| Leptin-Induced Apoptosis in Human Bone Marrow Stromal Cells:
Mitochondrial Dysfunction Pathway
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| 렙틴에 의한 사람 골수기질세포의 세포자멸:
미토콘드리아 기능장애 경로
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| 배성진,홍정수12,김승욱1,고정민,안정선2,김동관3,김기수 |
| 울산대학교 의과대학 서울아산병원 내과학교실, 아산생명과학연구소1, 흉부외과학교실3,
서울대학교 자연과학대학 생명과학부2
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| Abstract |
Background
Leptin, the Ob gene product, has recently emerged as a key regulator of bone mass. However, the mechanism mediating leptin effect remains controversial. We investigated leptin's effects in osteoblast-lineage primary human bone marrow stromal cells (hBMSC).
Methods
hBMSC were isolated from ribs that were discarded at the time of open thoracotomy in patients without metabolic bone disease. We analyzed the expression of leptin receptor (Ob-R) and its variants in hBMSC using RT-PCR and Western blotting. Leptin's effects on cell viability were measured by WST-1 assay, and alkaline phosphatase activity and osteocalcin secretion was determined. Apoptosis of hBMSC was determined by FACS analysis and Hoechst 33342 staining. After treatment of the cells with leptin, the activity of caspase-3, caspase-8 and caspase-9 was measured using commercial Kit. Active form of caspase-3, caspase-8 and caspase-9, release of cytochrome c into cytosol, and cleavage of poly (ADP-ribose) polymerase (PARP) were measured by Western blotting.
Results
Both the short and long forms of leptin receptors were detected in hBMSC. Leptin significantly decreased the viability of hBMSC. This cytotoxic effect was prevented by Z-Val-Ala-Asp- fluoromethylketone, a pan-caspase inhibitor, and by blocking Ob-R Ab, implicating that leptin-induced hBMSC death was caspase-dependent and specific to leptin receptor. Further investigation demonstrated that leptin activated caspase-3 and caspase-9, but not caspase-8, and increased the release of cytochrome c and the cleavage of poly (ADR-ribose) polymerase (PARP).
Conclusion
These data indicated that leptin induce apoptosis in hBMSC via cytochrome c pathway with activating of caspase-9 and caspase-3, and cleavage of RARP. To our knowledge, this is the first study demonstrating the direct detrimental effect of leptin on bone cells. |
| Key Words:
Leptin, Leptin receptor, Apoptosis, Bone, Human bone marrow stromal cells |
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