Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

Int J Mol Sci. 2016 Oct 10;17(10):1696. doi: 10.3390/ijms17101696.

Abstract

As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C β2 (PKCβ2) at Ser660. Inhibiting (protein kinase C β) PKCβ blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCβ2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection.

Keywords: (protein kinase C β) PKCβ; Caco-2; butyrate; myosin II regulatory light chain (MLC2); myosin light chain kinase (MLCK); tight junction.

MeSH terms

  • AMP-Activated Protein Kinases / metabolism
  • Blotting, Western
  • Butyric Acid / pharmacology*
  • Caco-2 Cells
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase / metabolism
  • Cardiac Myosins / antagonists & inhibitors
  • Cardiac Myosins / metabolism*
  • Humans
  • Immunoprecipitation
  • Myosin Light Chains / antagonists & inhibitors
  • Myosin Light Chains / metabolism*
  • Myosin-Light-Chain Kinase / antagonists & inhibitors
  • Myosin-Light-Chain Kinase / metabolism*
  • Phosphorylation / drug effects
  • Protein Kinase C beta / metabolism*
  • Tight Junctions / drug effects*
  • Tight Junctions / metabolism

Substances

  • Myosin Light Chains
  • myosin light chain 2
  • Butyric Acid
  • Protein Kinase C beta
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Myosin-Light-Chain Kinase
  • AMP-Activated Protein Kinases
  • Cardiac Myosins