Induction of monocyte chemotactic protein 1 in colonic epithelial cells by Entamoeba histolytica is mediated via the phosphatidylinositol 3-kinase/p65 pathway

Abstract

The role intestinal epithelial cells play in the pathogenesis of amebic colitis is poorly understood. Herein, we demonstrate that secreted and soluble ameba (Entamoeba histolytica) proteins (SAP) induce expression of the chemoattractant monocyte chemotactic protein (MCP) in the colonic epithelial cell lines Caco-2, T84, and LS174T. MCP-1 mRNA induction was both dose and time dependent, with peak induction occurring at 8 h and with 100 mug/ml of SAP. Significant increase in MCP-1 protein expression was observed after 12 h. SAP failed to activate any of the mitogen-activated protein kinase pathways or IkappaB kinase activity. Moreover, inhibiting the classical pathway of NF-kappaB activation did not affect SAP-induced MCP-1 expression. Instead, we find that SAP-induced MCP-1 expression is dependent on posttranslational modification of the NFkappaB p65 subunit. SAP induced phosphorylation of p65 and enhanced NF-kappaB transcriptional activity, which are phosphatidylinositol 3-kinase (PI3 kinase) dependent. Treatment with PI3 kinase inhibitor LY290004 significantly abrogated the activation of Akt, p65, and MCP-1 mRNA induction. We conclude that colonic epithelial cells play a role in the initiation of inflammation by secreting chemokines in response to soluble ameba components.

FIG. 1.

E. histolytica components induce MCP-1 mRNA and protein production from colonic epithelial cells. (A) Confluent Caco-2 and T84 cells were treated with different concentrations of SAP for 8 h. Total cellular RNA was extracted by the TRIzol method, and real-time PCR was performed as described in Materials and Methods. **, P < 0.01; ***, P < 0.001 (over untreated controls). (B) Epithelial cells were treated with 100 μl/mg of SAP for different time periods as indicated. Results in the last lane [Eh (6)] represent results from live E. histolytica amebas (2.5 × 10⁶) in transwells incubated with colonic cell monolayers for 6 h. Data represent changes (n-fold) of mRNA expression over that for the control in experiments repeated thrice with similar results. **, P < 0.01; ***, P < 0.001 (over untreated controls). (C) Caco-2 cells were treated with 100 μl/mg of SAP for different time periods, and whole-cell lysates were subjected to SDS-PAGE and Western blot analysis with MCP-1 antibody and actin antibodies. Ctl, control.

FIG. 2.

Ameba components do not activate IKK activity. (A) Caco-2 cells were treated or not (−) with MG132 for 2 h followed by SAP for 2 h. RNA was extracted and real-time PCR for MCP-1 was done as described in the text. Experiments were repeated three times. **, P < 0.01. NS, change not significant compared to results of treatment with SAP alone. (B) Caco-2 cells were treated with different concentrations of SAP for 30 min, cell lysates were immunoprecipitated using IKK-α antibody, and an in vitro kinase assay was done using glutathione S-transferase-IκB-α as the substrate. Equal quantities (20 μg) of cellular proteins were subjected to Western blotting to check the IKK-α levels. KA, kinase assay; IB, immunoblotting; Ctl, control. A representative blot from two experiments is shown. (C) Cells were treated with 100 μg/ml of SAP for different time periods and cell lysates subjected to SDS-PAGE and Western blotting with antibodies against phosphorylated IκB (P-IκB). The blot was stripped and reprobed with actin antibodies. A blot from one of three independent experiments is shown.

FIG. 3.

SAP induces NF-κB transactivation and p65 phosphorylation. (A) Caco-2 cells were transfected with pNF-κB-Luc plasmid with or without the positive control (p-MEKK), treated with different concentrations of SAP for 6 h, and assayed for luciferase activity as described in the text. Results are from three independent studies. *, P < 0.05; **, P < 0.01; RLU, relative luciferase units compared to values for the untreated cells; +ve, positive control. (B) Cells were treated with 100 μg/ml of SAP for different time periods, nuclear extracts were prepared as described in the text, and 10 μg/ml of protein was separated by SDS-PAGE and checked for p65 nuclear translocation. The blot was stripped and reprobed with histone antibody for normalization. A blot from one of three experiments is shown. Ctl, control. (C) Whole-cell lysates from cells treated as described above were subjected to Western blotting and probed with antibody specific to phosphorylated p65 (P-p65) and then with antibody against actin. Representative blots from two experiments, in which P-p65 levels are normalized to actin following densitometric scanning of blots, are shown. Expression in control cells was assigned an arbitrary value of 1, and expressions relative to this value are shown for treated cells.

FIG. 4.

E. histolytica activates Akt. (A) Caco-2 cells were treated with 100 μg/ml of SAP for different times, and cell lysates were checked for phosphorylation of Akt by Western blotting. Blots were reprobed with actin antibody for normalization. Blots from one of three studies are shown, and values are relative densitometric units of phosphorylated Akt (P-Akt) following normalization to actin. (B) E. histolytica (Eh) trophozoites (10⁶) were added to the transwell culture with confluent Caco-2 cells at the bottom as described in Materials and Methods, and cell lysates were extracted at different times and checked for phosphorylated Akt as described above. Actin expression was analyzed to check equal loading. Each experiment was repeated twice. Ctl, control.

FIG. 5.

SAP-induced p65 phosphorylation is dependent on PI3 kinase but is independent of MAP kinases. (A) Caco-2 cells were pretreated with PI3 kinase inhibitor LY294002 for 1 h prior to SAP or E. histolytica trophozoite (Eh Trohs) (in transwell) exposure for 2 h, and whole-cell lysates were checked for phosphorylated Akt (P-Akt) levels in relation to total Akt expression. (B) Lyates from cells pretreated or not (−) with LY294002 followed by SAP for 30 min were subjected to SDS-PAGE and Western blotting using antibodies against phosphorylated p65 (P-p65) and total p65. (C) Caco-2 cells were exposed to SAP for different time periods, and cell lysates were checked for activation of MAP kinases by use of phosphospecific antibodies against Jun N-terminal protein kinase, ERK, and p38 MAP kinase. Actin expression was checked for normalization. Ctl, control. (D) Cells were pretreated with 50 μM of PD98059 (ERK inhibitor) or SB203580 (p38 inhibitor) for 2 h before incubation with SAP for 30 min, and whole-cell lysates were checked for phosphorylation of p65. The blot was stripped and reprobed with antibody against total p65. For all, representative blots from two or three independent experiments are shown.

FIG. 6.

SAP-induced MCP-1 induction is PI3 kinase dependent. Caco-2 cells were pretreated or not (−) with different concentrations of LY294002 for 1 h followed by SAP treatment for 2 h. RNA was extracted and real-time PCR done as described in the text. The experiment was repeated three times, and increase (n-fold) of MCP-1 over the level for untreated cells is depicted in the graph (*, P < 0.01 [over control]; **, P < 0.05 [over that of SAP alone]).