Recent progress in hormone research最新文献

When the pituitary or hypothalamus becomes resistant to steroid negative feedback, a vicious cycle ensues, resulting in chronic hypersecretion of luteinizing hormone (LH) from the pituitary and steroids from the ovaries. In women, LH hypersecretion is implicated in infertility, miscarriages, and development of granulosa cell tumors. Progress in defining the underlying mechanisms of LH toxicity, however, has been limited by the lack of well-defined animal models. To that end, we have developed a new transgenic mouse model (alpha-LHbetaCTP) wherein LH hypersecretion occurs chronically and results in several dire pathological outcomes. Chronic hypersecretion of LH was achieved by introducing a transgene containing a bovine alpha subunit promoter fused to the coding region of a chimeric LHbeta subunit. The alpha subunit promoter directs transgene expression only to gonadotropes. The LHbeta chimera contains the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit linked to the carboxyl terminus of bovine LHbeta. This carboxyl extension extends the half-life of LH heterodimers that contain the chimeric beta subunit. In intact alpha-LHbetaCTP females, serum LH is elevated five- to ten-fold in comparison to nontransgenic littermates. Levels of testosterone (T) and estradiol (E2) also are elevated, with an overall increase in the T-to-E2 ratio. These transgenic females enter puberty precociously but are anovulatory and display a prolonged luteal phase. Anovulation reflects the absence of gonadotropin-releasing hormone (GnRH) and the inability to produce a pre-ovulatory surge of LH. The ovaries are enlarged, with reduced numbers of primordial follicles and numerous, giant, hemorrhagic follicles. Despite the pathological appearance of the ovary, females can be superovulated and mated. Although pregnancy occurs, implantation is compromised due to defects in uterine receptivity. In addition, pregnancy fails at midgestation, reflecting a maternal defect presumably due to estrogen toxicity. When the transgene is in a CF-1 background, all females develop granulosa cell tumors and pituitary hyperplasia by five months of age. They die shortly thereafter due to bladder atony and subsequent kidney failure. When the transgene is placed in other strains of mice, their ovaries develop a luteoma rather than a granulosa cell tumor and the pituitary develops pituitary hyperplasia followed by adenoma. In summary, alpha-LHbetaCTP mice provide a direct association between abnormal secretion of LH and development of a number of ovarian and pituitary pathological responses.

Apolipoprotein (apo) B, the protein component of low-density lipoproteins (LDLs), has been under intense investigation for the last three decades. During the first decade after its initial description, most reports dealt with the physical-chemical characterization of apoB in its natural environment (i.e., intact LDL particles). A few studies dealing with attempts to elucidate the primary structure of apoB were published at this time (Deutsch et al., 1978; Bradley et al., 1980). However, most of these, in retrospect, represented heroic efforts that were doomed to failure because of the huge size and insoluble nature of apoB, once it is separated from its lipid environment. Indeed, during the 1970s, there was no universal agreement on the true molecular weight of the protein, which was not established until sometime into the second decade of apoB research (Yang et al., 1986b). The next 10 years were punctuated by breakthroughs on three different fronts in our understanding of apoB. The first exciting discovery was that apoB exists in two forms, apoB-100 and apoB-48 (Kane et al., 1980; Elovson et al., 1981). The next breakthrough was the elucidation of the primary structure of apoB-100 by a combination of cDNA cloning (Chen et al., 1986; Knott et al., 1986; Yang et al., 1986a) and direct peptide sequencing (Yang et al., 1986a, 1989). This decade of renaissance in apoB research was concluded by the elucidation of the structure of apoB-48. More important in terms of basic cellular molecular biology was the discovery of RNA editing, when apoB-48 was found to be the translation product of an edited apoB mRNA (Chen et al., 1987; Powell et al., 1987). RNA editing had just been described for a kinetoplastid protozoa the year before (Benne et al., 1986). ApoB mRNA editing was the first instance of RNA editing described in a higher eukaryote (Chan and Seeburg, 1995; Grosjean and Benne. 1998). The last decade, which brings us to the present, has been marked by studies that benefited from the breakthroughs of the 1980s. which enabled many different laboratories to examine various aspects of apoB structure, function, and expression. The function of apoB in vivo was analyzed in different animal models (e.g., transgenic animals that overexpress apoB) (Linton et al., 1993; Callow and Rubin, 1995; Veniant et al., 1997) and in knockout animals that have no functional apoB (Farese et al., 1995,1996; Huang et al., 1995,1996). Furthermore, the structure-function relationship of apoB has been investigated in mice that express site-specific apoB mutants (Callow and Rubin, 1995; Veniant et al., 1997: Borén et al., 1998). A breakthrough in a related area led to the identification and cloning of microsomal triglyceride transfer protein (MTP) (Wetterau and Zilversmitt, 1984: Wetterau et al., 1992; Sharp et al., 1993) and the demonstration that MTP is essential for apoB production (Gordon et al., 1994; Leiper et al., 1994). The absence of MTP was found to lead to the co

