Gonadotropin Actions in Leydig Tumor Cells

描述（由申请人提供）：在啮齿类动物中进行的大量观察以及携带lutropin受体基因（LHR）缺失或功能获得突变的46XY个体的表型清楚地表明，该受体对间质细胞的增殖很重要，甚至可能参与这种细胞类型的转化。本文提出的实验旨在验证LHR激活促进间质细胞增殖和/或存活的信号级联反应的假设。我的实验室最近的结果表明，两种经典的有丝分裂/存活途径，细胞外调节激酶1/2 （ERK1/2）和磷脂酰肌醇3-激酶(PI3K)/Akt，在间质细胞中被LHR激活，并参与其增殖。我们对LHR激活PI3K的机制几乎一无所知，但我们已经证明LHR引发的ERK1/2级联的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，一种src家族激酶和表皮生长因子受体（EGFR）。我们现在提出确定lhr诱导的ERK1/2和PI3K/Akt级联激活的分子基础，并了解它们在间质细胞增殖、存活和分化中的作用。该实验将使用MA-10间质瘤细胞和未成熟大鼠间质细胞的原代培养物进行，并将分为五个具体目标。(1)明确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ERK1/2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ERK1/2级联激活的机制；(4)定义。PI3K/Akt通路参与间质细胞的增殖和存活，并表征LHR激活该通路的机制。(5)完成lhr诱导的间质细胞ERK1/2和PI3K/Akt活化的功能后果表征。一些男性生殖障碍，包括女性化和早熟，都与hLHR的生殖系或体细胞突变有关。在睾丸中，LHR仅在间质细胞中表达，这些突变不仅会引起破坏性的内分泌表现，还会影响间质细胞的数量，并可能与间质细胞腺瘤有关。我们的研究在试图了解LHR如何影响间质细胞的增殖，从而更好地理解间质细胞腺瘤和性分化障碍的病理生理方面是独特而新颖的。

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.