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. 2016 Oct 10;17(10):1696.
doi: 10.3390/ijms17101696.

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

Wei Miao  1   2   3 , Xiujuan Wu  4   5   6 , Kang Wang  7   8   9 , Wenjing Wang  10   11   12 , Yumei Wang  13   14   15 , Zhigang Li  16   17   18 , Jingjing Liu  19   20   21 , Li Li  22   23   24 , Luying Peng  25   26   27
Affiliations

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

Wei Miao et al. Int J Mol Sci. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of sodium butyrate (NaB) on GPR109A and store-operated calcium entry. Caco-2 monolayers were incubated in a low-calcium medium for 16 h, and then cell monolayers were switched to Caco-2 culture medium alone or with (A) 2 mmol/L of NaB, 2 mmol/L of niacin, 100 nmol/L of mepenzolate bromide (MB) or both NaB and MB, respectively. TERs of the monolayers were detected at the time points of 0, 2, 4 and 8 h; (B) 2 mmol/L of NaB, 10 μmol/L of SKF-96365 and NaB/SKF-96365, respectively. TERs of the monolayers were detected at 0, 2, 4 and 8 h; (C) Dot plot diagram of [Ca2+]i change under the condition of 2 mmol/L of NaB, 5 μmol/L of SKF-96365 and NaB/SKF-96365, respectively; (D) Summary of the data in (C) which represents the peak values during the examined time interval. Data are expressed as means of relative Fluo-8 fluorescence intensity ± SE (Standard Error), n = 3. The asterisks denote a significant difference between chemical-treated groups and controls groups as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference (p < 0.05) between NaB and NaB/SKF-96365. MB—Mepenzolate bromide.
Figure 2
Figure 2
Effects of NaB on the interaction between MLCK and calmodulin as well as phosphorylation levels of MLC2 in Caco-2 cell monolayers. (A) After Ca2+ switch, Caco-2 cells were cultured in normal Caco-2 medium with or without 2 mmol/L of NaB. Co-Immunoprecipitation (Co-IP) of MLCK and calmodulin was performed at 0, 4 or 8 h, respectively; (B) The quantification of MLCK immunoreactive signals by normalized to calmodulin signals in (A); (C) The change of TERs after Ca2+ switch under the condition of 2 mmol/L of NaB, or 250 μmol/L of Permeant inhibitor of MLC kinase (PIK) at 0, 2, 4 and 8 h, respectively; (D) Total cell lysates from untreated cells or those treated with 2 mmol/L of NaB or 250 μmol/L of PIK were subjected to immunoblotting for pSer19-MLC2, total MLC2 and GAPDH, respectively; (E) MLC2 activity was expressed as the ratio of the phosphorylated form of the MLC2 to total MLC2. Values are means ± SE, n = 3. The asterisks denote a significant difference between chemical-treated groups and controls as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference (p < 0.05) between NaB and NaB/PIK. PIK-Permeant inhibitor of MLC kinase.
Figure 3
Figure 3
Effects of NaB on PKCβ. (A) The change of TERs after Ca2+ switch under different conditions (medium alone, or with 2 mmol/L of NaB, or with 5 μmol/L of LY-333531 or LY-333531/NaB) for 0, 2, 4 and 8 h; (B) Western blotting at indicated conditions: medium alone; 2 mmol/L of NaB; 5 μmol/L of LY-333531; 1 mmol/L of AICAR; 10 μmol/L of Compound C; (C) The ratio of phosphorylated PKCβ1 to total PKCβ1 was quantified in (B); (D) The ratio of phosphorylated PKCβ2 to total PKCβ2 was quantified in (B). Data represent mean ± SE, n = 3. The asterisks denote a significant difference between chemical-treated groups and control group as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference between indicated groups, p < 0.05. Ctr—control; LY—LY-333531; AI—AICAR; CC—Compound C.
Figure 4
Figure 4
The phosphorylation change of MLC2 and PKCβ2 during TJs assembly induced by NaB. (A) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 10 umol/L of SKF-96365; (B,C) The ratio of phosphorylated MLC2 to MLC2 or phosphorylated PKCβ2 to PKCβ2 in (A); (D) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 5 μmol/L of LY-333531; (E) The ratio of phosphorylated MLC2 to MLC2 in (D); (F) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 10 μmol/L of Compound C; (G) The ratio of phosphorylated MLC2 to MLC2 in (F). All experiments were performed at 8 h after calcium switch. Data represent mean ± SE, n = 3. The asterisks denote a significant difference between chemical-treated groups and control group as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference between indicated groups, p < 0.05. CC—Compound C.
Figure 5
Figure 5
Proposed diagram showing the mechanism of NaB on reassembly of tight junctions in Caco-2 monolayers. NaB appears to activate Store-Operated Ca2+ Channel (SOCC) which conducts the Ca2+ influx and then activates CaMKKβ/AMPK, resulting in PKCβ2 and MLCK/MLC2 pathways to mediate barrier function recovery. The black arrows indicate active effects, and the line with bar at the end indicates inhibition effect. The red arrow indicates reduction, and the green arrows indicates increase.
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