Soft substrate induces apoptosis by the disturbance of Ca²⁺ homeostasis in renal epithelial LLC-PK1 cells

Different rigidities of adhesive collagen substrate affect cellular functions with unclear mechanisms. Here, we cultured a renal epithelial cell line (LLC-PK1) and a tumor cell line (HeLa)on substrates of different rigidities and compared the cell type-specific responses. The culture dish was coated with a very thin layer of collagen gel (control group) or overlaid with collagen gel (soft substrate). LLC-PK1 cells contracted as they grew on collagen gel and the apoptotic bodies obviously appeared with time. The protein levels of procaspase-12 and its downstream target procaspase-3 were decreased when LLC-PK1 cells cultured on collagen gel. μ-calpain was activated on collagen gel. Collage gel also induced the cleavage of α-spectrin which resulted in the disorganization of actin cytoskeleton. In contrast, there was no significant change in cytochrome c revelation, mitochondrial membrane potential, and the protein levels of procaspase-8 and procaspase-9. Moreover, soft substrate caused elevated cytosolic Ca , Ca²⁺ overload in ER and upregulation of capacitative calcium entry. Ca²⁺ chelator or channel blocker partially rescued the collagen-gel induced apoptosis by inhibiting μ-calpain activation. In contrast, for HeLa cells cultured either on collagen gel or on gel-coated dish, there was no significant change in positive Annexin V staining, no activation of procaspase-12 and no cleavage of μ-calpain. Thus, soft substrate induces apoptosis in LLC-PKI cells by the disturbance of Ca ²⁺ homeostasis.