Kruppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells

Abstract

Lipopolysaccharide (LPS) is a bacterially-derived endotoxin that elicits a strong proinflammatory response in intestinal epithelial cells. It is well established that LPS activates this response through NF-kappaB. In addition, LPS signals through the mitogen-activated protein kinase (MAPK) pathway. We previously demonstrated that the Krüppel-like factor 5 [KLF5; also known as intestine-enriched Krüppel-like factor (IKLF)] is activated by the MAPK. In the current study, we examined whether KLF5 mediates the signaling cascade elicited by LPS. Treatment of the intestinal epithelial cell line, IEC6, with LPS resulted in a dose- and time-dependent increase in KLF5 messenger RNA (mRNA) and protein levels. Concurrently, mRNA levels of the p50 and p65 subunits of NF-kappaB were increased by LPS treatment. Pretreatment with the MAPK inhibitor, U0126, or the LPS antagonist, polymyxin B, resulted in an attenuation of KLF5, p50 and p65 NF-kappaB subunit mRNA levels from LPS treatment. Importantly, suppression of KLF5 by small interfering RNA (siRNA) resulted in a reduction in p50 and p65 subunit mRNA levels and NF-kappaB DNA binding activity in response to LPS. LPS treatment also led to an increase in secretion of TNF-alpha and IL-6 from IEC6, both of which were reduced by siRNA inhibition of KLF5. In addition, intercellular adhesion molecule-1 (ICAM-1) levels were increased in LPS-treated IEC6 cells and this increase was associated with increased adhesion of Jurkat lymphocytes to IEC6. The induction of ICAM-1 expression and T cell adhesion to IEC6 by LPS were both abrogated by siRNA inhibition of KLF5. These results indicate that KLF5 is an important mediator for the proinflammatory response elicited by LPS in intestinal epithelial cells.

Figure 1

Northern and western blot analyses of KLF5 mRNA and protein in IEC6 cells in response to LPS. IEC6 cells were treated from 0 to 24 h with 5 µg/ml E.coli 0111:B4 LPS (A) or water control (B). Twenty micrograms of total RNA from each time point was analyzed by northern blot analysis using labeled cDNA probe for KLF5 or GAPDH (as a loading control.). (C) IEC6 cells were treated for 2 h with a range of LPS concentrations from 0 to 10 µg/ml. Blots were hybridized with cDNA probe for KLF5 or GAPDH. For protein measurement, IEC6 cells were treated from 0 to 12 h with 5 µg/ml LPS (D) or water control (E). Fifty micrograms of protein were loaded per lane and examined by western blot using antibodies against KLF5 or actin (as a loading control).

Figure 2

Northern blot analyses of the effects of LPS, PMXB and U0126 on expression of KLF5 and the p65 and p50 subunits of NF-κB in IEC6 cells. (A) IEC6 cells were treated with 5 µg/ml LPS for the time periods specified before extraction of RNA. Twenty micrograms of RNA were loaded in each lane and probed with cDNA encoding KLF5, and the p65 and p50 subunits of NF-κB. GAPDH was used as a loading control. (B) IEC6 cells were pretreated with 10 µg/ml PMXB for 30 min (lanes 3 and 4) and then treated with 5 µg/ml LPS (lanes 2 and 4) or water control (lanes 1 and 3) for 2 h before being analyzed for the mRNA levels of KLF5, and the p65 and p50 subunits of NF-κB. (C) IEC6 cells were pretreated with water control (lanes 1 and 2), the vehicle, DMSO (lanes 3 and 4), or the MAPK inhibitor, U0126 (lanes 5 and 6), for 30 min and followed by treatment with 5 µg/ml LPS (lanes 2, 4 and 6) or water control (lanes 1, 3 and 5) for 2 h before northern analyses for the mRNA stated. GAPDH was used as a loading control in both panels.

Figure 3

Inhibition of KLF5 expression by siRNA abrogates induction of NF-κB subunit levels and binding activity in response to LPS. (A) IEC6 cells were transfected by electroporation with non-specific (NS) siRNA or KLF5-specific siRNA. Mock-transfected cells were used as control. Twenty-four hours following transfection, cells were treated with 5 µg/ml LPS or water control for 2 h, followed by northern blot analysis for the various mRNAs indicated. (B) Nuclear extracts were prepared from mock-transfected IEC6 cells or cells transfected with non-specific (NS) siRNA or KLF5-specific siRNA that have been treated with 5 µg/ml LPS or water control for 2 h. Electrophoretic mobility shift assay (EMSA) was then performed with a labeled consensus NF-κB binding sequence using 5 µg nuclear extracts per lane. In the last lane (Comp.), 150-fold excess of unlabeled NF-κB probe was included in the reaction containing nuclear extracts from mock-transfected and LPS-treated cells. The same nuclear extracts were also analyzed for the content of KLF5 or actin by western blotting, as shown below the EMSA. (C) IEC6 cells were pretreated with 10 µM of the NF-κB inhibitor, TLCK, or water control for 1 h and then treated with 5 µg/ml LPS or water control for 2 h before being analyzed for the mRNA levels for KLF5 or GAPDH by northern hybridization.

Figure 4

Increased secretion of TNF-α and IL-6 in IEC6 cells in response to LPS requires KLF5. IEC6 cells were treated with water control (open bars) or 5 µg/ml LPS (filled bars) for the periods of time specified. The amounts of TNF-α (A) and IL-6 (B) in the supernatants were determined by ELISA. N = 4 in all experiments. *P < 0.05 compared to control. Mock-transfected IEC6 cells or IEC6 cells transfected with non-specific (NS) siRNA or KLF5-specific siRNA were treated with water control (open bars) or 5 µg/ml LPS (filled bars) for 48 h. The amounts of TNF-α (C) and IL-6 (D) in the supernatants were determined by ELISA. N = 4 in all experiments. *P < 0.05 compared to control.

Figure 5

Increased expression of ICAM-1 and adhesion of Jurkat T cells to IEC6 cells in response to LPS requires KLF5. (A) IEC6 cells were treated with 5 µg/ml LPS for the time periods specified before protein extraction. Fifty micrograms of protein were loaded in each lane and analyzed for the content of KLF5, ICAM-1 or actin by western blotting. (B) Fluorescently labeled Jurkat E6.1 lymphocytes were co-cultured with mock-, non-specific (NS) siRNA or KLF5-specific siRNA-transfected IEC6 cells that were pretreated with either water control (open bars) or 5 µg/ml LPS (filled bars) for 24 h. After 2 h of co-culturing, cells were washed, lysed and attachment of Jurkat cells to IEC6 cells determined by a spectrophotometer. Results are expressed as relative fluorescence units (RFU). N = 6 in all experiments. *P < 0.01 compared to control. (C) Western blot analysis was performed using the same set of cells that were used in (B) for the protein content of KLF5, ICAM-1 or actin.

Figure 6

A model for the role of KLF5 in mediating the proinflammatory response in IEC6 cells elicited by LPS. Treatment of IEC6 cells by LPS activates MAP kinase activity, which leads to induction of KLF5 mRNA and protein levels. The increased KLF5 then transcriptionally activates expression of the p65 and p50 subunits of NF-κB with a subsequent increase in NF-κB binding activity, leading to increased production of TNF-α, IL-6 and ICAM-1, and subsequent proinflammatory response.