Recent Progress In Hormone Research[JOURNAL]
Sun
200 papers
Microarray analysis and identification of novel molecules involved in insulin-like growth factor-1 receptor signaling and gene expression.
Dupont J, Dunn SE, Barrett JC … +1 more , LeRoith D
Recent Prog Horm Res · 2003 · PMID 12795426 · Publisher ↗
The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-1R) are members of the same subfamily of receptor tyrosine kinases. The two receptors phosphorylate many of the same substrates and activate th...
The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-1R) are members of the same subfamily of receptor tyrosine kinases. The two receptors phosphorylate many of the same substrates and activate the same signaling modules, including the mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3' kinase (PI3K) signaling pathways. Although the IR and IGF-1R share some redundant functions in metabolism, cell growth, differentiation, and apoptosis, they also exhibit distinct physiological roles. Some of these may be due to differences in tissue distribution, receptor structure, formation of hybrid receptors, or mechanisms of ligand binding. However, the divergent effects of insulin and IGF-1 also may be explained by specificity in the intracellular signals generated by insulin and IGF-1. In particular, the IR and IGF-1R are capable of triggering their own biological responses by using specific or preferential substrates, molecular adapters, or signaling pathways. In a recent study, we used cDNA microarray analysis to identify genes differentially regulated by insulin and IGF-1. Mouse NIH-3T3 fibroblasts expressing either the wild-type human IGF-1R or IR were stimulated with either IGF-1 or insulin, respectively. We identified 39 genes differentially regulated by insulin and IGF-1. Most of these genes had not been reported previously to be responsive to insulin or IGF-1. The genes induced by IGF-1 generally were involved in mitogenesis or differentiation, while the genes found to be induced by insulin did not conform to any particular category. In a separate study, immortalized breast epithelial cells were stimulated with IGF-1 and a cDNA microarray analysis was used to generate a profile of IGF-1-regulated genes. A number of genes known to be involved in angiogenesis were found to be regulated by IGF-1. These results strongly suggest that this technology may be extremely useful in identifying groups of genes that are specifically regulated by different ligands and their activated receptors.
Investigation of the transcriptional changes underlying functional defects in the mammary glands of prolactin receptor knockout mice.
Ormandy CJ, Naylor M, Harris J … +5 more , Robertson F, Horseman ND, Lindeman GJ, Visvader J, Kelly PA
Recent Prog Horm Res · 2003 · PMID 12795425 · Publisher ↗
Knockout (KO) mice have been created that carry null mutations of genes encoding molecules essential for prolactin (PRL) release, PRL, the receptor for prolactin (PRLR), and various members of the receptor's signaling pa...
Knockout (KO) mice have been created that carry null mutations of genes encoding molecules essential for prolactin (PRL) release, PRL, the receptor for prolactin (PRLR), and various members of the receptor's signaling pathway. This allowed an in vivo genetic analysis of the role of PRL in target organ function. In PRLKO and PRLRKO mice, mammary ductal side branching was absent, terminal end bud (TEB)-like structures persisted at the ductal termini well into maturity, and no alveolar buds formed along the ductal tree. Transplants of recombined mammary glands formed from stromal and epithelial elements with and without PRLR showed normal development, while supplementation of progesterone levels in PRLKO animals restored ductal side branching. During pregnancy, PRLR heterozygous animals initially showed normal ductal and alveolar development. However, alveolar development stalled during late pregnancy, preventing successful lactation. This defect could be rescued by the loss of a single allele of the suppressor of cytokine signaling (SOCS) 1 gene. Transplants of recombined glands containing PRLRKO epithelium and wild-type (WT) stroma formed alveolar buds during pregnancy but showed no lobuloalveolar development. Recombinations of WT epithelium and PRLRKO stroma showed normal development, demonstrating that a direct action of the lactogenic hormones is confined to the epithelium, to promote lobuloalveolar development. Transcript profiling of epithelial transplants expressing or not expressing PRLR was used during early pregnancy to investigate the transcriptional response to lactogens underlying this defect. Such profiling has identified a number of genes with well-characterized roles in mammary development, in addition to a number of novel transcripts.
