAFLS is reduced by the inhibitors of ERK, p38MAPK, and MSKs. RAFLS were treated with or without TNFα (80 ng/ml) for 8 h before stimulation with 5 μM LPA for 5 min. Where indicated, cell samples were pre-treated for 30 min with the indicated inhibitors or their combination prior to stimulation with LPA. Left (A,C,E) are Western blots representative of at least three independent experiments with similar results. Right (B,D,F) are the densitometry quantification analysis. Data were normalized with respect to GAPDH as a loading control. The non-treated sample (NT) was set to 100% for comparison between experiments. Data are the mean value ± SEM. The resulting values in (B,D) were subjected to a two-way ANOVA, Sidak’s multiple comparison test. The values in F were subjected to a one-way ANOVA, Dunnett’s multiple comparison test for selected groups. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

FIGURE 7

Priming of RAFLS with TNFα enhances LPA-mediated cytokine and chemokine secretion. RAFLS were treated with or without TNFα (80 ng/ml) for 8 h before stimulation with 5 μM LPA for 24 h. The supernatants were collected and the levels of IL-8 (A), IL-6 (B), IP-10 (C), RANTES (D), and MCP-1 (E) were monitored using a Luminex immunoassay as described in “Materials and Methods.” Data are the mean value ± SEM from at least three independent experiments. The values were subjected to Student’s t-test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 8

The sensitivity of LPA-mediated IL-8 secretion to signaling inhibitors is altered after priming of RAFLS with TNFα. RAFLS were treated with or without TNFα (80 ng/ml) for 8 h before stimulation with 5 μM LPA for 24 h. Where indicated, cell samples were pre-treated for 30 min with the indicated concentrations of the p38MAPK (A), ERK1/2 (B), Rho kinase (C), MSK (D), PI3K (E), or NF-κB (F) inhibitor prior to stimulation with LPA. The supernatants were collected and the IL-8 ELISA was performed. Data are the mean value ± SEM (pg/ml) from 3 (B–D,F) or 6 (E) independent experiments. The values were subjected to Student’s t-test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 9

Combinations of signaling inhibitors differentially impact LPA-mediated IL-8, IL-6, IP-10, MCP-1 and RANTES secretion in TNFα-primed cells. RAFLS were treated with or without TNFα (80 ng/ml) for 8 h before stimulation with 5 μM LPA for 24 h. Where indicated, cells were pre-treated for 30 min with the indicated concentrations of the p38MAPK (A), ERK1/2 (B), Rho kinase (C), MSK (D), PI3K (E), or NF-kB (F) inhibitor prior to stimulation with LPA. The supernatants were collected and the levels of IL-8 (A), IL-6 (B), IP-10 (C), MCP-1 (D), and RANTES (E) were monitored using a Luminex immunoassay as described in “Materials and Methods.” The level of cytokine/chemokine produced by TNFα-primed RAFLS stimulated with LPA was set to 100% for comparison between experiments. Data are the mean value ± SEM from 4 (A,B,E) or 3 (C,D) independent experiments. The values were subjected to a one-way ANOVA, Dunnett’s multiple comparison test. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 10

Proposed signaling pathways involved in LPA-induced IL-8 secretion in TNFα-primed RAFLS.