Lipopolysaccharide-promoted proliferation of Caco-2 cells is mediated by c-Src induction and ERK activation

Abstract

As a major component of the cell wall of Gram-negative bacteria, lipopolysaccharide (LPS) can be released into the bloodstream to cause a spectrum of pathophysiological reactions. Despite the fact that colon epithelium cells in situ are continuously exposed to LPS, their biological responses as provoked by LPS as well as the underlying mechanisms are poorly defined. In the present study, we observed that LPS directly stimulated growth of Caco-2 cells as well as enhanced the amounts of c-Src, which could be partly attributable to increased c-src transcript. Parallel to LPS-induced c-Src expression was FAK activation and ERK activation. Remarkably, activation of ERK and cellular proliferation by LPS could be inhibited by PP2, the specific Src inhibitor, implicating the essential role of c-Src in this process. To our knowledge, this is the first report indicating that LPS can increase cellular growth via upregulation of c-Src in colon epithelial cells.

Keywords: Caco-2;; ERK; LPS;; c-Src;.

Fig. 1

LPS accelerates proliferation in Caco-2 cells. Caco-2 cells (5 × 10⁵) were plated at the beginning. After 18 h, cells were incubated with or without 10 μg/ml LPS for various times as indicated. Total number of control and LPS-treated cells were counted and plotted. The results were shown in means ± SD for three independent experiments performed in triplicate. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001 as compared to its control counterpart.

Fig. 2

LPS treatment leads to the enhancement of protein tyrosyl phosphorylation in Caco-2 cells. Caco-2 cells were treated with or without 10 μg/ml LPS for various times as indicated. Equal amounts of lysates (100 μg) from each sample were resolved by SDS-PAGE and probed with anti-pTyr Ab. Similar results were repeated three times and the representative was demonstrated. The protein markers (KDa) were shown on the left.

Fig. 3

Expression of c-Src is increased in LPS-stimulated Caco-2 cells. (A) Caco-2 cells were treated with or without LPS (10 μg/ml) for various times as indicated. Equal amounts of lysates (100 μg) from each sample were resolved by SDSPAGE and probed with antibodies against c-Src and actin. (B) Densitometric quantification of c-Src expression normalized for actin protein levels; values were means ± SD from three measurements of (A).

Fig. 4

The enhancement of transcript abundance of c-src in LPS-treated Caco-2 cells. (A) Caco-2 cells were treated with various concentration of LPS for 96 h. Total RNA was then harvested. Semi-quantitative RT-PCR analysis of c-src was performed as described in “Materials and Methods”. β-actin was utilized as an internal control for amplification of src efficiency. (B) Densitometric quantification of c-src transcript normalized for β-actin; values were mean ± SD from three measurements of (A).

Fig. 5

LPS increases Y-397 phosphorylated FAK and ERK activation in Caco-2 cells. Caco-2 cells were incubated with LPS (10 μg/ml) for various times as indicated. Their content of Y-397 phosphorylated FAK, FAK, phosphorylated ERK and ERK was analyzed by direct Western blot analysis, with specific antibody against Pi-Y397 FAK, FAK, phosphorylated ERK (E10), and ERK, respectively.

Fig. 6

PP2 suppresses the LPS-increased ERK activation and proliferation in Caco-2 cells. Caco-2 cells were pretreated with PP2 (10 μM) for 20 min and then stimulated with or without LPS (10 μg/ml) for 96 h. (A) The content of phosphorylated ERK and ERK were determined by direct Western blot analysis with specific antibody against phosphorylated ERK (E10), and ERK, respectively. (B) Caco-2 cells (5 × 10⁵) were handled as described in (A). After a 96-h incubation, cells of each treatment were harvested and counted. The results are shown as means ± SD of triplicate experiments. ^*** P < 0.001 as compared to Ctrl. ^### P < 0.001 as compared to LPS-treated cells. Similar results were obtained at least twice.