LG4–5 domains of laminin‐211 binds α‐Dystroglycan to allow myotube attachment and prevent anoikis

Abstract

Poly(2-hydroxyethyl methacrylate) (PolyHEMA) prevents cell attachment was used here to study anoikis, the process where cells die when unattached or attached to an inappropriate matrix, in mouse C 2 C 12 myotubes. A method was developed to efficiently embed proteins into PolyHEMA and the effect on cultured myotubes was determined. Myotubes grown on PolyHEMA-coated plates fail to attach to the surface and remain as rounded, suspended cells, undergo dramatic increases in apoptosis and necrosis, and the number of viable cells decreases,. Incorporation of merosin (laminin-211) or the short laminin globular (LG4-5) modules of the laminin α2 chain C-terminus (called 2E3) that binds α-dystroglycan diminishes both apoptosis and necrosis and increases viability while bovine serum albumin had a much lesser effect, showing the specificity of this effect for these matrix proteins. One sarcolemma receptor for laminin-binding is α-dystroglycan. An antibody which binds α-dystroglycan but which does not block laminin-binding (VIA4) had little effect on apoptosis or viability on merosin or 2E3 embedded plates while another antibody (IIH6) which specifically blocks binding dramatically decreased viability and increased apoptosis. When merosin or 2E3 are added to culture media rather than embedded on plates these can also increase viability and decrease apoptosis even though the cells remain in suspension, though the effect is not as great as found for the embedded proteins where the cells attach. Thus, we conclude that the binding of a small LG4-5 modules of laminin-211 to α-dystroglycan is important in preventing anoikis and that attachment plus binding is necessary for maximal cell survival.

Procaspase-3 regulates fibronectin secretion and influences adhesion, migration and survival independent of catalytic function

“…To test this possibility, we examined the role of adhesion in the cell death of wild-type and Casp3 2/2 MEFs. Cells were seeded in the presence or absence of serum on plates coated with or without polyHEMA, which blocks adhesion and results in the induction of anoikis (Munoz et al, 2010). Consistent with previous findings, Casp3 2/2 MEFs were significantly protected from serum-withdrawal-induced cell death when compared with wild-type MEFs at all time-points analyzed (Fig.…”

Physiological aspects of cardiac tissue engineering

“…Here freshly isolated cardiac cells are mixed with natural, gel-forming, extracellular matrixes that fulfill the main purpose of keeping cells in a 3-D space until they form cell-cell contacts and generate their own extracellular matrix, thereby forming a tissue. The hydrogels also appear to protect isolated cells from a process termed anoikis, cell death due to loss of cell-cell contacts (41,57). In the various hydrogel approaches currently pursued, the macroscopic form of the construct is defined by respective casting molds and the techniques to fix the developing tissue to holders (Fig.…”

Influence of stress on extracellular matrix and integrin biology

“…Alternatively, external signals may also regulate anoıkis. For example, it has been shown that some specific laminin domains, laminin-proteoglycan complexes or collagen IV are highly efficient to block anoıkis (Weber et al, 2008;Salo et al, 2009;Munoz et al 2010). Resistance to anoıkis is a prerequisite for epithelial-mesenchymal transition, and acquisition of invasion capacity.…”