Research Thrust 2: Effect of Mechanical Load on Cell-Cell Adhesion Pathology
Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease that affects the skin and mucous membranes and serves as an excellent model for the study of human organ-specific autoimmunity. PV is characterized by the presence of autoantibodies (autoAbs) directed against keratinocyte surface antigens, primarily the desmosomal cadherins desmoglein 3 (Dsg3) and Dsg1. Binding of these autoAbs leads to desmosome internalization, keratin retraction, cell dissociation, and ultimately blister formation. The bulk of research in PV to date has focused on immune mechanisms upstream of autoAb binding. Consequently, therapeutic interventions in PV have mostly relied on non-specific immunosuppression, which is often accompanied by detrimental long-term side effects. However, the biophysical aspects of the cell behavior after autoAb binding are only recently being explored thanks to the advancement of innovative technologies applied to fundamental biological questions at the cellular level.
In a collaborative work, we showed that keratinocyte (HaCaT) cell monolayers are significantly less fragmented after being subjected to cyclic stretch when treated with anti-Dsg3 Abs in conventional dispase based dissociation assays, a clear indication that periodic mechanical stress exposure leads to cell-cell adhesion stability (Figure 2a-b). This protective effect is further underscored by the fact that cyclic mechanical stretch suppresses anti-Dsg3 Ab-induced p38 phosphorylation (Figure 2c-d), a major mechanotransduction pathway in PV pathology. More importantly, we showed applied cyclic stretch can enhance the cortical actin network (Figure 2e-f), stabilize the E-cadherin (E-cad) based adherens junction (Figure 2e-f) and thus mitigate the anti-Dsg3 Ab-induced cell-cell dissociation, via the RhoA pathway (Figure 2g-h).
Mechanical stretch has been shown to induce a spectrum of physiological transformations in many cell types. The most widely reported effects are stress fiber reorganization in endothelial cells and in muscles cells through Rho-dependent stress fiber assembly from myosin light chain phosphorylation. But our data shows for the first time that mechanical stretch-induced cellular physiological changes can provide a protective mechanism for cell-cell adhesion to maintain stability against autoAb-induced desmosome disassembly. The seemingly counterintuitive finding of the protective effect of externally applied mechanical stress in a disease state may represent the consequences of enhancing normal homeostatic mechanisms that are operative under physiological conditions. We hypothesize that this mechanism is facilitated by the same set of pathways that prepare keratinocytes for proliferation and for wound healing.
Modulation of mechanical stress mitigates anti-Dsg3 antibody-induced dissociation of cell-cell adhesion. a. Dispase-based dissociation assay shows reduced cell fragmentation after cyclic mechanical stretch (CS) with the presence of AK23 mAb (anti-Dsg3 Ab). Dissociation assay images of HaCaT monolayers after culture in regular medium, treated with AK23 and mechanical stretch (CS). b. Quantitative result of dissociation assay images. c. Cyclic mechanical stretch suppresses anti-Dsg3 Ab-induced p38 phosphorylation shown from Western blot data with control, 4 hours of AK23 treatment with two concentrations, as well as AK23 treatment with mechanical stretch. d. The ratio of band intensity from c, p-p38/p38, as compared with the control. e. Staining of E-cadherin (E-cad) and F-actin with AK23 and AK23+cyclic mechanical stretch (CS). f. Quantitative data of the relative intensity of E-cad and F-actin staining at the cell-cell border with the AK23, static stretch (SS) and cyclic stretch (CS) treatments. g. Staining of the RhoA molecule with AK23 and AK23 + cyclic stretch. h. Quantitative data of the relative intensity of RhoA at the cell-cell boarder with AK23, SS and CS combinations. Scale bars: e, 50 µm; inset, 20 µm; g, 50 µm.
Jin, Xiaowei, Jordan Rosenbohm, Eunju Kim, Amir Monemian Esfahani, Kristina Seiffert‐Sinha, James K. Wahl III, Jung Yul Lim, Animesh A. Sinha, and Ruiguo Yang. Modulation of Mechanical Stress Mitigates Anti‐Dsg3 Antibody‐Induced Dissociation of Cell–Cell Adhesion. Advanced Biology: 2000159:1-12.