SIRT1 regulation of apoptosis of human chondrocytes.
OBJECTIVE
SIRT1 is known to inhibit apoptosis and to promote survival of various types of cells. However, the roles of SIRT1 in apoptosis of human chondrocytes have never been reported. We undertook this study to investigate the relationship of SIRT1 to apoptosis of human chondrocytes, which is a characteristic feature of osteoarthritis (OA).


METHODS
The expression of SIRT1 in human chondrocytes was examined by reverse transcription-polymerase chain reaction, immunoblotting, and immunohistology of human cartilage samples. The expression of SIRT1 under catabolic, mechanical, and nutritional stresses was investigated by immunoblotting. To examine the effect of SIRT1 on apoptosis, SIRT1 was inhibited by small interfering RNA (siRNA) and activated by resveratrol during nitric oxide (NO)-induced apoptosis. TUNEL staining and immunoblotting of cleaved poly(ADP-ribose) polymerase (PARP) were performed to detect apoptosis. To examine the mechanisms of apoptosis, we used immunoblotting to determine the levels of cleaved caspases and mitochondria-related apoptotic signaling proteins, Bax and Bcl-2, in the mitochondrial fraction.


RESULTS
SIRT1 expression was confirmed in human chondrocytes and human cartilage samples. All catabolic, mechanical, and nutritional stresses inhibited SIRT1 expression. SIRT1 inhibition by siRNA for SIRT1 increased the percentage of TUNEL-positive cells and increased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. In contrast, treatment with resveratrol decreased the percentage of TUNEL-positive cells and decreased the amounts of cleaved PARP and cleaved caspases 3 and 9 induced by NO. Furthermore, in the mitochondrial fraction, SIRT1 inhibition by siRNA for SIRT1 increased the amount of Bax but reduced the amount of Bcl-2, while resveratrol reduced the amount of Bax but increased the amount of Bcl-2.


CONCLUSIONS
These results indicate that SIRT1 regulates apoptosis in human chondrocytes through the modulation of mitochondria-related apoptotic signals. Further research on SIRT1 might contribute to resolving the pathogenesis of OA.
Takayama
Koji
K
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 70501 Kusunoki-cho, Kobe, Japan.
Ishida
Kazunari
K
Matsushita
Takehiko
T
Fujita
Norifumi
N
Hayashi
Shinya
S
Sasaki
Ken
K
Tei
Katsumasa
K
Kubo
Seiji
S
Matsumoto
Tomoyuki
T
Fujioka
Hiroyuki
H
Kurosaka
Masahiro
M
Kuroda
Ryosuke
R
D000328:Adult; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001549:Benzamides / Q000494:pharmacology; D016022:Case-Control Studies; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D009284:Naphthols / Q000494:pharmacology; D009536:Niacinamide / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology; D020370:Osteoarthritis, Knee / Q000378:metabolism* / Q000473:pathology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D000077185:Resveratrol; D056564:Sirtuin 1; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D001549:Benzamides; D004791:Enzyme Inhibitors; D009284:Naphthols; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D013267:Stilbenes; D051028:bcl-2-Associated X Protein; C439060:sirtinol; D009536:Niacinamide; D009569:Nitric Oxide; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053453:Caspase 9; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D037761:Sirtuins; D000077185:Resveratrol