Mitochondrial involvement in fas-mediated apoptosis of human lumbar disc cells.
BACKGROUND
Two main pathways of Fas-mediated apoptosis have been identified: the Type-I (death-inducing signaling complex) pathway and the Type-II (mitochondrial) pathway. While apoptotic cell death has been implicated in lumbar degenerative disc disease, we are not aware of any studies in which surgically removed discs from live humans have been examined to determine which of the two pathways is involved in the apoptosis of disc cells. As an initial step in the development of therapies to inhibit inappropriate or premature apoptosis of disc cells, our objective was to determine which pathway is involved.


METHODS
We examined thirty-two samples of herniated lumbar disc tissue with use of immunohistochemical staining and Western blot analysis to determine the presence of several proteins, including caspase-8 (associated with the Type-I pathway); BID (BH3 interacting domain death agonist), cytochrome-c, and caspase-9 (associated with the Type-II pathway); and caspase-3 (an executioner of apoptosis). The TUNEL (terminal deoxynucleotidyl transferase [TDT]-mediated dUTP nick end labeling) assay was performed to confirm the occurrence of apoptosis of the disc cells.


RESULTS
The proteins associated with the Type-II pathway (BID, cytochrome-c, and caspase-9) stained positively in all samples. Although the protein associated with the Type-I pathway (caspase-8) was not detected on immunohistochemical analysis, a small amount of caspase-8 was detected on Western blot analysis. However, the expression of Type-II proteins was still higher than the expression of caspase-8 on Western blot analysis. The expression of caspase-3 was identified in all samples with immunohistochemical and Western blot analysis. TUNEL-positive disc cells were identified in all samples.


CONCLUSIONS
The results of the present study suggest that human disc cells are Type-II cells, which undergo apoptotic cell death through mitochondrial involvement.
Park
Jong-Beom
JB
Department of Orthopaedic Surgery, Uijongbu St. Mary's Hospital, The Catholic University of Korea School of Medicine, 65-1 Kumho-dong, Uijongbu-si, Kyunggi-do, Seoul 480-130, Korea. spinepjb@catholic.ac.kr
Lee
Jin-Kyung
JK
Park
Sung-Jin
SJ
Kim
Ki-Won
KW
Riew
K Daniel
KD
D000328:Adult; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D002352:Carrier Proteins / Q000502:physiology; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007403:Intervertebral Disc / Q000166:cytology*; D007405:Intervertebral Disc Displacement / Q000503:physiopathology*; D008159:Lumbar Vertebrae / Q000166:cytology*; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000502:physiology; D019014:fas Receptor / Q000502:physiology*
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D019014:fas Receptor; D045304:Cytochromes c; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases