Antisense oligonucleotide of clusterin mRNA induces apoptotic cell death and prevents adhesion of rat ASC-17D Sertoli cells.
Clusterin has been known to play important roles in cell-cell and/or cell-substratum interactions. Recently we reported the transient expression of clusterin in pancreatic endocrine cells during the early developmental stages and suggested a role in aggregating the endocrine cells for islet formation. In the present study, we have investigated the involvement of clusterin in cell-substratum interaction by the inhibition of clusterin synthesis using antisense oligonucleotide. The expression of clusterin was transiently increased as early as 2-8 h after plating the ASC-17D Sertoli cells to the culture flask, which was the period of cell attachment. In addition, up-regulation of clusterin mRNA was so much greater when the Sertoli cells were plated on the petri dish for the bacterial culture instead of in a animal cell culture flask that therefore, the cells failed to attach to it. These findings suggested that interruption of cell to plate substratum interaction might lead to over-expression of clusterin from Sertoli cells to induce cell to cell aggregation or, perhaps, to re-establish attachment with the substratum. Transfection of ASC-17D Sertoli cells with a 20-base antisense oligonucleotide against clusterin mRNA resulted in extracellular release of LDH and DNA fragmentation. Sertoli cell death by antisense oligonucleotide of clusterin was sequence specific and dose dependent. Treatment of antisense oligonucleotide induced a marked reduction of synthesis for clusterin protein, but not for clusterin mRNA expression, suggesting the translational suppression of clusterin by antisense oligonucleotide. Further, microscopic observation showed that more noticeable cell death was induced by treating the antisense prior to plating the cells than by treating after cell attachment to the plate. From these results, we speculate that down-regulation of clusterin expression in the anchorage-dependent Sertoli cells prevents them from attaching to the plate, and therefore induces cell death.
Kang
S W
SW
Department of Pharmacology, College of Medicine, Korea University, Seoul.
Lim
S W
SW
Choi
S H
SH
Shin
K H
KH
Chun
B G
BG
Park
I S
IS
Min
B H
BH
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D002448:Cell Adhesion; D002460:Cell Line; D051152:Clusterin; D053938:DNA Fragmentation; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D018832:Molecular Chaperones; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012708:Sertoli Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D016376:Oligonucleotides, Antisense; D012313:RNA