Regulation of hematopoietic stem cell self-renewal.
Reya T
Recent Prog Horm Res · 2003 · PMID 12795424 · Publisher ↗
Every day, billions of new blood cells are produced in the body, each one derived from a hematopoietic stem cell (HSC). Because most mature blood stem cells have a limited life span, the ability of HSCs to perpetuate the...
Every day, billions of new blood cells are produced in the body, each one derived from a hematopoietic stem cell (HSC). Because most mature blood stem cells have a limited life span, the ability of HSCs to perpetuate themselves through self-renewal and generate new blood cells for the lifetime of an organism is critical to sustaining life. A key problem in hematopoietic stem cell biology is how HSC self-renewal is regulated. Recent evidence suggests that signaling pathways classically involved in embryonic development--such as the Wnt signaling pathway--play an important role in regulating stem cell self-renewal. The Wnt signaling pathway has been shown to regulate stem cell fate choice in a variety of organs, including the skin, the nervous system, and the hematopoietic system. In the hematopoietic system, stimulation of hematopoietic progenitors and stem cells with soluble Wnt proteins or downstream activators of the Wnt signaling pathway leads to their expansion. Future studies focusing on the mechanism of action of the Wnt signaling pathway and its interaction with other pathways are needed to gain further insight into the regulation of stem cell self-renewal, not only in the hematopoietic system but also in a variety of other tissues.
Dynamic changes in gene expression during human trophoblast differentiation.
Handwerger S, Aronow B
Recent Prog Horm Res · 2003 · PMID 12795423 · Publisher ↗
The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental developme...
The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental development in an in vitro model system in which highly purified cytotrophoblast cells aggregate spontaneously and fuse to form a multinucleated syncytium that expresses placental lactogen, human chorionic gonadotropin, and other proteins normally expressed by fully differentiated syncytiotrophoblast cells. Of the 6918 genes present on the Incyte Human GEM V microarray that we analyzed over a 9-day period, 141 were induced and 256 were downregulated by more than 2-fold. The dynamically regulated genes fell into nine distinct kinetic patterns of induction or repression, as detected by the K-means algorithm. Classifying the genes according to functional characteristics, the regulated genes could be divided into six overall categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tags and function unknown. Gene expression changes within key functional categories were tightly coupled to the morphological changes that occurred during trophoblast differentiation. Within several key gene categories (e.g., cell and tissue structure), many genes were strongly activated, while others with related function were strongly repressed. These findings suggest that trophoblast differentiation is augmented by "categorical reprogramming" in which the ability of induced genes to function is enhanced by diminished synthesis of other genes within the same category. We also observed categorical reprogramming in human decidual fibroblasts decidualized in vitro in response to progesterone, estradiol, and cyclic AMP. While there was little overlap between genes that are dynamically regulated during trophoblast differentiation versus decidualization, many of the categories in which genes were strongly activated also contained genes whose expression was strongly diminished. Taken together, these findings point to a fundamental role for simultaneous induction and repression of mRNAs that encode functionally related proteins during the differentiation process.
Paired-like repression/activation in pituitary development.
Olson LE, Dasen JS, Ju BG … +2 more , Tollkuhn J, Rosenfeld MG
Recent Prog Horm Res · 2003 · PMID 12795422 · Publisher ↗
Pituitary gland development is controlled by signals that guide expression of specific combinations of transcription factors that dictate serial determination and differentiation events. One class of factors is paired-li...
Pituitary gland development is controlled by signals that guide expression of specific combinations of transcription factors that dictate serial determination and differentiation events. One class of factors is paired-like homeodomain factors. Two that have been investigated are the repressor Hex1/Rpx and activator prophet of Pit-1 (Prop-1), which exert selective roles during pituitary development. The opposing actions of these factors provide one aspect of pituitary organogenesis.
