TNF-α Modulation of Intestinal Tight Junction Permeability Is Mediated by NIK/IKK-α Axis Activation of the Canonical NF-κB Pathway

Abstract

Tumor necrosis factor (TNF)-α, a key mediator of intestinal inflammation, causes an increase in intestinal epithelial tight junction (TJ) permeability by activating myosin light chain kinase (MLCK; official name MYLK3) gene. However, the precise signaling cascades that mediate the TNF-α-induced activation of MLCK gene and increase in TJ permeability remain unclear. Our aims were to delineate the upstream signaling mechanisms that regulate the TNF-α modulation of intestinal TJ barrier function with the use of in vitro and in vivo intestinal epithelial model systems. TNF-α caused a rapid activation of both canonical and noncanonical NF-κB pathway. NF-κB-inducing kinase (NIK) and mitogen-activated protein kinase kinase-1 (MEKK-1) were activated in response to TNF-α. NIK mediated the TNF-α activation of inhibitory κB kinase (IKK)-α, and MEKK1 mediated the activation of IKK complex, including IKK-β. NIK/IKK-α axis regulated the activation of both NF-κB p50/p65 and RelB/p52 pathways. Surprisingly, the siRNA induced knockdown of NIK, but not MEKK-1, prevented the TNF-α activation of both NF-κB p50/p65 and RelB/p52 and the increase in intestinal TJ permeability. Moreover, NIK/IKK-α/NF-κB p50/p65 axis mediated the TNF-α-induced MLCK gene activation and the subsequent MLCK increase in intestinal TJ permeability. In conclusion, our data show that NIK/IKK-α/regulates the activation of NF-κB p50/p65 and plays an integral role in the TNF-α-induced activation of MLCK gene and increase in intestinal TJ permeability.

Figure 1

Time course effect of TNF-α on Caco-2 NIK and MEKK-1 activation. A: TNF-α (10 ng/mL) causes a time-dependent increase in Caco-2 NIK phosphorylation (total NIK was used for equal protein loading). B: TNF-α causes a time-dependent increase in MEKK-1 phosphorylation (total MEKK-1 was used for equal protein loading). MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-κB-inducing kinase; TNF, tumor necrosis factor.

Figure 2

Effect of siRNA-induced NIK and MEKK-1 knockdown on TNF-α–induced increase in Caco-2 TJ permeability. A: NIK siRNA transfection results in a near complete depletion in NIK protein expression. B: NIK silencing prevents the TNF-α–induced drop in Caco-2 TER. C: NIK silencing by siRNA transfection prevents the TNF-α–induced increase in inulin flux. D: MEKK-1 siRNA transfection results in a near complete depletion in MEKK-1 protein expression. E: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced drop in Caco-2 TER. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced increase in inulin flux. G: NIK inhibitor (4H-isoquinoline-1,3-dione; 50 μmol/L) prevents the TNF-phosphorylation of NIK. H: NIK inhibitor prevents the TNF-α–induced drop in Caco-2 TER. Data are expressed as means ± SEM. n = 4. **P < 0.005 versus control; ^††P < 0.005 versus TNF-α treatment. C, no siRNA controls; inh, inhibitor; MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-κB-inducing kinase; NT, non-targeted siRNA; TER, transepithelial electrical resistance; TJ, tight junction; TNF, tumor necrosis factor.

Figure 3

Effect of 10 ng/mL TNF-α on Caco-2 NF-κB pathway (p65 and p52) activation. A: TNF-α causes a time-dependent degradation in IκB-α expression. B: TNF-α treatment causes a significant increase in Caco-2 NF-κB p65 binding to the DNA probe as assayed by ELISA-based DNA binding assay of NF-κB p65. C: TNF-α treatment causes a significant increase in Caco-2 NF-κB p52 binding to the DNA probe as assayed by ELISA-based DNA binding assay of NF-κB p52. D: NF-κB p65 siRNA transfection prevents the TNF-α–induced drop in Caco-2 TER. E: NF-κB p65 siRNA transfection prevents the TNF-α–induced increase in inulin flux. F: NF-κB p52 siRNA transfection does not prevent the TNF-α–induced drop in Caco-2 TER. G: NF-κB p52 induced silencing by siRNA transfection does not prevent the TNF-α–induced increase in inulin flux. Data are expressed as means ± SEM. n = 4. **P < 0.005, ****P < 0.0001 versus control; ^††P < 0.005 versus TNF-α treatment. C, no siRNA controls; ELISA, enzyme-linked immunosorbent assay; IκB, inhibitory κB; NT, non-targeted siRNA; TER, transepithelial electrical resistance; TNF, tumor necrosis factor.

Figure 4

Effect of siRNA-induced MAP3 kinase knockdown on TNF-α activation of NF-κB p65. A: NIK siRNA transfection prevents the TNF-α–induced degradation of IκB-α as assessed by Western blot analysis. B: NIK silencing inhibits the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. C: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced degradation of IκB-α. D: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. Data are expressed as means ± SEM. ****P < 0.0001 versus control; ^††P < 0.005, ^††††P < 0.0001 versus TNF-α treatment. C, no siRNA controls; ELISA, enzyme-linked immunosorbent assay; IκB, inhibitory κB; MAP3 kinase, mitogen-activated protein kinase, kinase, kinases; MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-κB-inducing kinase; NT, non-targeted siRNA; TNF, tumor necrosis factor.