Somatotrope function requires consideration of both growth hormone (GH) secretion and cellular proliferation. The regulation of these processes is, to a large extent, controlled by three hypothalamic hormones: GH-releasing hormone (GHRH), somatostatin (SRIF), and an as-yet-unidentified GH secretagogue (GHS). Each binds to G protein-linked membrane receptors through which signaling occurs. Our laboratory has used a series of genetic and transgenic models with perturbations of individual components of the GH regulatory system to study both somatotrope signaling and proliferation. Impaired GHRH signaling is present in the lit mouse, which has a GHRH receptor (R) mutation, and the dw rat, which has a post-receptor signaling defect. Both models also have impaired responses to a GHS, implying an interaction between the two signaling systems. The spontaneous dwarf rat (SDR), in which a mutation of the GH gene results in total absence of the hormone, shows characteristic changes in the hypothalamic regulatory hormones due to an absence of GH feedback and alterations in the expression of each of their pituitary receptors. Treatment of SDRs with GHRH and a GHS has allowed demonstration of a stimulatory effect of GHRH on GHRH-R, GHS-R, and SRIF type 2 receptor (SSTR-2) expression and an inhibitory effect on SSTR-5 expression. GH also modifies the expression of these receptors, though its effects are seen at later time periods and appear to be indirect. Overall, the results indicate a complex regulation of GH secretion in which somatotrope receptor, as well as ligand expression, exerts an important physiological role. Both the SDR and the GH-R knockout (ko) mouse have small pituitaries and decreased somatotropes, despite elevated GHRH secretion and intact GHRH-R signaling. Introduction of the hGHRH transgene into GH-R ko mice confirmed that the proliferative effects of GHRH require GH/insulin-like growth factor-I (IGF-I) action. The results offer new insights into factors participating in somatotrope proliferation.

Hormones from the hypothalamus mediate interactions between the nervous and endocrine systems by controlling the activity of specific target cells in the anterior pituitary gland. The hypothalamic peptide, growth hormone-releasing hormone (GHRH), acts on pituitary somatotroph cells to stimulate their proliferation during development and to regulate their ability to produce and secrete growth hormone (GH). These actions are mediated by a recently identified receptor for GHRH that belongs to family B-III of the G protein-coupled receptor superfamily. The rat GHRH receptor is expressed predominantly in the pituitary gland and in somatotroph cells. To investigate this tissue- and cell-specific expression, the receptor gene has been cloned and characterized. The receptor gene promoter is selectively expressed in pituitary cells and is regulated by the pituitary-specific transcription factor Pit-1. There is a sexual dimorphism in GHRH receptor expression in the rat pituitary, suggesting regulation by gonadal steroids. In addition, glucocorticoids are potent positive regulators of GHRH receptor gene expression. Substantial evidence points to an important role for GHRH in regulating the proliferation and functional activity of the somatotroph cell. This is best observed in the dwarf little mouse, which harbors a mutation in the extracellular domain of the GHRH receptor that abolishes the receptor's hormone-binding and signaling properties, resulting in severe somatotroph hypoplasia. Complementary studies in transgenic mice overexpressing the ligand GHRH reveal corresponding somatotroph hyperplasia. Consistent with these observations, GHRH potently activates the MAP kinase pathway in pituitary somatotroph cells. To better understand the hormone-binding and signaling properties of the GHRH receptor, mutant and chimeric receptors have been analyzed to define domains important for GHRH interaction. The GHRH receptor signals predominantly through cAMP-dependent pathways; however, a variant form of the GHRH receptor with an insertion into the third intracellular domain, generated through alternative RNA processing, binds GHRH but fails to signal, suggesting potential modulation of receptor function at a post-transcriptional level. This chapter will integrate these basic investigations of GHRH and its receptor with current information on the involvement of the GHRH signaling system in human diseases of GH secretion and growth.

