Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene

Abstract

Lipopolysaccharides (LPSs) are a major component of Gram-negative bacterial cell wall and play an important role in promoting intestinal inflammatory responses. Recent studies have shown that physiologically relevant concentrations of LPS (0 to 2000 pg/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability without causing cell death. However, the intracellular pathways and the mechanisms that mediate LPS-induced increase in intestinal TJ permeability remain unclear. The aim was to delineate the intracellular pathways that mediate the LPS-induced increase in intestinal permeability using in vitro and in vivo intestinal epithelial models. LPS-induced increase in intestinal epithelial TJ permeability was preceded by an activation of transforming growth factor-β-activating kinase-1 (TAK-1) and canonical NF-κB (p50/p65) pathways. The siRNA silencing of TAK-1 inhibited the activation of NF-κB p50/p65. The siRNA silencing of TAK-1 and p65/p50 subunit inhibited the LPS-induced increase in intestinal TJ permeability and the increase in myosin light chain kinase (MLCK) expression, confirming the regulatory role of TAK-1 and NF-κB p65/p50 in up-regulating MLCK expression and the subsequent increase in TJ permeability. The data also showed that toll-like receptor (TLR)-4/myeloid differentiation primary response (MyD)88 pathway was crucial upstream regulator of TAK-1 and NF-κB p50/p65 activation. In conclusion, activation of TAK-1 by the TLR-4/MyD88 signal transduction pathway and MLCK by NF-κB p65/p50 regulates the LPS-induced increase in intestinal epithelial TJ permeability.

Figure 1

Effect of pharmacologic inhibition of NF-κB on lipopolysaccharide (LPS)-induced increase in intestinal epithelial tight junction permeability. A: Filter-grown Caco-2 monolayers were treated with 300 pg/mL LPS for a 5-day experimental period. Pharmacologic inhibition of NF-κB by 10 μm ammonium pyrrolidinedithiocarbamate (PDTC) inhibited the LPS (physiological dose of 300 pg/mL)-induced drop in Caco-2 transepithelial electrical resistance. B: Pharmacologic inhibition of NF-κB by PDTC prevented the LPS-induced increase in inulin flux. NF-κB inhibitor PDTC was added 1 hour before LPS treatment. All experimental treatments were renewed every 24 hours for the 5-day experimental period. Data are expressed as means ± SEM. n = 4 independent experiments. ^∗∗∗P < 0.001 versus control; ^†††P < 0.001 versus LPS.

Figure 2

Time course effect of lipopolysaccharide (LPS) on Caco-2 NF-κB pathway (p65 and p52) activation. A: LPS at the concentration of 300 pg/mL caused significant increase in p65 protein expression in Caco-2 cells on day 3 to 3.5. The expression of p52 and p100 did not change with LPS treatment. β-Actin was used as an internal control for protein loading. B: Relative densitometry analysis for p65 protein levels. C: Confocal immunofluorescence of Caco-2 cells treated with 300 pg/mL LPS (3 to 3.5 days) indicated p65 (red) translocation to the nucleus (blue) (arrowheads). D: P52 (green) did not change after LPS treatment (arowheads). E: LPS caused degradation of inhibitory κ B (IκB)-α expression (3 to 3.5 days), as assessed by Western blot analysis. β-Actin was used as an internal control for protein loading. F: Densitometry analysis of LPS treatment showed significant decrease in IκB-α level on day 3 to 3.5 compared with control untreated cells. G and H: LPS-treated Caco-2 cells (3 to 3.5 days) analyzed by flow cytometry for phospho-IκB-α and phospho-p65, respectively, showed increased expression compared with control untreated cells. Gray indicates isotope control (Cont); blue, control untreated; red, LPS treated day 3. [Mean fluorescence intensity (MFI): pIκB-α, 175 ± 12.12, versus 292 ± 25.63; pp65, 3307 ± 154.7, versus 4329.3 ± 169.4]. Data are expressed as means ± SEM. n = 3 independent experiments. ^∗∗∗P < 0.001 versus control; ^†††P < 0.01 versus LPS (day 4); ^‡‡‡P < 0.01 versus LPS (day 3.5). Scale bars = 5 μm (C and D).

Figure 3

Effect of lipopolysaccharide (LPS) on inhibitory κ B kinases (IKKs) and intestinal epithelial tight junction permeability. A: LPS at 300 pg/mL in Caco-2 cells caused significant increase in activation of both IKK-α and IKK-β on day 3 to 3.5 after LPS treatment. β-Actin was used as an internal control (C; Con; Cont) for protein loading. Densitometry analysis of LPS treatment showed significant increase in IKK-α and IKK-β, respectively, on day 3 and 3.5 compared with control untreated cells. B and C: siRNA transfection of IKK-α and IKK-β in Caco-2 cells inhibited the protein expression of IKK-α and IKK-β. D: siRNA transfection of IKK-α in Caco-2 cells partially prevented the LPS-induced increase in inulin flux. E: siRNA transfection of IKK-β in Caco-2 cells inhibited the LPS-induced increase in inulin flux. Data are expressed as means ± SEM. n = 6 experiments (B, C, and E). ^∗∗∗P < 0.001 versus control untreated cells; ^†††P < 0.001 versus control (siNT); ^‡‡‡P < 0.001 versus nontarget (NT) siRNA; ^§§§P < 0.001 versus LPS.

