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. Mu-calpain was activated on collagen gel. Collage gel also induced the cleavage of alpha-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(2+), Ca(2+) overload in ER and upregulation of capacitative calcium entry. Ca(2+) chelator or channel blocker partially rescued the collagen-gel induced apoptosis by inhibiting mu-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 mu-calpain. Thus, soft substrate induces apoptosis in LLC-PK1 cells by the disturbance of Ca(2+) homeostasis.
Chiu
Wen-Tai
WT
Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan.
Wang
Yao-Hsien
YH
Tang
Ming-Jer
MJ
Shen
Meng-Ru
MR
D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D053454:Caspase 12 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D024042:Collagen Type I / Q000737:chemistry / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005782:Gels; D006367:HeLa Cells; D006706:Homeostasis; D006801:Humans; D007668:Kidney / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D018374:LLC-PK1 Cells; D013049:Spectrin / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology
D000199:Actins; D024042:Collagen Type I; D005782:Gels; D013049:Spectrin; D002154:Calpain; D053454:Caspase 12; D053148:Caspase 3; C449002:mu-calpain; D002118:Calcium