Caspase-3 and -6 expression and enzyme activity in hen granulosa cells.
We have cloned and sequenced cDNAs corresponding to the complete coding regions of the chicken homologues to mammalian caspase-3 and caspase-6. Both caspases are included among members of the cysteine protease (caspase) family that are most closely identified with mediating apoptosis. The deduced amino acid sequences for chicken caspase-3 and -6 show 65% and 68% identity with the respective human sequences, with complete conservation found within the QACRG active peptide region. Both caspase-3 and -6 are widely expressed within various tissues from the hen. Within the ovary, levels of caspase-3 and caspase-6 mRNA and protein do not change significantly in theca tissue during follicle development. On the other hand, procaspase-3 and -6 protein levels are elevated by 2- to 5-fold in preovulatory, compared to prehierarchal (6- to 8-mm diameter), follicle granulosa cells. Nevertheless, the function of this family of cell death-inducing proteins requires activation of the proenzyme caspase, which occurs after cleavage at predictable sites within the N-terminal domain. Accordingly, it was determined that okadaic acid, a pharmacologic inducer of apoptotic cell death in cultured apoptosis-resistant, preovulatory follicle granulosa cells, induced both caspase-3- and caspase-6-like activity within 8-16 h of treatment. By comparison, spontaneous apoptotic cell death that occurs in apoptosis-sensitive, prehierarchal follicle granulosa cells after short-term suspension culture is accompanied by a more rapid increase (within 2 h) in both caspase-3- and -6-like activity. Treatment with 8-bromo-cAMP, which has previously been shown to attenuate, or at least slow, the onset of apoptosis in prehierarchal follicle granulosa cells, mitigates this suspension culture-induced increase in caspase activity. While the present results provide further support for the relationship between caspase activation and apoptotic cell death in hen granulosa cells, the molecular ordering of enzymatic events and the caspase-specific substrates remain to be elucidated.
Johnson
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Department of Biological Sciences, The University of Notre Dame, Notre Dame, Indiana 46556, USA. johnson.128@nd.edu
Bridgham
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D053148:Caspase 3; D053178:Caspase 6; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics* / Q000378:metabolism*; D002645:Chickens / Q000235:genetics*; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010060:Ovulation; D012333:RNA, Messenger; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013799:Theca Cells / Q000201:enzymology
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D012333:RNA, Messenger; C110923:acetyl-valyl-isoleucyl-aspartyl-aldehyde; D019319:Okadaic Acid; D015124:8-Bromo Cyclic Adenosine Monophosphate; C505407:CASP3 protein, human; C505514:CASP6 protein, human; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases