Comparison of Effects of Protein Kinase A, Mitogen-activated Protein Kinase, and Cyclin-dependent Kinase Blockers on Rabbit Ovarian Granulosa Cell Functions

Abstract

The aim of the present study was to define the role of protein kinase A (PKA)-, mitogen-activated protein kinase (MAPK)-, and cyclin-dependent kinase (CDK)-dependent pathways in the control of ovarian cell functions. The effects of PKA, MAPK, and CDK blockers (KT 5720, PD 98059, and olomoucine, respectively), given at doses of 0.001–10.0 μg/ml medium on functions of cultured rabbit granulosa cells were examined. Expression of PKA, MAPK/ERK1,2, secretory activity (IGF-I output), and proliferation (proliferating cell nuclear antigen, PCNA) in these cells were determined by RIA, immunocytochemistry and Western blotting. A PKA inhibitor, KT 5720 suppressed the expression of PKA and MAPK/ERK1,2, the IGF-I release, and the ratio of PCNA-positive cells in granulosa cells. A MAPK blocker, PD 98059 reduced the expression of MAPK/ERK1,2 (but not PKA), the IGF-I release, and percentage of PCNA-positive cells. A CDK blocker, olomoucine, increased the PKA expression, decreased the expression of MAPK/ERK1,2 and PCNA, but did not affect the IGF-I release. These observations confirm the involvement of PKs in control of basic ovarian functions and demonstrate the involvement of PKA in stimulation of ovarian cell proliferation and MAPK (but not CDK) and in promotion of ovarian IGF-I release. Different activity and specificity of the PKA, MAPK, and CDK blockers in their effects on PCNA and IGF-I suggests different biological role of these PKs in control of proliferative and secretory functions of rabbit ovarian cells.

