A number of observations made in rodents as well as the phenotype of 46XY individuals harboring loss-of- or gain-of-function mutations of the lutropin receptor gene (LHR) clearly show that this receptor is important for the proliferation of the Leydig cells and may even be involved in the transformation of this cell type. The experiments proposed herein are designed to test the hypothesis that the LHR activates signaling cascades that promote the proliferation and/or survival of Leydig cells. Recent results from my laboratory have shown that two classic mitogenic/survival pathways, extracellular regulated kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt, are activated by the LHR in Leydig cells and that they are involved in their proliferation. We know virtually nothing about the mechanisms by which the LHR activates PI3K, but we have shown that the LHR-provoked activation of the ERK1/2 cascade involves two independent pathways. One requires the cAMP-activated protein kinase A (PKA) and the other requires Fyn, a Src-family kinase and the epidermal growth factor receptor (EGFR). We now propose to define the molecular basis of the LHR-induced activation of the ERK1/2 and PI3K/Akt cascades and to understand their roles in the proliferation, survival and differentiation of Leydig cells. The proposed experiments will be pursued using MA-10 Leydig tumor cells and primary cultures of immature rat Leydig cells and will be divided into five specific aims. (1) Define the mechanisms by which the LHR activates Fyn. (2) Complete the characterization of the involvement of EGF-like growth factors in the hCG- provoked ERK1/2 phosphorylation in Leydig cells (3) Define the mechanisms by which protein kinase A (PKA) mediates the LHR-provoked activation of the ERK1/2 cascade in Leydig cells;(4) Define the . involvement of the PI3K/Akt pathway in the proliferation and survival of Leydig cells and characterize the mechanisms by which the LHR activates this pathway. (5) Complete the characterization the functional consequences of the LHR-induced ERK1/2 and PI3K/Akt activation in Leydig cells. Some male reproductive disorders including feminization and precious puberty are associated with germ line or somatic mutations of the hLHR. In the testes the LHR is expressed exclusively in Leydig cells and these mutations not only cause disruptive endocrine manifestations but they also influence the number of Leydig cells, and can be associated with Leydig cell adenomas. Our studies are unique and novel in attempting to understand how does the LHR affect the proliferation of Leydig cells and thus to gain a better understanding of the pathophysiology of Leydig cell adenomas and disorders of sexual differentiation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA040629-27
Application #
7817086
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Sathyamoorthy, Neeraja
Project Start
1998-02-15
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
27
Fiscal Year
2010
Total Cost
$406,558
Indirect Cost
Name
University of Iowa
Department
Pharmacology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Matzkin, Maria Eugenia; Yamashita, Soichi; Ascoli, Mario (2013) The ERK1/2 pathway regulates testosterone synthesis by coordinately regulating the expression of steroidogenic genes in Leydig cells. Mol Cell Endocrinol 370:130-7
Tai, Ping; Ascoli, Mario (2011) Reactive oxygen species (ROS) play a critical role in the cAMP-induced activation of Ras and the phosphorylation of ERK1/2 in Leydig cells. Mol Endocrinol 25:885-93
Yamashita, Soichi; Tai, Ping; Charron, Jean et al. (2011) The Leydig cell MEK/ERK pathway is critical for maintaining a functional population of adult Leydig cells and for fertility. Mol Endocrinol 25:1211-22
Tai, Ping; Shiraishi, Koji; Ascoli, Mario (2009) Activation of the lutropin/choriogonadotropin receptor inhibits apoptosis of immature Leydig cells in primary culture. Endocrinology 150:3766-73
Galet, Colette; Ascoli, Mario (2008) Arrestin-3 is essential for the activation of Fyn by the luteinizing hormone receptor (LHR) in MA-10 cells. Cell Signal 20:1822-9
Shiraishi, Koji; Ascoli, Mario (2008) A co-culture system reveals the involvement of intercellular pathways as mediators of the lutropin receptor (LHR)-stimulated ERK1/2 phosphorylation in Leydig cells. Exp Cell Res 314:25-37
Shiraishi, Koji; Ascoli, Mario (2007) Lutropin/choriogonadotropin stimulate the proliferation of primary cultures of rat Leydig cells through a pathway that involves activation of the extracellularly regulated kinase 1/2 cascade. Endocrinology 148:3214-25
Ascoli, Mario (2007) Potential Leydig cell mitogenic signals generated by the wild-type and constitutively active mutants of the lutropin/choriogonadotropin receptor (LHR). Mol Cell Endocrinol 260-262:244-8
Galet, Colette; Ascoli, Mario (2006) A constitutively active mutant of the human lutropin receptor (hLHR-L457R) escapes lysosomal targeting and degradation. Mol Endocrinol 20:2931-45
Shiraishi, Koji; Ascoli, Mario (2006) Activation of the lutropin/choriogonadotropin receptor in MA-10 cells stimulates tyrosine kinase cascades that activate ras and the extracellular signal regulated kinases (ERK1/2). Endocrinology 147:3419-27

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