Thrombopoietin regulates proliferation, apoptosis, secretory activity and intracellular messengers in porcine ovarian follicular cells: involvement of protein kinase A

Abstract

Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their interrelationships within ovarian cells. We investigated the effect of TPO treatment (0, 1, 10 or 100 ng/ml) on the following characteristics of cultured porcine ovarian follicles, determined using SDS-PAGE and Western blotting, immunocytochemistry, RIA and ELISA: the expression of intracellular peptides associated with proliferation (PCNA), apoptosis (Bax), tyrosine kinase (TK, phosphotyrosine), Cdc2/p34 kinase, PKA and the transcription factor CREB-1, and the secretion of progesterone, androstenedione, estradiol-17beta, oxytocin, inhibin A, inhibin B, IGF-I, transforming growth factor-2beta (TGF-2beta) and IGF-binding protein 3 (IGFBP-3). The involvement of PKA-dependent pathways was examined by evaluating the effect of a PKA blocker (KT5720, 1 microg/ml), either alone or in combination with TPO, on the parameters listed above. A TPO-induced increase in expression of PCNA, Bax, PKA, TK, Cdc2/p34 and CREB was observed. Furthermore, TPO was able to inhibit androstenedione, estradiol, TGF-2beta and IGFBP-3 secretion, and to stimulate oxytocin, inhibin A, inhibin B and IGF-I secretion. Progesterone secretion was not stimulated. The PKA blocker KT5720, when given alone, reduced the expression of Bax and TGF-2beta, augmented the expression of PKA, CREB and oxytocin, but did not influence the secretion of progesterone, androstenedione, estradiol, IGFBP-3, inhibins A and B or IGF-I. When given together with TPO, the PKA blocker prevented or reversed the action of TPO on PKA, CREB, androstenedione, estradiol, IGFBP-3, oxytocin, but not its effect on Bax, TGF-2beta or inhibin B. On the other hand, treatment with KT5720 augmented the effect of TPO on progesterone, inhibin A and IGF-I. These results provide the first evidence that TPO may be a potent regulator of ovarian function (e.g. proliferation, apoptosis and the secretion of peptide hormones, steroids, growth factors and growth factor-binding protein, as well as of the expression of some intracellular messengers). Furthermore, they demonstrated the importance of PKA in controlling these functions and in mediating the effects of TPO on ovarian cells. It remains possible that other (TK- and Cdc2/p34-dependent) intracellular mechanisms are also involved in mediating TPO action on the ovary.

Protein kinases and ovarian functions

“…Although not all known PKs are demonstrated in the ovary, presence of members of MAPK, CDK, TK, PI3K, Akt, PKA, AK, HER kinases, mTOR and activin receptor like kinase (ALK) families in ovarian follicular granulosa cells has been well documented (Sirotkin et al, 2000a(Sirotkin et al, , 2004Shimada et al, 2001;Makarevich et al, 2002Makarevich et al, , 2004bHunzicker-Dunn and Maizels, 2006;Kumar et al, 2007;Sawada et al, 2007;Wang and Tsang, 2007;Dupont et al, 2008). Furthermore, observations of effects of activators and blockers of PKs indicate presence and importance of a number of other PKs in control of ovarian functions and in mediating effect of hormones on these functions (see below).…”

“…There are reports on the action of GH on ovarian PKA and MAPK Sirotkin, 2005; Fig. 3), leptin on PKA (Sirotkin et al, 2008b), ghrelin on PKA, MAPK, and CDK/p34 (Sirotkin andGrossmann, 2007b, 2008), oxytocin on PKA and MAPK (Makarevich et al, 2004b), IGF-I, IGF-II and EGF on PKA, MAPK/ERK1,2, CDK/p34 Sirotkin andGrossmann, 2003, 2006), PI3K, p70S6K, and mTOR (Hunzicker-Dunn and Maizels, 2006), thrombopoietin on PKA, TKs, and CDK/p34 (Sirotkin et al, 2004;Fig. 2).…”

“…GH Sirotkin, 2005) and oxytocin (Makarevich et al, 2004b) can prevent apoptosis and stimulate release of ovarian hormones via PKA and MAPKs. Leptin (Sirotkin and Grossmann, 2007a,b;Sirotkin et al, 2004Sirotkin et al, , 2008b and ghrelin (Sirotkin andGrossmann, 2007b, 2008) regulate ovarian cell proliferation, apoptosis and secretory activity through PKA-, MAPK-, and CDK-dependent intracellular mechanisms. IGF-I, IGF-II, and EGF can promote ovarian cell proliferation and secretory activity through cAMP/PKA, cGMP/PKG, MAPK-, PI3/Akt and glycogen synthase kinase (GSK)-dependent pathways (Makarevich et al, 2000Richards et al, 2002;Sirotkin andGrossmann, 2003, 2006;Dupont et al, 2008).…”

Protein Kinase A Represses Skeletal Myogenesis by Targeting Myocyte Enhancer Factor 2D

“…As a classical effector of the G-protein-coupled receptor, responding to increased intracellular cAMP, protein kinase A (PKA) is likewise involved in regulation of diverse biological processes, including cell proliferation (41,68,69), muscle differentiation (11,33,74), and neuronal survival (27,34,64). Inactive PKA is a heterotetramer composed of two catalytic and two regulatory subunits.…”

Interactions of the Protein Kinase A Signaling Pathway: Implications for the Treatment of Endocrine and Other Tumors

“…The data suggest that PKA has a role in mediating the effect of TPO on proliferation and apoptosis in the ovary. However, the exact mechanism involved is yet to be determined (Sirotkin et al, 2004). It is well established that leptin, a product of adipose tissue, has direct effects on the control of ovarian steroidogenesis, prostaglandin secretion and IGFBP-3 and IGG-1 release.…”