granulosa - PCNA - IGF-I - ERK1,2 kinase - PKA - CDC2 kinase

References

• 1 Sirotkin AV, Ovcharenko D, Benèo A, Mlynèek M. Protein kinases controlling PCNA and p53 expression in human ovarian cells.  Funct Integr Genomics. 2009;  9 185-295
• 2 Cohen P. Protein kinases – the major drug targets of the twenty-first century?.  Nat Rev Drug Disc. 2002;  1 309-315
• 3 Chiaradonna F, Balestrieri C, Gaglio D, Vanoni M. RAS and PKA pathways in cancer: new insight from transcriptional analysis.  Front Biosci. 2008;  13 5257-5278
• 4 Torii K, Nishizawa K, Kawasaki A, Yamashita Y, Katada M, Ito M, Nishimoto I, Terashita K, Aiso S, Matsuoka M. Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways.  Cell Signal. 2008;  20 1256-1266
• 5 Lu CC, Tsai SC. The cyclic AMP-dependent protein kinase A pathway is involved in progesterone effects on calcitonin secretion from TT cells.  Life Sci. 2007;  81 1411-1420
• 6 Sedej S, Rose, Rupnik M. cAMP increases Ca2+-dependent exocytosis through both PKA and Epac2 in mouse melanotrophs from pituitary tissue slices.  J Physiol. 2005;  15 799-813
• 7 Makarevich A, Sirotkin A, Chrenek P, Bulla J, Hetenyi L. The role of IGF-I, cAMP/protein kinase A and MAP-kinase in the control of steroid secretion, cyclic nucleotide production, granulosa cell proliferation and preimplantation embryo development in rabbits.  J Steroid Biochem Mol Biol. 2000;  73 123-133
• 8 Cheadle C, Nesterova M, Watkins T, Barnes KC, Hall JC, Rosen A, Becker KG, Cho-Chung YS. Regulatory subunits of PKA define an axis of cellular proliferation/differentiation in ovarian cancer cells.  BMC Med Genomics. 2008;  1 43
• 9 Viegas LR, Hoijman E, Beato M, Pecci A. Mechanisms involved in tissue-specific apopotosis regulated by glucocorticoids.  J Steroid Biochem Mol Biol. 2008;  109 273-278
• 10 Hsueh AJ, Eisenhauer K, Chun SY, Hsu SY, Billig H. Gonadal cell apoptosis.  Recent Progr Horm Res. 1996;  51 433-455
• 11 Amsterdam A, Sasson R, Keren-Tal I, Aharoni D, Dantes A, Rimon E, Land A, Cohen T, Dor Y, Hirsh L. Alternative pathways of ovarian apoptosis: death for life.  Biochem Pharmacol. 2003;  66 1355-1362
• 12 Paton AC, Collins WP. Differentiation processes of granulosa cells.  Oxford Rev Reprod Biol. 1992;  14 169-223
• 13 Hillier SG, Tetsuka M. Role of androgens in follicle maturation and atresia.  Baillieres Clin Obstet Gynaecol. 1997;  11 249-260
• 14 Sirotkin AV, Makarevich AV. GH regulates secretory activity and apoptosis in cultured bovine granulose cells through the activation of the cAMP/protein kinase A system.  J Endocrinol. 1999;  163 317-327
• 15 Sirotkin AV, Makarevich AV, Hetényi L. Effect of growth hormone and inhibitors of protein kinase A on IGF-I, oxytocin and progesterone release by cultured bovine granulosa cells.  Ann Endocrinol (Paris). 2000;  61 154-158
• 16 Makarevich AV, Sirotkin AV, Genieser HG. Action of protein kinase A regulators on secretory activity of porcine granulosa cells in vitro.  Anim Reprod Sci. 2004a;  81 125-136
• 17 Sirotkin AV. Control of reproductive processes by growth hormone: extra- and intracellular mechanisms.  Vet J. 2005;  170 307-317
• 18 Peluso JJ. Multiplicity of progesterone's actions and receptors in the mammalian ovary.  Biol Reprod. 2006;  75 2-8
• 19 Makarevich AV, Sirotkin AV. Presumptive mediators of growth hormone action on insulin-like growth factor I release by porcine ovarian granulosa cells.  Biol Sign Recept. 2000;  9 248-254
• 20 Izadyar F, Colenbrander B, Bevers MM. Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through the cyclic adenosine 3′,5′-monophosphate signaling pathway.  Biol Reprod. 1997;  57 1484-1489
• 21 Greely M, Sreenan JM. Effect of adenylyl cyclase activation on intracellular and extracellular cAMP and cGMP in preimplantation cattle blastocysts.  J Reprod Fert. 1999;  116 355-361
• 22 Hillier SG. Gonadotropic control of ovarian follicular growth and development.  Mol Cell Endocrinol. 2001;  179 39-46
• 23 McKenna SD, Pietropaolo M, Tos EG, Clark A, Fischer D, Kagan D, Bao B, Chedrese PJ, Palmer S. Pharmacological inhibition of phosphodiesterase 4 triggers ovulation in follicle-stimulating hormone-primed rats.  Endocrinol. 2005;  146 208-214
• 24 Hunzicker-Dunn M, Maizels ET. FSH signaling pathways in immature granulosa cells that regulate target gene expression: branching out from protein kinase A.  Cell Signal. 2006;  18 1351-1359
