AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability.

ABSTRACT

The intestinal epithelial tight junctions (TJs) provide barrier against paracellular permeation of lumenal antigens. Defects in TJ barrier such as increased levels of pore-forming TJ protein CLDN2 (claudin-2) is associated with inflammatory bowel disease. We have previously reported that starvation-induced macroautophagy/autophagy enhances the TJ barrier by degrading pore-forming CLDN2. In this study, we examined the molecular mechanism underlying autophagy-induced CLDN2 degradation. CLDN2 degradation was persistent in multiple modes of autophagy induction. Immunolocalization, membrane fractionation, and pharmacological inhibition studies showed increased clathrin-mediated CLDN2 endocytosis upon starvation. Inhibition of clathrin-mediated endocytosis negated autophagy-induced CLDN2 degradation and enhancement of the TJ barrier. The co-immunoprecipitation studies showed increased association of CLDN2 with clathrin and adaptor protein AP2 (AP2A1 and AP2M1 subunits) as well as LC3 and lysosomes upon starvation, signifying the role of clathrin-mediated endocytosis in autophagy-induced CLDN2 degradation. The expression and phosphorylation of AP2M1 was increased upon starvation. In-vitro, in-vivo (mouse colon), and ex-vivo (human colon) inhibition of AP2M1 activation prevented CLDN2 degradation. AP2M1 knockout prevented autophagy-induced CLDN2 degradation via reduced CLDN2-LC3 interaction. Site-directed mutagenesis revealed that AP2M1 binds to CLDN2 tyrosine motifs (YXXФ) (67-70 and 148-151). Increased baseline expression of CLDN2 and TJ permeability along with reduced CLDN2-AP2M1-LC3 interactions in ATG7 knockout cells validated the role of autophagy in modulation of CLDN2 levels. Acute deletion of Atg7 in mice increased CLDN2 levels and the susceptibility to experimental colitis. The autophagy-regulated molecular mechanisms linking CLDN2, AP2M1, and LC3 may provide therapeutic tools against intestinal inflammation.Abbreviations Amil amiloride; AP2 adaptor protein complex 2; AP2A1 adaptor related protein complex 2 subunit alpha 1; AP2M1 adaptor related protein complex 2 subunit mu 1; ATG7 autophagy related 7; CAL calcitriol; Cas9 CRISPR-associated protein 9; Con control; CPZ chlorpromazine; DSS dextran sodium sulfate; EBSS Earle's balanced salt solution; IBD inflammatory bowel disease; TER trans-epithelial resistance; KD knockdown; KO knockout; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; MßCD Methyl-ß-cyclodextrin; MET metformin; MG132 carbobenzoxy-Leu-Leu-leucinal; MTOR mechanistic target of rapamycin kinase; NT non target; RAPA rapamycin; RES resveratrol; SMER small-molecule enhancer 28; SQSTM1 sequestosome 1; ST starvation; ULK1 unc-51 like autophagy activating kinase 1; WT wild type.