It is becoming increasingly clear that astroglial cells are active participants in the process by which information is generated and disseminated within the central nervous system (CNS). In the hypothalamus, astrocytes regulate the secretory activity of neuroendocrine neurons. They contribute to facilitating sexual development by stimulating the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls sexual development, from LHRH neurons. Astrocytes secrete several growth factors able to stimulate LHRH secretion. Two members of the epidermal growth factor (EGF) family--transforming growth factor alpha (TGFalpha) and the neuregulins (NRGs)-are produced in hypothalamic astrocytes and elicit LHRH secretion indirectly, via activation of receptor complexes formed by three members of the EGF receptor family, also located on astrocytes. Activation of these receptors results in the production of at least one neuroactive substance, prostaglandin E2 (PGE2), which stimulates LHRH secretion upon binding to specific receptors on LHRH neurons. Overexpression of TGFalpha in the hypothalamus accelerates puberty, whereas blockade of either TGFalpha or NRG actions delays the process, indicating that both peptides are physiological components of the neuroendocrine mechanism that controls sexual maturation. An increase in hypothalamic expression of at least two of the erbB receptors is initiated before the pubertal augmentation of gonadal steroid secretion and is completed on the day of the first preovulatory surge of gonadotropins. This secondary increase is brought about by gonadal steroids. Estrogen and progesterone facilitate erbB-mediated glia-to-LHRH neuron communication by enhancing astrocytic gene expression of at least one of the EGF-related ligands (TGFalpha) and two of the receptors (erbB-2 and erbB-4). They also facilitate the LHRH response to PGE2 via induction of PGE2 receptors in LHRH neurons. A search for genes that may act as upstream regulators of the pubertal process resulted in the identification of two potential candidates: Oct-2, a POU domain gene originally described in cells of the immune system, and TTF-1, a member of the Nkx family of homeodomain transcriptional regulators required for diencephalic morphogenesis. The hypothalamic expression of both genes increases during juvenile development before the first hormonal manifestations of puberty take place. Their mRNA transcripts are localized to specific hypothalamic cellular subsets, where they appear to regulate different, but interactive, components of the neuronal-glial complex controlling LHRH secretion. While Oct-2 transactivates the TGFalpha promoter, TTF-1 does so to the erbB-2 and LHRH genes but inhibits preproenkephalin promoter activity, suggesting that both transcriptional regulators may act coordinately in the normal hypothalamus to activate genes involved in facilitating the advent of puberty and repress those restraining sexual development.

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its co-regulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-regulated transcription factor that mediates the effects of estrogens and anti-estrogens (e.g., tamoxifen) in breast cancer and other estrogen target cells. This chapter presents our recent work on several aspects of estrogen action and the function of the ER: 1) elucidation of ER structure-function relationships and development of ligands that are selective for one of the two ER subtypes, ERalpha or ERbeta; 2) identification of ER-selective co-regulators that potentiate the inhibitory effectiveness of anti-estrogens and dominant-negative ERs and modulate the activity of estrogens; 3) characterization of genes that are regulated by the anti-estrogen-ER versus the estrogen-ER complex; and 4) elucidation of the intriguing pharmacology of these ER complexes at different gene regulatory sites. These findings indicate that different residues of the ER hormone-binding domain are involved in the recognition of structurally distinct estrogens and anti-estrogens and highlight the exquisite precision of the regulation of ER activities by ligands, with small changes in ligand structure resulting in major changes in receptor character. Studies also explore the biology and distinct pharmacology mediated by ERalpha and ERbeta complexed with different ligands through different target genes. The upregulation of the anti-oxidant detoxifying phase II enzyme, quinone reductase, by the anti-estrogen-occupied ER, mediated via the electrophile response element in the QR gene, may contribute to the beneficial antioxidant effects of anti-estrogens in breast cancer and illustrates the activation of some genes by ER via non-estrogen response element sequences. The intriguing biology of estrogen in its diverse target cells is thus determined by the structure of the ligand, the ER subtype involved, the nature of the hormone-responsive gene promoter, and the character and balance of co-activators and co-repressors that modulate the cellular response to the ER-ligand complex. The continuing development of novel ligands and the study of how they function as selective agonists or antagonists through ERalpha or ERbeta should allow optimized tissue selectivity of these agents for hormone replacement therapy and treatment and prevention of breast cancer.

Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. The closest homolog of Apo2L is CD95 (Fas/Apo1) ligand, to which it has 24% amino acid sequence identity. Similar to CD95L, Apo2L activates rapid apoptosis in many types of cancer cells; however, whereas CD95L mRNA expression is restricted mainly to activated T cells, natural killer cells, and immune-privileged sites, Apo2L mRNA occurs in a wide variety of tissues. Most normal cells appear to be resistant to Apo2L's cytotoxic action, suggesting the existence of mechanisms that can protect against apoptosis induction by Apo2L. The first receptor described for Apo2L, called death receptor 4 (DR4), contains a cytoplasmic "death domain"; DR4 transmits the apoptosis signal carried by Apo2L. We have identified three additional receptors that bind to Apo2L. One receptor, called DR5, contains a cytoplasmic death domain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs are expressed in many normal tissues and tumor cell lines. The second receptor, designated decoy receptor 1 (DcR1), is a phospholipid-anchored cell-surface protein that lacks a cytoplasmic tail. The third receptor, called DcR2, is structurally similar to DR4 and DR5 but has a truncated cytoplasmic death domain and does not transmit a death signal. The mRNAs for DcR1 and DcR2 are expressed in multiple normal tissues but in few tumor cell lines. Transfection experiments indicate that DcR1 and DcR2 act as decoys that prevent Apo2L from inducing apoptosis through DR4 and DR5. These decoy receptors thus represent a novel mechanism for regulating sensitivity to a pro-apoptotic cytokine directly at the cell's surface. The preferential expression of these inhibitory receptors in normal tissues suggests that Apo2L may be useful as an anticancer agent that induces apoptosis in cancer cells while sparing normal cells.

MEN1 is a syndrome of parathyroid adenomas, gastrinomas, prolactinomas, and other endocrine tumors. Collagenomas and facial angiofibromas are newly recognized but common skin expressions. Many tumors in MEN1 are benign; however, many entero-pancreatic neuroendocrine tumors and foregut carcinoid tumors are malignant. MEN1 is thus the expression of a cancer gene but without available prevention or cure for malignancy. Hereditary (as compared to sporadic) endocrine tumors show early onset age and multiplicity, because each cell of the body has "one hit" by inheritance. Multiple neoplasia syndromes with endocrine tumor(s) all include nonendocrine components; their known defective genes seem mainly to disturb cell accumulation. Hereditary neoplasia/hyperplasia of one endocrine tissue reflects a defect that is tissue selective and directed at cell secretion. Though the hereditary endocrine neoplasias are rare, most of their identified genes also contribute to common sporadic endocrine neoplasms. Hereditary tumors may be caused by activation of an oncogene (e.g., RET) or, more often, by inactivation of a tumor suppressor gene (e.g., P53, MEN1). Recently, MEN1 was identified by positional cloning. This strategy included narrowing the gene candidate interval, identifying many or all genes in that interval, and testing the newly identified candidate genes for mutation in MEN1 cases. MEN1 was identified because it showed mutation in 14 of 15 MEN1 cases. NIH testing showed germline MEN1 mutations in 47 of 50 MEN1 index cases and in seven of eight cases with sporadic MEN1. Despite proven capacity to find germline MEN1 mutation, NIH testing found no MEN1 mutation among five families with isolated hyperparathyroidism, suggesting that this often arises from mutation of other gene(s). Analogous studies in Japan found that familial isolated pituitary tumors also did not show MEN1 germline mutation. MEN1 mutation testing can now be considered for cases of MEN1 and its phenocopies and for asymptomatic members of families with known MEN1 mutation. Germline MEN1 testing does not have the urgency of RET testing in MEN2a and 2b, as MEN1 testing does not commonly lead to an important intervention. Somatic MEN1 mutation was found in sporadic tumors: parathyroid adenoma (21%), gastrinoma (33%), insulinoma (17%), and bronchial carcinoid (36%). For each of these, MEN1 was the known gene most frequently mutated. MEN1 has a widely expressed mRNA that encodes a protein (menin) of 610 amino acids. The protein sequence is not informative about domains or functions. The protein was mainly nuclear. Menin binds to JunD, an AP-1 transcription factor, inhibiting JunD's activation of transcription. Most of the germline and somatic MEN1 mutations predict truncation of menin, a likely destructive change. Inactivating MEN1 mutations in germline and in sporadic neoplasms support prior predictions that MEN1 is a tumor suppressor gene. Germline MEN1 mutation underlies all or most cases