Figure 4

Effects of genetic knockdown of p65/p50 and p52/p100 on lipopolysaccharide (LPS)-induced increase in intestinal epithelial tight junction permeability. A: p65 siRNA transfection resulted in a near-complete depletion of p65. Relative densitometry analysis of p65 protein expression levels. B: The siRNA of p50 transfection resulted in a near-complete depletion of p50 protein expression. Relative densitometry of p50 protein expression levels. C: The p52 siRNA transfection resulted in a near-complete depletion of p52 as shown in densitometry analysis. D: p100 siRNA transfection resulted in a near-complete depletion of p100, as shown in densitometry analysis. E: p65 and p50 siRNA transfection in Caco-2 monolayers inhibited the LPS-induced drop in Caco-2 cell transepithelial electrical resistance (TER). F: p65 and p50 siRNA transfection inhibited the LPS-induced increase in inulin flux. G: p52 and p100 siRNA transfection in Caco-2 monolayers did not prevent the LPS-induced drop in TER. H: p52 and p100 siRNA transfection did not prevent the LPS-induced increase in inulin flux. The Western blot analysis was performed 72 hours after all siRNA transfections. n = 4 experiments (A–D and F); n = 3 experiments (H). ^∗∗∗P < 0.001 versus control; ^†††P < 0.001 versus LPS. Con, control; NT, nontarget.

Figure 5

Role of transforming growth factor-β–activating kinase (TAK)-1 in the lipopolysaccharide (LPS)-induced activation of canonical NF-κB pathway and Caco-2 tight junction permeability. A: LPS treatment at the concentration of 300 pg/mL caused activation of phospho-TAK-1. B and C: LPS treatment did not induce phosphorylation of NF-κB–inducing kinase (NIK) or mitogen-activated kinase kinase (MEKK)-1 at day 3 after LPS exposure compared with untreated Caco-2 cells. D: Confocal immunofluorescence of Caco-2 cells treated with 300 pg/mL LPS for 5 days indicated increase in pTAK-1 (green) on day 3 and 3.5 after LPS treatment. Nucleus, blue. E: TAK-1, NIK, and MEKK-1 siRNA transfections in Caco-2 cells significantly reduced TAK-1, NIK, and MEKK-1 protein expression, as analyzed by Western blot analysis and relative densitometry, respectively. F: TAK-1 siRNA transfection prevented the LPS-induced drop in Caco-2 cell transepithelial electrical resistance (TER). G: TAK-1 siRNA transfection inhibited the LPS-induced increase in Caco-2 inulin flux. H: NIK or MEKK-1 siRNA in transfection in Caco-2 cells did not prevent the LPS-induced drop in Caco-2 TER. Data are expressed as means ± SEM. n = 4 experiments (A). ^∗∗∗P < 0.001 versus control; ^††P < 0.01 versus LPS day 3; ^‡‡P < 0.01 versus LPS day 3.5; ^§§P < 0.01 versus nontarget (NT) siRNA. Scale bars = 5 μm. C, control.

Figure 6

Effect of toll-like receptor (TLR)-4, myeloid differentiation primary response (MyD)88, and transforming growth factor-β–activating kinase (TAK)-1 siRNA on lipopolysaccharide (LPS)-induced activation of NF-κB canonical pathway in Caco-2 cells. A: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced degradation of inhibitory κ B (IκB)-α protein expression compared with nontarget (NT) siRNA-transfected LPS-treated cells. Relative densitometry analysis of IκB-α protein levels. B: TLR-4 siRNA and MyD88 siRNA transfection in Caco-2 cells prevented LPS-induced increase in TAK-1 phosphorylation. Relative densitometry analysis of pTAK-1 protein levels. C: Confocal immunofluorescence showed that TLR-4, MyD88, and TAK-1 siRNA transfection of Caco-2 monolayers prevented the LPS-induced p65 (red) translocation to the nucleus (blue) (arrowhead) at 3 day after LPS exposure D: TLR-4 siRNA and MyD88 siRNA transfection in Caco-2 cells prevented LPS-induced degradation of IκB-α protein expression. Densitometry of IκB-α protein levels. E: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced activation of IκB kinase (IKK)-α and IKK-β compared with nontarget (NT) siRNA-transfected LPS-treated cells. Relative densitometry analysis of IKK-α and IKK-β is also shown. n = 4 experiments. ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control; ^††P < 0.01, ^†††P < 0.001 versus LPS. Scale bars: 5 μm (C). C, control.

