Desmosome Dysregulation and MLC Phosphorylation Contribute to Loss of Barrier Integrity in DRA KO mice

Abstract

DRA or SLC26A3 is the key transporter for chloride absorption in the mammalian intestine. Our recent studies demonstrated that DRA deficiency increased paracellular permeability and altered tight and adherens junction protein expression in mouse colon. However, the mechanisms underlying increased paracellular permeability and the role of intercellular junctional proteins (desmosomes) that play a critical role in the maintenance of intestinal barrier integrity in DRA KO mice are not well understood. Aim The current studies were undertaken to examine the role of desmosomes and Rho‐ROCK‐MLC phosphorylation pathway in colonic barrier integrity in DRA KO mice.MethodDRA KO and WT mice (M/F, 8‐10 weeks old) were used as an in‐vivo model. Protein expression was measured by immunoblotting and immunofluorescence, whereas mRNA expression was measured by QRT‐PCR and Rho A activity was examined utilizing the G‐LISA kit.ResultsOur results showed that the mRNA expression of desmosome proteins, desmoglein‐2 (DSG2) and desmocollin‐2 (DSC2) was significantly reduced (50‐70%, p<0.05) in DRA KO colons compared to wild‐type mice. Similarly, DSG2 protein expression was also significantly downregulated in DRA KO mice colon (~ 50%, p<0.05) as assessed by immunoblotting and immunostaining. We also examined the role of Rho‐ROCK‐MLC2 phosphorylation pathway in DRA KO mice. Interestingly, DRA KO mice colon showed a significant increase in Rho A protein expression (~5‐fold, p<0.01) and Rho A activity (~ 3.5‐fold, p<0.05) compared to WT. Furthermore, DRA KO mice also exhibited a significant decrease in ezrin phosphorylation (a downstream target of Rho A signaling) (~ 2‐fold, p<0.05) compared to WT mice. In addition, another downstream target of the Rho A pathway, MYPT‐1 (Myosin Phosphatase Target Subunit 1), also showed a significant decrease (~ 60%, p<0.05) in its protein expression in DRA KO mice. MYPT‐1 downregulation in DRA KO mice was associated with a significant increase in MLC‐2 phosphorylation (~ 2‐fold, p<0.05), which may have contributed to barrier dysfunction in colon of DRA KO mice. Since, our recent studies showed microbial dysbiosis in DRA KO mice, it was of interest to examine the role of gut microbiome by co‐housing WT and KO mice for 4 weeks for microbial exchange. Interestingly, co‐housed DRA KO mice also showed a significant but lower increase in Rho A expression (~ 3‐fold, p<0.05) and downregulation of MYPT‐1 expression (~ 60%, p<0.05) compared to co‐housed WT mice indicating that the regulation of Rho A and MYPT‐1 expression was only partly dependent on microbial dysbiosis observed in DRA KO mice.ConclusionOur studies highlight the novel role of desmosome dysregulation and increased MLC phosphorylation in compromised colonic barrier integrity in DRA KO mice.