FIGURE 5:

Dsc2 controls the expression of integrins, Pkp3 and Rap1 activity in vitro. (A, B) Loss of Dsc2 in SKCO15 cells results in reduced expression of beta 1 and beta 4 integrins. (A) Whole cell lysates from subconfluent monolayer of SKCO15 KD for Dsc2 (Dsc2 KD) or control cells were subjected to SDS–PAGE and immunoblot against Dsc2 and integrin subunits alpha 2, alpha 6, beta 1, and beta 4. Calnexin was used as loading control. Western blot images are representative of four independent experiments. (B) Histograms represent densitometric values normalized to controls from four independent experiments with independently transduced SKCO15 cell culture. Bar graphs are mean ± SEM. Significance is determined by two-tailed Student’s t test. ***p ≤ 0.001, *p = 0.05. (C, D) Loss of Dsc2 resulted in pull down of less active Rap1 in comparison to control cells. (C) Subconfluent monolayers of SKCO15 KD for Dsc2 (Dsc2 KD) or control cells were subjected to RalGDS pull down followed by SDS–PAGE. GTP-bound Rap1 was detected by Western blotting with anti-Rap1 antibodies. Densitometric quantification of immunoblots of active Rap1 by RalGDS pull down normalized to control. (D) Pkp3 was evaluated in whole cell lysates of SKCO15 KD for Dsc2 (Dsc2 KD) or control cells. Densitometric quantification of immunoblots of Plakophilin 3 normalized to control. (C, D) WB images are representative of three independent experiments with independently transduced SKCO15 cell culture. Bar graphs are mean values from three independent experiments ± SEM. Significance is determined by two-tailed Student’s t test. **p = 0.01. (E) Model summarizing the role of intestinal epithelial-expressed Dsc2 in mucosal repair after injury. Dsc2 regulates intestinal mucosal wound closure by controlling integrin expression, cell–matrix adhesion dynamics and cell migration. Top panel depicts baseline condition with IEC expressing Dsc2 (red) and Dsg2 (blue) forming mature Desmosome (DM) complexes that promote cell cohesion and contribute to collective migration and wound healing. Signaling pathways downstream of DM in parallel to those from the AJC activate the small GTPase Rap1 influencing integrin function, thereby promoting cell adhesion to the extracellular matrix (ECM) and cell migration. Bottom panel shows the consequences of Dsc2 absence on IEC that results in reduced expression of Pkp3. Loss of Dsc2 is also associated with reduced cell cohesion, decreased levels of integrin β1 and β4, and reduced activity of Rap1. AJ, adherens junction; DP, Desmoplakin, IF, intermediate filaments; Integrins are depicted as αβ complexes; PG, Plakoglobin; TJ, tight junction.