Figure 7

Effect of genetic knockdown of (p65/p50) canonical pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) protein expression. A: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced increase in MLCK mRNA. B: Similarly, TAK-1 siRNA transfection in Caco-2 cells also inhibited LPS-induced increase in MLCK and phosphoMLC protein expression by Western blot analysis and was shown by relative densitometry of MLCK protein. C: p65 siRNA transfection in Caco-2 cells prevented LPS-induced increase in MLCK mRNA. D: p50 and p65 siRNA transfection in Caco-2 cells caused marked inhibition of LPS-induced increase in MLCK protein expression, as analyzed by Western blot analysis and relative analysis of MLCK protein levels by densitometry. n = 3 experiments (A); n = 4 experiments (B and D). ^∗∗P < 0.01 versus nontarget siRNA control; ^††P < 0.01 versus LPS. C, control; NT, nontarget.

Figure 8

Activation of p65/p50 canonical pathway by lipopolysaccharide (LPS) in mice enterocytes and effect of NF-κB inhibitors on LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of canonical p65/p50 pathway, as assessed by degradation of inhibitory κ B (IκB)-α protein expression on day 3 in mice enterocytes. Densitometry of IκB-α protein levels. B: The immunoblot analysis from LPS-treated mice enterocytes revealed significant increase in nuclear p65 protein expression on day 3 compared with untreated mice enterocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lamin B were used as loading controls for cytoplasmic (cyto) and nuclear (nuc) fractions, respectively C: Confocal immunofluorescence of mouse intestines treated with LPS (0.1 mg/kg body weight) on day 3 indicated p65 (red) (arrowheads) translocation to the nucleus (blue) compared with control (C) mouse enterocytes. D: NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 10 mg/kg body weight), and Bay-11 (5 mg/kg body weight) pretreatment prevented the LPS-induced increase in 10K dextran flux. PDTC and Bay-11 were dissolved in dimethyl sulfoxide and injected 1 hour before LPS treatment. Data are expressed as means ± SEM. n = 4 experiments (A and C); n = 3 experiments (B). ^∗∗P < 0.01 versus control vehicle; ^††P < 0.01 versus LPS. Scale bars = 5 μm.

Figure 9

Lipopolysaccharide (LPS) activated transforming growth factor-β–activating kinase (TAK)-1 in vivo, and TAK-1 inhibition prevented the LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of TAK-1 (phopshoTAK-1) expression by day 3. Densitometry of phosphoTAK-1 protein levels. B: Confocal immunofluorescence showed increase in phopshoTAK-1 expression (green) (arrowheads) (nucleus, blue) in the intestines (enterocytes) of mice treated with LPS (0.1 mg/kg body weight). C: Pretreatment with TAK-1 inhibitor (Inh), oxozeaenol (5 mg/kg body weight), prevented the LPS-induced increase in 10K dextran flux. Oxozeaenol was dissolved in dimethyl sulfoxide and injected intraperitoneally, 1 hour before LPS injection. n = 3 experiments (A and B). ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus control; ^†††P < 0.001 versus LPS. Scale bars = 5 μm. C, control.

Figure 10

Lipopolysaccharide (LPS)-induced activation of NF-κB canonical pathway was inhibited in toll-like receptor (TLR)-4^−/− and myeloid differentiation primary response (MyD)88^−/− mice and with transforming growth factor-β–activating kinase (TAK)-1 inhibition. A: LPS i.p. injections (0.1 mg/kg body weight) in TLR-4^−/− and MyD88^−/− mice did not induce inhibitory κ B (IκB)-α degradation (day 3) compared with TLR4^+/+ and MyD88^+/+ control (C) mice, respectively. Densitometry of IκB-α protein. B: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced degradation of IκB-α expression (day 3) compared with vehicle- or LPS-treated mice. Densitometry of IκB-α protein levels. n = 3 experiments. ^∗∗P < 0.01 versus control; ^††P < 0.01 versus LPS.

Figure 11

Effect of inhibition of NF-κB pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) expression in mouse enterocytes. A: NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. B: NF-κB inhibitor Bay-11 also prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or /LPS-treated mice. Densitometry of MLCK protein levels. C: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. n = 3 experiments. ^∗∗P < 0.01 versus control; ^††P < 0.01 versus LPS. C, control.

Figure 12

Proposed scheme of the intracellular pathways involved in lipopolysaccharide (LPS)-induced activation of canonical NF-κB pathway. LPS treatment results in activation of the toll-like receptor (TLR)-4 signal transduction and myeloid differentiation primary response (MyD)88-dependent signaling cascade. Activation of TLR-4/MyD88 signal transduction pathway leads to the activation of IL-1 receptor-associated kinase (IRAK)-4 and phosphorylation of transforming growth factor-β–activating kinase (TAK)-1, leading to the activation of canonical NF-κB pathway of both inhibitory κ B (IκB) kinases (IKKs), IKK-α and IKK-β, which further leads to degradation of IκB-α and nuclear translocation of NF-κB p65/p50. The activation of TAK-1 and canonical NF-κB p65/p50 causes up-regulation of myosin light chain kinase (MLCK) gene and protein expression, ultimately increasing tight junction (TJ) permeability in vitro and in vivo.