Identification of protein kinases that control ovarian hormone release by selective siRNAs.
The goal of this study was to identify protein kinases (PKs) that control the secretory activity of human ovarian cells. Cultured ovarian granulosa cells were transfected with 264 siRNA constructs that selectively block the expression of 88 known PKs. The efficiency of transfection and of silencing marker molecules (glyceraldehyde 3-phosphate dehydrogenase, GAPDH and CDC2/p34 PK) was validated by fluorescence microscopy, real-time reverse transcription-PCR, and immunocytochemistry. Release of steroid hormones (progesterone, P(4)) and IGF1 was determined by RIA. siRNA suppressed the expression of marker molecules by up to 84%. P(4) release was suppressed after inhibiting 34 individual PKs and was stimulated after inhibiting 12 PKs. Blocking nine individual PKs inhibited IGF1 release, while the inactivation of 17 others stimulated IGF1 release. Together, these results demonstrate that the release of both steroid and peptide hormones by human ovarian cells is controlled by a large number of PKs, and that siRNA constructs may be useful tools for further defining the role of PKs in controlling ovarian secretory function.
Sirotkin
Alexander V
AV
Institute of Genetics and Reproduction, Animal Production Research Centre, Hlohovecká, LuZianky near Nitra, Slovakia. sirotkin@scpv.sk
Ovcharenko
Dmitriy
D
Mlyncek
Milos
M
D000818:Animals; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D042341:Gonadal Hormones / Q000378:metabolism*; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D010053:Ovary / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*
D042341:Gonadal Hormones; D034741:RNA, Small Interfering; D007334:Insulin-Like Growth Factor I; D011494:Protein Kinases; D016203:CDC2 Protein Kinase