Recent progress in hormone research最新文献

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. Wnt family genes encode secreted glycoproteins that act as ligands for membrane receptors belonging to the Frizzled family of proteins. Wnt-1 originally was found as a proto-oncogene that was upregulated in tumors caused by the mouse mammary tumor virus. The Drosophila homologue of Wnt-1, wingless, is a segment polarity gene that regulates body patterning of the fly embryo. In Xenopus, the Wnt pathway regulates formation of the ventral-dorsal axis. Although Wnt proteins are expressed widely in mammals, the function of the Wnt signaling pathway in normal adult mammalian tissues is not understood. Downstream components of the Wnt pathway, APC (adenomatous polyposis coli) and beta-catenin, clearly are involved in human cancer. There are also several reports that Wnt ligands are highly expressed in tumors. Wnt stabilizes cytoplasmic beta-catenin and activates beta-catenin/Lef-1 (lymphoid enhancer factor), Tcf (T-cell factor)-dependent gene transcription. This regulation of cytosolic beta-catenin is mediated by glycogen synthase kinase-3 (GSK-3) activity but in neither case is the mechanism known. The mechanism by which Wnt inhibits GSK-3 is unknown. Recent studies have shown that some of the intracellular signaling molecules that mediate the Wnt pathway are in complexes, including Dishevelled (Dsh or Dvl), GSK-3beta, and APC protein. However, little is known about how Wnt or other upstream stimuli regulate these complexes to stabilize beta-catenin. We took a variety of approaches to identify new components of the Wnt pathway. Using an expression-cloning technique, we isolated casein kinase I (CKI)epsilon as a positive regulator of beta-catenin in the Wnt pathway. Overexpression of CKIepsilon mimics Wnt by stabilizing beta-catenin, thereby increasing expression of beta-catenin-dependent genes. Inhibition of endogenous CKIepsilon attenuated gene transcription stimulated by Wnt or by Dsh. CKIepsilon forms a complex with Axin and the other downstream components of the Wnt pathway. CKIepsilon is a positive regulator of the Wnt pathway and a possible functional link between upstream signals and the intracellular Axin signaling complex that regulates beta-catenin. In separate experiments, we have identified a Dishevelled-associated kinase (DAK) that binds to Dsh and regulates its functions. Dsh is required for two different pathways, the Wnt pathway and planar polarity pathway in Drosophila. DAK dramatically enhances the function of Dsh in the Wnt pathway and inhibits its function in the planar polarity pathway. This chapter will discuss these newly identified components of the Wnt pathway.

Members of the transforming growth factor beta (TGFbeta) superfamily are polypeptide growth factors that exhibit diverse effects on normal cell growth, adhesion, mesenchymal-epithelial interactions, cell differentiation, and programmed cell death. This chapter will discuss the work of ourselves and others on one member of this large superfamily, Müllerian inhibiting substance (MIS, or anti-Müllerian hormone, AMH) and its role in reproductive tract development and the adult gonad. Using recombinant MIS protein, it is possible to begin unraveling the molecular mechanism of duct involution in the embryo. Our recent results suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions. In addition to the developmental effects of MIS in secondary sexual differentiation, expression studies of the MIS ligand and the MIS type II receptor (MISIIR) suggest a potential regulatory role for MIS in adult germ cell maturation and gonadal function. Recent data from others suggest that MIS may act in a paracrine manner to block differentiation of interstitial cells of the adult gonad by repressing all or some steps of steroidogenesis. Our studies are highly suggestive of direct repression of steroidogenic enzyme gene expression by activation of the MIS signaling pathway. Thus, for the first time, an opportunity to define fully target genes and components of the MIS signaling pathway may be possible.

Growth hormone (GH) has long been known to be a primary determinant of body height and an important regulator of body metabolism, yet the cellular and molecular bases for these effects of GH are only beginning to be understood. In 1993, GH receptor (GHR) was first observed to bind to the tyrosine kinase JAK2. GH increased JAK2's affinity for GHR, potently activated JAK2, and stimulated the phosphorylation of tyrosines within JAK2 and the cytoplasmic domain of GHR. In the intervening six years, a variety of signaling molecules have been identified that are tyrosyl phosphorylated in response to GH, presumably by the activated JAK2. These signaling molecules include 1) the latent cytoplasmic transcription factors--designated signal transducers and activators of transcription (Stats)--that have been implicated in the regulation of a variety of GH-dependent genes; 2) Shc proteins that lead to activation of the Ras-MAP kinase pathway: and 3) insulin receptor substrate (IRS) proteins that bind and thereby activate phosphatidylinositol 3' kinase and presumably other proteins. Recently, we have identified two additional signaling molecules for GH that bind to JAK2 and are phosphorylated on tyrosines in response to GH: SH2-B and signal regulated protein (SIRP). Based upon amino acid sequence analysis, SH2-B is presumed to be a cytoplasmic adapter protein. It binds with high affinity via its SH2 domain to phosphorylated tyrosines within JAK2. GH-induced binding of SH2-B to JAK2 via this site potently activates JAK2, leading to enhanced tyrosyl phosphorylation of Stat proteins and other cellular proteins. Because of its other potential protein-protein interaction domains and its recruitment and phosphorylation by kinases that are not activated by SH2-B, SH2-B is thought likely to mediate other, more-specific actions of GH, as yet to be determined. SIRP is a transmembrane protein that is now known to bind to integrin-associated protein. It appears to bind directly to JAK2 by a process that does not require tyrosyl phosphorylation, although is itself highly phosphorylated on tyrosines in response to GH. The phosphorylated SIRP recruits one or more molecules of the tyrosine phosphatase SHP2 that, in turn, de-phosphorylates SIRP and most likely JAK2. Thus, SIRP is predicted to be a negative regulator of GH action. It seems likely that the diverse actions of GH will be found to require coordinated interaction of all of these signaling proteins with each other as well as with other signaling molecules that are activated by GH and the numerous other ligands that are present at cells during a response to GH.

