TNFα-induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, Akt and NFκB signaling

Abstract

SummaryTNFa-mediated tight junction defects contribute to diarrhea in inflammatory bowel diseases (IBDs). In our study, the signaling pathways of the TNFa effect on barrier-or pore-forming claudins were analyzed in HT-29/B6 human colon monolayers. Berberine, a herbal therapeutic agent that has been recently established as a therapy for diabetes and hypercholesterinemia, was able to completely antagonize the TNFa-mediated barrier defects in the cell model and in rat colon. Ussing chamber experiments and two-path impedance spectroscopy revealed a decrease of paracellular resistance after TNFa to 11±4%, whereas transcellular resistance was unchanged. The permeability of the paracellular marker fluorescein was increased fourfold. Berberine alone had no effect while it fully prevented the TNFa-induced barrier defects. This effect on resistance was confirmed in rat colon. TNFa removed claudin-1 from the tight junction and increased claudin-2 expression. Berberine prevented TNFa-induced claudin-1 disassembly and upregulation of claudin-2. The effects of berberine were mimicked by genistein plus BAY11-7082, indicating that they are mediated via tyrosine kinase, pAkt and NFkB pathways. In conclusion, the anti-diarrheal effect of berberine is explained by a novel mechanism, suggesting a therapeutic approach against barrier breakdown in intestinal inflammation.

Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes

“…When we examined other abundant MDCK II cell claudins, including claudin-1, 23, 24 and 27 by Phos-tag SDS PAGE and immunoblot, we saw little evidence for the degree of constitutive phosphorylation seen in claudin-2 (not shown). Why claudin-2 is constitutively phosphorylated is unclear, but as mentioned previously, it is a highly regulated member of the claudin family (Ridyard et al, 2007;Fries et al, 2008;Schulzke et al, 2009;Amasheh et al, 2010;Suzuki et al, 2011). In addition, it is also normally associated with other cell structures aside from tight junctions; depending on cell or tissue type, it is present to a variable extent on the lateral cell membrane (Enck et al, 2001) and in MDCK II cells (this study) (Guillemot et al, 2008;Dukes et al, 2012), a fraction is normally associated with intracellular vesicles.…”

“…It forms a cation-selective pore (Amasheh et al, 2002;Yu et al, 2009) and its transcription is physiologically regulated by cytokines (Amasheh et al, 2010;Suzuki et al, 2011). Claudin-2 levels are increased in the gut in several models of inflammation (Ridyard et al, 2007;Fries et al, 2008;Weber et al, 2008;Schulzke et al, 2009) and in Crohn's disease (Zeissig et al, 2007) and its expression is associated with increased paracellular leakiness for cations and a decrease in transepithelial electrical resistance (TER).…”

Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation

“…[28][29][30] Under inflamed conditions, cytokines such as tumor necrosis factor-a (TNFa) and interleukin (IL)-13 are increased and induce an upregulation of claudin-2 as shown in cell culture studies. 29,[31][32][33] For example, the pro-inflammatory cytokine TNFa caused an increase in claudin-2 expression via phosphatidylinositol-3-kinase (PI3K) signaling in the colonic HT-29/B6 cell line 32,34 , while IL-13 did the same in T84 colon cells (Fig. 1).…”

“…77 Although potential changes in the expression of claudin proteins were not reported, all 3 cytokines are known to have significant effects on TJ protein expression, including the regulation of claudin-2. 30,32,34,78 A more direct link between claudin-2 and the induction of colitis is found in SAMP1/YitFc mice, which show an increase in claudin-2 expression and intestinal permeability that precedes the onset of a spontaneous Crohn's disease-like ileitis. 79 …”

“…34 Berberine is used in traditional Eastern medicine for the treatment of diarrhea and gastroenteritis. The alkaloid has been shown to cause a downregulation of claudin-2 which is associated with the antidiarrheal effect.…”