Figure 5

Time course effect of TNF-α on Caco-2 IKK catalytic subunit activation. A: TNF-α (10 ng/mL) causes a time-dependent increase in Caco-2 IKK-α and IKK-β phosphorylation. B: IKK-α siRNA transfection completely prevents the TNF-α–induced drop in Caco-2 TER. C: siRNA-induced knockdown of IKK-α prevented the TNF-α–induced increase in inulin flux. D: IKK-β siRNA transfection does not prevent the TNF-α–induced drop in Caco-2 TER. E: siRNA-induced knockdown of IKK-β does not inhibit the TNF-α–induced increase in inulin flux. Data are expressed as means ± SEM. n = 4. **P < 0.005 versus control; ^††P < 0.005 versus TNF-α treatment. C, no siRNA controls; IKK, inhibitory κB kinase; NT, non-targeted siRNA; TER, transepithelial electrical resistance; TNF, tumor necrosis factor.

Figure 6

Effect of siRNA IKK subunit knockdown on TNF-α–induced activation of NF-κB p65. A: siRNA-induced knockdown of IKK-α completely abolishes the TNF-α–induced degradation of IκB-α. B: IKK-α siRNA transfection inhibits the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. C: IKK-β siRNA transfection does not prevent the TNF-α–induced degradation of IκB-α. D: siRNA-induced knockdown of IKK-β does not inhibit the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. ****P < 0.0001 versus control; ^††††P < 0.0001 versus TNF-α treatment. C, no siRNA controls; ELISA, enzyme-linked immunosorbent assay; IκB, inhibitory κB; IKK, inhibitory κB kinase; NT, non-targeted siRNA; TNF, tumor necrosis factor.

Figure 7

Effect of siRNA-induced MEKK-1 and NIK knockdown on TNF-α activation of IKK-α. A: siRNA-induced knockdown of NIK prevents the TNF-α–induced phosphorylation of IKK-α as assessed by Western blot analysis. B: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced phosphorylation of IKK-α. Effect of siRNA induced knockdown of NIK and MEKK-1 on TNF-α–induced increase in MLCK gene activity and protein expression. C: siRNA-induced knockdown of NIK results in a complete inhibition of TNF-α–induced increase in MLCK promoter activity. D: siRNA-induced knockdown of NIK prevents the TNF-α–induced increase in MLCK mRNA levels. E: NIK silencing by siRNA transfection prevents the TNF-α–induced increase in MLCK protein expression. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced increase in MLCK promoter activity. G: Knocking-down MEKK-1 by siRNA does not prevent the TNF-α–induced increase in MLCK mRNA levels. H: Knocking-down MEKK-1 by siRNA does not affect the TNF-α–induced increase in MLCK protein expression. I: Knocking-down NIK by siRNA prevents the TNF-α–induced phosphorylation of p38 kinase (siNIK: siRNA NIK transfection). J: Knocking-down NIK by siRNA does not inhibit the TNF-α–induced phosphorylation of ERK1/2. K: Knocking-down p38 kinase by siRNA does not inhibit the TNF-α–induced degradation of IκB-α. *P < 0.05, **P < 0.005 versus control; ^††P < 0.005, ^†††P < 0.001 versus TNF-α treatment. C, no siRNA controls; ERK, extracellular signal-related kinase; IKK, inhibitory κB kinase; MEKK-1, mitogen-activated protein kinase kinase kinase-1; MLCK, myosin light chain kinase; NIK, NF-κB-inducing kinase; NT, nontargeted siRNA; siNIK: siRNA NIK transfection; sip38: siRNA p38 kinase transfection; T, TNF-α for 30 minutes; TNF, tumor necrosis factor.

Figure 8

Effect of TNF-α activation of NF-κB pathway in mouse intestinal permeability. A: TNF-α (5 μg) causes an increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. B: TNF-α causes a time-dependent increase in IκB-α degradation in mouse intestinal tissue, starting at 2 hours and continuing up to 24 hours as assessed by Western blot analysis. C: TNF-α caused a time-dependent increase in phosphorylation of NIK and MEKK-1 in mouse intestinal tissue as assessed by Western blot analysis. **P < 0.01. IκB, inhibitory κB; MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-κB-inducing kinase; TNF, tumor necrosis factor.

Figure 9

Effect of TNF-α on MLCK expression in vivo. A: TNF-α causes an increase in mouse intestinal tissue MLCK mRNA transcript as assessed by real-time PCR; TNF-α 24 hours of treatment. B: TNF-α causes a time-dependent increase in mouse intestinal tissue MLCK protein expression as assessed by Western blot analysis. C: NIK siRNA transfection in vivo results in a near-complete knockdown of NIK expression in mouse intestinal tissue. D: NIK siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. E: NIK siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. F: MEKK-1 siRNA transfection in vivo results in a near-complete knockdown of MEKK-1 expression in mouse intestinal tissue. G: siRNA-induced knockdown of MEKK-1 in vivo does not prevent the TNF-α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. H: MEKK-1 siRNA transfection in vivo does not prevent the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. **P < 0.01, ***P < 0.001 versus control; ^††P < 0.005 versus TNF-α treatment. MEKK-1, mitogen-activated protein kinase kinase kinase-1; MLCK, myosin light chain kinase; NIK, NF-κB-inducing kinase; TNF, tumor necrosis factor.

Figure 10

Effect of siRNA-induced silencing of NIK and MEKK-1 on NF-κB signaling pathway and mouse intestinal permeability. A: NIK siRNA transfection in vivo prevents the TNF-α–induced degradation of IκB-α in mouse intestinal tissues. B: MEKK-1 siRNA transfection in vivo does not inhibit the TNF-α–induced degradation of IκB-α in mouse intestinal tissues. C: NF-κB p65 siRNA transfection in vivo prevents the TNF–α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. D: NF-κB p65 siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. ***P < 0.001 versus control; ^††P < 0.005 versus TNF-α treatment. IκB, inhibitory κB; MEKK-1, mitogen-activated protein kinase kinase kinase-1; MLCK, myosin light chain kinase; NIK, NF-κB-inducing kinase; TNF, tumor necrosis factor.