Gonadotropin Actions in Leydig Tumor Cells

描述（由申请方提供）：在啮齿动物中进行的大量观察以及携带促黄体激素受体基因（LHR）功能丧失或获得突变的46 XY个体的表型清楚地表明，该受体对Leydig细胞的增殖很重要，甚至可能参与该细胞类型的转化。本文提出的实验旨在检验LHR激活促进Leydig细胞增殖和/或存活的信号级联的假设。本实验室最近的研究结果表明，两个经典的促有丝分裂/生存途径，细胞外调节激酶1/2（ERK 1/2）和磷脂酰肌醇3-激酶（PI 3 K）/Akt，被激活的LHR在Leydig细胞，他们参与了他们的增殖。我们对LHR激活PI 3 K的机制几乎一无所知，但我们已经证明LHR引起的ERK 1/2级联激活涉及两个独立的途径。一种需要cAMP激活的蛋白激酶A（PKA），另一种需要Fyn、Src家族激酶和表皮生长因子受体（EGFR）。我们现在建议确定LHR诱导的ERK 1/2和PI 3 K/Akt级联激活的分子基础，并了解它们在Leydig细胞增殖，存活和分化中的作用。将使用MA-10 Leydig肿瘤细胞和未成熟大鼠Leydig细胞的原代培养物进行拟议的实验，并将分为五个具体目标。(1)定义LHR激活Fyn的机制。(2)完成EGF样生长因子参与hCG诱导的间质细胞ERK 1/2磷酸化的表征;（3）明确蛋白激酶A（PKA）介导LHR诱导的间质细胞ERK 1/2级联激活的机制;（4）明确PI 3 K/Akt通路参与Leydig细胞的增殖和存活，并表征LHR激活该通路的机制。(5)完成Leydig细胞中LHR诱导的ERK 1/2和PI 3 K/Akt激活的功能后果的表征。一些男性生殖障碍，包括女性化和早熟与hLHR的生殖系或体细胞突变有关。在睾丸中，LHR仅在Leydig细胞中表达，这些突变不仅会导致破坏性内分泌表现，而且还会影响Leydig细胞的数量，并可能与Leydig细胞腺瘤相关。我们的研究是独特的和新颖的，试图了解LHR如何影响Leydig细胞的增殖，从而更好地了解Leydig细胞腺瘤和性分化障碍的病理生理学。

DESCRIPTION (provided by applicant): 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.