Regulation of OTULIN ubiquitination by RNF6 alleviates asthma via mitigating epithelial-mesenchymal transition‑like...

In asthma, chronic inflammation and injurious microenvironment drive bronchial epithelial cells to undergo epithelial-mesenchymal transition (EMT)‑like phenotypic changes. This process disrupts the epithelial barrier, and facilitates extracellular matrix deposition, thereby constituting a pivotal mechanism underlying irreversible airway remodeling. Ring Finger Protein 6 (RNF6), an E3 ubiquitin ligase, was highly expressed in lung tissues of asthma mice. The combined Label-free and Co-Immunoprecipitation (Co-IP)/Mass Spectrometry proteomic profile revealed that OTU Deubiquitinase With Linear Linkage Specificity (OTULIN) was the downstream protein of RNF6. Here, we investigated whether RNF6 modulated OTULIN to participate in partial EMT progression of bronchial epithelial cells. Ovalbumin-induced asthma mouse model was established and BEAS-2B cells were treated with Transforming Growth Factor-Beta 1 (TGF-β1, 10 ng/mL) to construct in vitro model. Histologic analyses revealed that RNF6 knockdown ameliorated airway wall thickening, goblet cell metaplasia and inflammatory cell infiltration of lung tissues of asthma mice. Moreover, silencing RNF6 suppressed EMT‑like phenotypic changes of TGF-β1-stimulated BEAS-2B cells. Thereafter, the interaction between RNF6 and OTULIN was verified by Co-IP and immunofluorescence assays. The addition of cycloheximide and MG132 was used to identify that RNF6 downregulation inhibited the ubiquitination and degradation of OTULIN. Furthermore, overexpressed OTULIN was able to impede TGF-β1-triggered EMT‑like phenotypic changes. Interestingly, downregulated OTULIN reversed the weakened partial EMT progression caused by knocked down RNF6. In summary, RNF6 promoted the ubiquitination and degradation of OTULIN, thereby inducing partial EMT and ultimately exacerbating asthma. Thus, silencing RNF6 might be developed as a potential therapeutical strategy for asthma.