Hsp70 prevents nitric oxide-induced apoptosis in articular chondrocytes.
OBJECTIVE
To deliver and overexpress the hsp70 gene in cultured chondrocytes to investigate its effect on nitric oxide (NO)-induced apoptosis of chondrocytes.


METHODS
Primary chondrocyte cultures were established from rabbit joints. The cells were transduced with an empty adenovirus vector (Ax1w) or an adenovirus vector harboring the hsp70 E-tag fusion gene (AxSHEwt). Apoptosis was induced by sodium nitroprusside (SNP) dihydrate, which generates NO, or by staurosporine, which is a proapoptotic agent dependent upon Bax or Bak protein. Cell viability and apoptosis induction were estimated by lactate dehydrogenase (LDH) assay, Hoechst 33342 staining, or the TUNEL method. To study Hsp70, cytochrome c, and caspase 3, Western blot analyses were performed.


RESULTS
The AxSHEwt-transduced cells escaped apoptosis, as revealed by the LDH assay, Hoechst 33342 staining, and the TUNEL method. A massive amount of the tagged Hsp70 was demonstrated in the AxSHEwt-transduced chondrocytes but not in control cells. Hsp70 did not affect the cytosolic cytochrome c level, but appeared to have obstructed the activation of caspase 3.


CONCLUSIONS
Experimentally overexpressed Hsp70 almost completely inhibited NO- or staurosporine-induced apoptosis in primary chondrocytes.
Terauchi
Ryu
R
Department of Orthopaedic Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Takahashi
Kenji A
KA
Arai
Yuji
Y
Ikeda
Takumi
T
Ohashi
Suzuyo
S
Imanishi
Jiro
J
Mazda
Osam
O
Kubo
Toshikazu
T
D000260:Adenoviruses, Human / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001562:Benzimidazoles; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D011817:Rabbits; D013194:Staining and Labeling / Q000379:methods; D019311:Staurosporine / Q000494:pharmacology; D014161:Transduction, Genetic
D001562:Benzimidazoles; D061945:Caspase Inhibitors; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D009599:Nitroprusside; D009569:Nitric Oxide; D007770:L-Lactate Dehydrogenase; C505407:CASP3 protein, human; D053148:Caspase 3; D019311:Staurosporine; C017807:bisbenzimide ethoxide trihydrochloride