Connexin 31.1 degradation requires the Clathrin‐mediated autophagy in NSCLC cell H1299

Abstract

Connexins have relative short half-lives. Connexin 31.1 (Cx31.1) was newly reported to be down-regulated in non-small cell lung cancer cell lines, and displayed tumour-suppressive properties. However, no reports describing how a cell regulates Cx31.1 level were found. In this study, Cx31.1 was suggested to be degraded through both ubiquitin–proteasome system (UPS) and autophagy. Blockage of UPS with MG-132 increased Cx31.1 level, but could not inhibit the degradation of Cx31.1 completely. In H1299 cells stably expressing Cx31.1, Cx31.1 reduced when autophagy was induced through starvation or Brefeldin A treatment. Knockdown of autophagy-related protein ATG5 could increase the cellular level of Cx31.1 both under normal growth condition and starvation-induced autophagy. Colocalization of Cx31.1 and autophagy marker light chain 3 (LC3) was revealed by immunofluorescence analysis. Coimmunoprecipitation and immunofluorescence showed that Cx31.1 might interact with clathrin heavy chain which was newly reported to regulate autophagic lysosome reformation (ALR) and controls lysosome homoeostasis. When clathrin expression was knockdown by siRNA treatment, the level of Cx31.1 increased prominently both under normal growth condition and starvation-induced autophagy. Under starvation-induced autophagy, LC3-II levels were slightly accumulated with siCla. treatment compared to that of siNC, which could be ascribed to that clathrin knockdown impaired the late stage of autophagy, ALR. Taken together, we found autophagy contributed to Cx31.1 degradation, and clathrin might be involved in the autophagy of Cx31.1.

Cx31.1 expression is modulated in HaCaT cells exposed to UV‐induced damage and scrape‐wounding

“…Cx31.1 was found to be responsive to retinoic acid (Hatakeyama, Mikami, Habano, & Takeda, 2011), where reduced Cx31.1 was thought to be due to terminal differentiation inhibition. Additionally, in small cell lung cancer models, Cx31.1 expression is regulated by autophagy (Zhu et al, 2015).…”

Cross-over endocytosis of claudins is mediated by interactions via their extracellular loops

“…The targeting of Cldns via a clathrin-dependent mechanism shares similarities with the internalization of some gap junction proteins. Connexin 31.1 can also be targeted by clathrin for degradation, via autophagy, in cell lines of non-small cell lung cancer [ 46 ]. For other connexins, the existence of small double-membrane vesicles has been proposed in cervix carcinoma cells [ 47 ].…”

Role of autophagy in the regulation of epithelial cell junctions

“…73 Clathrin pits are also implicated in the autophagy mediated connexin Cx31.1 degradation. 74 Autophagy machinery also regulates caveolae. Under basal conditions, caveolin-1 (Cav-1) binds to ATG5 and ATG12 in the cytosol while formation of ATG5-ATG12 complex during starvation leads to rapid dissociation of Cav-1 from ATG5-ATG12 conjugates.…”