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

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Abstract
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 Ca2+, 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‐PK1 cells by the disturbance of Ca2+ homeostasis. J. Cell. Physiol. 212: 401–410, 2007.