Activation of cells by agents that stimulate inositol trisphoshate (IP3) formation causes, via IP3 receptor (IP3R) activation, the release of Ca2+ from internal stores and also the entry of Ca2+ via plasma membrane cation channels, referred to as capacitative Ca2+ entry or CCE channels. Trp proteins have been proposed to be the unitary subunits forming CCE channels; however, there is no definitive proof for this hypothesis. We have now identified amino acid sequences of a Trp and of an IP3R that interact to form stable complexes. These complexes appear to form in vivo, as evidenced by co-immunoprecipitation of Trp with IP3R and by the fact that expression of the respective interacting sequences modulates development of CCE brought about by store depletion. The finding that a Trp-interacting sequence of IP3R interferes with natural CCE leads us to conclude that Trp proteins are, indeed, structural members of CCE channels. We conclude further that direct coupling of IP3R to Trp is a physiological mechanism by which cells trigger CCE in response to signals that stimulate phosphoinositide hydrolysis and IP3 formation. Pros and cons of various CCE activation models are discussed.

The development of a vascular supply is essential not only for organ development and differentiation during embryogenesis but also for wound healing and reproductive functions in the adult Folkman, 1995). Angiogenesis is also implicated in the pathogenesis of a variety of disorders: proliferative retinopathies, age-related macular degeneration, tumors, rheumatoid arthritis, and psoriasis (Folkman, 1995; Garner, 1994). Several potential regulators of angiogenesis have been identified, including fibroblast growth factor-a (aFGF), bFGF, transforming growth factor-alpha (TGF-alpha), TGF-beta, hepatocyte growth factor/scatter factor (HGF/SF), tumor necrosis factor-alpha (TNF-alpha), angiogenin, and interleukin-8 (IL-8) (Folkman and Shing, 1992; Risau, 1997). More recently, the angiopoietins, the ligands of the Tie-2 receptor (Suri et al., 1996; Maisonpierre et al., 1997), have been identified. Vascular endothelial growth factor (VEGF) is an endothelial-cell-specific mitogen. The finding that VEGF was potent and specific for vascular endothelial cells and, unlike bFGF, freely diffusible, led to the hypothesis that this molecule plays a unique role in the regulation of physiological and pathological angiogenesis (Ferrara and Henzel, 1989: Leung et al., 1989). Over the last few years, several additional members of the VEGF gene family have been identified, including placenta growth factor (PIGF) (Maglione et al., 1991,1993), VEGF-B (Olofsson et al., 1996), VEGF-C (Joukov et al., 1996; Lee et al., 1996), and VEGF-D (Orlandini et al., 1996. Achen et al., 1998). There is compelling evidence that VEGF plays an essential role in the development and differentiation of the cardiovascular system (Ferrara and Davis-Smyth, 1997).

During development of the mammalian pituitary gland, specific hormone-producing cell types, critical in maintaining homeostasis, emerge in a spatially and temporally specific fashion from an ectodermal primordium. We have investigated the molecular basis of generating diverse cell phenotypes from a common precursor, providing in vivo and in vitro evidence that development of these cell types involves at least four sequential phases of signaling events and the action of a gradient at an ectodermal boundary. In the first phase, we hypothesize that this notochord induces invagination of Rathke's pouch from the oral ectoderm. This is followed by appearance of an ectodermal boundary, formed with exclusion of Shh from the nascent pouch. Next, signals from the ventral diencephalon--expressing BMP4, Wnt5a, FGF10, and FGF8--in concert with Shh represent critical in vivo signals for pituitary determination. Subsequently, a dorsal-ventral BMP2 signal gradient emanates from a ventral pituitary organizing center, forming at the boundary to oral ectoderm region from which Shh expression is selectively excluded. In concert with a dorsal FGF8 signal, this creates opposing gradients that generate overlapping patterns of specific transcription factors that underlie cell lineage specification events. The mechanisms by which these transient gradients of signaling molecules lead to the appearance of four ventral pituitary cell types appear to involve the reciprocal interactions of two transcription factors, Pit-1 and GATA-2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as a molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA-binding-independent function of Pit-1, suppressing the ventral GATA-2-dependent gonadotrope program by inhibiting GATA-2 binding to gonadotrope- but not thyrotrope-specific genes. This indicates that both DNA-binding-dependent and-independent actions of abundant determining factors contribute to generate distinct cell phenotypes. In the fourth phase, temporally specific loss of the BMP2 signal is required to allow terminal differentiation. The consequence of these sequential organ and cellular determination events is that each of the pituitary cell types--gonadotropes, thyrotropes, somatotropes, lactotropes, corticotropes, and melanotropes appears to be determined, in a ventral to dorsal gradient, respectively, apparently based on a combinatorial code of transcription factors induced by the gradient of specific signaling molecules.