Cell-Cell Contact Preserves Cell Viability via Plakoglobin

Abstract

Control over cell viability is a fundamental property underlying numerous physiological processes. Cell spreading on a substrate was previously demonstrated to be a major factor in determining the viability of individual cells. In multicellular organisms, cell-cell contact is likely to play a significant role in regulating cell vitality, but its function is easily masked by cell-substrate interactions, thus remains incompletely characterized. In this study, we show that suspended immortalized human keratinocyte sheets with persisting intercellular contacts exhibited significant contraction, junctional actin localization, and reinforcement of cell-cell adhesion strength. Further, cells within these sheets remain viable, in contrast to trypsinized cells suspended without either cell-cell or cell-substrate contact, which underwent apoptosis at high rates. Suppression of plakoglobin weakened cell-cell adhesion in cell sheets and suppressed apoptosis in suspended, trypsinized cells. These results demonstrate that cell-cell contact may be a fundamental control mechanism governing cell viability and that the junctional protein plakoglobin is a key regulator of this process. Given the near-ubiquity of plakoglobin in multicellular organisms, these findings could have significant implications for understanding cell adhesion, modeling disease progression, developing therapeutics and improving the viability of tissue engineering protocols.

Characterization of Partially Lifted Cell Sheets

“…17 Our data are consistent with the notion that the cell's ability to convert physical perturbations into signaling cascades is based on the integrity of the junctional-cytoskeletal linkages. 18,[33][34][35][36] Moreover, stretch is a well-known potent stimulus for many cell functions. 36,38 On stretching, applied physical force may stiffen the intercellular junctional-cytoskeletal network through mechanical strain-stiffening 40,41 or signalingmediated reinforcement.…”

“…18,22 Briefly, cells were plated on 0.005" thick silicone sheeting (SMI, Saginaw, MI) in a 100-mm diameter custom-made stretch device chamber, expanded and propagated in keratinocyte serum-free media (abbreviated ker-sfm, media, and supplements from Invitrogen, Carlsbad, CA, unless otherwise stated), and supplemented with recombinant epidermal growth factor (rEGF) (0.2 ng/mL) and BPE (25 mg/mL), CaCl 2 (0.4 mM; Sigma, St. Louis, MO), and penicillin/streptomycin. To grow cells to high confluency, cells were switched to a medium consisting of a 1:1 mixture of ker-sfm and a medium DF-K, with the latter consisting of a 1:1 mixture of Dulbecco's-modified Eagle's medium (DMEM) and Ham's F-12, supplemented with rEGF (0.2 ng/mL) and BPE (25 mg/ mL), l-glutamine (1.5 mM), and penicillin/streptomycin.…”

“…A previously established shear test protocol was performed to test cell sheet cohesion and, thus, cell-cell adhesive strength. 18,26,29,30 A second custom equiaxial stretch apparatus, referred to as the microscope stage-top stretch apparatus, was used for simultaneous stretching and imaging. This device consisted of a moving plate that is moved vertically by an actuator against a ring-shaped piston which generates uniform strain on the membrane.…”

“…18,[33][34][35][36] Plakoglobin, a key molecular constituent that resides in both adherens junctions and desmosomes, is essential for maintaining cellular viability in substrate-free conditions. 18 Further, plakoglobin exhibits stretch-induced up-regulation under conventionally plated conditions. 26,37 Plakoglobin was, thus, examined using the same four conditions as actin to assess its role in modulating cell properties when cell-cell interactions dominate.…”

“…[18][19][20] Third, most cell sheets are generated for immediate use and not conditioning-without supporting scaffolds, these sheets are too fragile to endure in vitro handling or significant manipulation. A recent study on characterizing the mechanics of cultured cell monolayers has begun shedding light on this topic.…”