Several genes have been recognized in Drosophila that regulate circadian rhythms. Homologues of these genes have now been found in mice and humans, suggesting a mechanism that is conserved throughout the animal kingdom. For some of these genes and their products, molecular oscillations are produced in certain cells of the Drosophila and mammalian brain. Two genes, period and timeless, are transcribed with a circadian rhythm that is regulated by activities derived from their encoded proteins, PER and TIM. Nuclear localization of these proteins downregulates per and tim transcription by suppressing the activities of two transcription factors, dCLOCK and dBMAL1. Cycles in this feedback regulation are promoted by events that regulate the accumulation, physical interaction, and nuclear translocation of PER and TIM proteins. PER and TIM must physically associate to enter the nucleus and their cytoplasmic interaction is delayed by a kinase encoded by the clock gene, double-time. This kinase directs PER phosphorylation, which leads to PER degradation. Effects of the kinase are blocked once PER is complexed to TIM. These interactions prolong the interval of per and tim transcription by ensuring that PER/TIM complexes from only after TIM has accumulated for several hours.

Phytoestrogens are compounds found in a wide variety of plant foods that historically are said to exhibit estrogen-like activity and, more recently, have been reported to display both estrogenic and anti-estrogenic effects. Population-based studies have been interpreted to suggest that consumption of a phytoestrogen-rich diet is protective against breast, prostate, and bowel cancer and cardiovascular disease and ameliorates estrogen-deficiency symptoms in postmenopausal women. Consequently, there is a global movement towards increased consumption of phytoestrogen-rich foods and tabletized concentrated isoflavone extracts are being heavily promoted. Evaluating the effects and hence the potential benefits and risks of phytoestrogens is a complex task. The interindividual diversity and complexity in dietary phytoestrogen absorption and metabolism make the bioactivity of these compounds unpredictable. Epidemiological studies of relationships between phytoestrogens and cancer and cardiovascular disease that take into account confounding factors are scarce. Results of many of the in vitro and in vivo studies are conflicting and confusing. These compounds do not simply mimic the effects of human steroidal estrogen but rather demonstrate both similar and divergent actions. The ultimate actions of these compounds in specific cells are determined by many factors, including the relative levels of estrogen receptor (ER) alpha and ER beta and the diverse cocktail of co-activators and co-repressors present in any given cell type. Therefore, effects vary according to the phytoestrogen studied, cell line, tissue, species, and response being evaluated. Overall, it is naive to assume that exposure to these compounds is always good; inappropriate or excessive exposure may be detrimental. Extensive documentation of the specific intracellular effects of the various phytoestrogens in different tissues, the relationships between timing and duration of exposure and disease, and results from prospective randomized studies in humans of their clinical effects and potential side effects are essential. Only then can widespread recommendations regarding the dietary and pharmacological intake of these compounds be made.