• 25 Sirotkin AV, Makarevich AV, Kwon HB, Kotwica J. Involvement of MAP kinase in the mediation of GH action on ovarian granulosa cells.  Mol Cell Endocrinol. 2003;  205 193-199
• 26 Makarevich AV, Sirotkin AV, Franek J, Kwon HB, Bulla J. The role of oxytocin, protein kinase A, and ERK-related MAP-kinase in the control of porcine ovarian follicle functions.  Exp Clin Endocrinol Diab. 2004b;  112 108-114
• 27 Petley T, Graff K, Jiang W, Yang H, Florini J. Variation among cell types in the signalling pathways by which IGF-I stimulates specific cellular responses.  Horm Metab Res. 1999;  31 70-76
• 28 Jones SM, Kazlauskas A. Growth factor-dependent signalling and cell cycle progression.  FEBS Lett. 2001;  490 110-116
• 29 Seger R, Krebs EG. The MAPK signaling cascade.  FASEB J. 1995;  9 726-735
• 30 Sirotkin AV, Grossmann R. Role of tyrosin kinase- and MAP kinase-dependent intracellular mechanisms in control of ovarian functions in the domestic fowl (Gallus domesticus) and in mediating effects of IGF-II.  J Reprod Dev. 2003;  49 99-106
• 31 Kishimoto T. Cell reproduction: induction of M-phase events by cyclin-dependent cdc2 kinase.  Int J Dev Biol. 1994;  38 185-191
• 32 Fernandez A, Cavadore JC, Demaille J, Lamb N. Implications for cAMP-dependent kinase in the maintenance of the interphase state.  Prog Cell Cycle Res. 1995;  1 241-253
• 33 D’Angiolella V, Costanzo V, Gottesman ME, Avvedimento EV, Gautier J, Grieco D. Role for cyclin-dependent kinase 2 in mitosis exit.  Curr Biol. 2001;  11 1221-1226
• 34 Naryzhny SN, Lee H. Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages.  Electrophoresis. 2001;  22 1764-1775
• 35 Duckworth BC, Weawer JS, Ruderman JV. G2 arrest in xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A.  Proc Natl Acad Sci USA. 2002;  99 16794-16799
• 36 Rhee K, Wolgemuth DJ. Cdk family genes are expressed not only in dividing but also in terminally differentiated mouse germ cells, suggesting their possible function during both cell division and differentiation.  Dev Dyn. 1995;  204 406-420
• 37 Lee K-Y, Rosales JL, Lee B-C, Chung S-H, Fukui Y, Lee N-S, Lee KY, Jeong Y-G. Cdk5/p35 expression in the mouse ovary.  Mol Cells. 2004;  17 17-22
• 38 Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors.  Biochem J. 2000;  351 95-105
• 39 Sirotkin AV, Grossmann R. The role of protein kinase A and cyclin-dependent (cdc2) kinase in the control of basal and IGF-II-induced proliferation and secretory activity of chicken ovarian cells.  Anim Reprod Sci. 2006;  92 169-181
• 40 Vesely J, Havlicek L, Strnad M, Blow JJ, Donella-Deana A, Pinna L, Letham DS, Kato J, Detivaud L, Leclerc S. Inhibition of cyclin-dependent kinases by purine analogues.  Eur J Biochem. 1994;  224 771-786
• 41 Abraham RT, Acquarone M, Andersen A, Asensi A, Belle R, Berger F, Bergounioux C, Brunn G, Buquet-Fagot C, Fagot D. Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases.  Biol Cell. 1995;  83 105-120
• 42 Wesierska-Gadek J, Gueorguieva M, Kramer MP, Ranftler C, Sarg B, Lindner H. A new, unexpected action of olomoucine, a CDK inhibitor, on normal human cells: up-regulation of CLIMP-63, a cytoskeleton-linking membrane protein.  J Cell Biochem. 2007;  102 1405-1419
• 43 Sirotkin AV, Grossmann R. The role of ghrelin and some intracellular mechanisms in controlling the secretory activity of chicken ovarian cells.  Comp Biochem Physiol Part A. 2007;  147 239-246
• 44 Olsen MK, Reszka AA, Abraham I. KT5720 and U-98017 inhibit MAPK and alter the cytoskeleton and cell morphology.  J Cell Physiol. 1998;  176 525-536
• 45 Webster J, Prager D, Melmed S. Insulin-like growth factor-I activation of extracellular signal-related kinase-1 and -2 in growth hormone-secreting cells.  Mol Endocrinol. 1994;  8 539-544
• 46 Berisha B, Schams D. Ovarian function in ruminants.  Dom Anim Endocrinol. 2005;  29 305-317
• 47 Makarevich A, Sirotkin A, Taradajnik T, Chrenek P. Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro.  J Ster Biochem Mol Biol. 1997;  63 329-337
• 48 Keppens S. Effect of genistein on both basal and glucagon-induced levels of cAMP in rat hepatocytes.  Biochem Pharmacol. 1995;  50 1303-1304

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