Analysis of gene expression in the normal and malignant cerebellum.
Wechsler-Reya RJ
Recent Prog Horm Res · 2003 · PMID 12795421 · Publisher ↗
The developing nervous system consists of a small number of multipotent precursors that undergo extensive proliferation to generate the neurons and glia that make up the adult brain. Elucidating the mechanisms that contr...
The developing nervous system consists of a small number of multipotent precursors that undergo extensive proliferation to generate the neurons and glia that make up the adult brain. Elucidating the mechanisms that control the growth and differentiation of these cells is important not only for understanding normal neural development but also for understanding the etiology of central nervous system tumors. A particularly striking example of this is in the cerebellum. Recent studies have suggested that the Sonic hedgehog-Patched signaling pathway plays a critical role in regulating the proliferation of cerebellar granule cell precursors and is also a major target of mutation in the cerebellar tumor medulloblastoma. In light of these observations, identification of additional genes that control cerebellar growth and differentiation is likely to provide important insight into the basis of cerebellar tumors. Similarly, analysis of gene expression in medulloblastoma will no doubt shed light on previously unknown signaling pathways that regulate normal cerebellar development. The advent of high-throughput gene expression analysis techniques--such as adapter-tagged competitive polymerase chain reaction (ATAC-PCR), serial analysis of gene expression (SAGE), and DNA microarrays--makes identification of such genes faster and easier than ever before. This review summarizes recent studies of gene expression in the cerebellum and discusses the value of such approaches for understanding development and tumorigenesis in this tissue.
Overlapping but distinct profiles of gene expression elicited by glucocorticoids and progestins.
Wan Y, Nordeen SK
Recent Prog Horm Res · 2003 · PMID 12795420 · Publisher ↗
Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biol...
Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biological functions. For example, progestins are associated with the incidence and progression of breast cancer, whereas glucocorticoids are growth suppressive in mammary cancer cells. To understand the mechanisms that engender biological specificity, we have employed two systematic approaches to identify genes that are differentially regulated by the two hormones. The first strategy is to utilize Affymetrix oligonucleotide arrays to compare glucocorticoid- and progestin-regulated gene expression in a human breast cancer cell line. This global analysis reveals that the two hormones regulate overlapping but distinct sets of genes, including 31 genes that are differentially regulated. Surprisingly, the set of differentially regulated genes was almost as large as the set of genes regulated by both hormones. Examination of the set of differentially regulated genes suggests mechanisms behind the distinct growth effects of the two hormones in breast cancer. The differential regulation of four genes representing different regulatory patterns was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses. Treatment with cycloheximide or mifepristone (RU486) indicates that the regulation is a primary, receptor-mediated event. The second strategy is to employ a retroviral promoter trap and Cre/loxP-mediated, site-specific recombination to identify genes that are differentially regulated by glucocorticoids and progestins. A mouse fibroblast cell line (4F) stably expressing both glucocorticoid receptor (GR) and progesterone receptor (PR) and containing a single copy of a multifunctional selection plasmid was generated. This line was transduced with a self-inactivating retroviral promoter trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Integration of the provirus places Cre expression under the control of genomic flanking sequence. Activation of Cre expression from integration into active genes results in a permanent switch between the selectable marker genes that convert the cells from neomycin resistant to hygromycin resistant. Selection for hygromycin resistance after hormone treatment yields recombinants in which Cre sequences in the U3 region are expressed from hormone-inducible, upstream cellular promoters. Because Cre-mediated recombination is a permanent event, the expression of the selectable marker genes is independent of ongoing Cre expression. Thus, this system permits the identification of genes that are transiently or weakly induced by hormone. Detailed analyses of genes identified in these studies will furnish a mechanistic understanding of differential regulation by glucocorticoids and progestins.
Regulation of a distinctive set of genes in glucocorticoid-evoked apoptosis in CEM human lymphoid cells.
Thompson EB, Johnson BH
Recent Prog Horm Res · 2003 · PMID 12795419 · Publisher ↗
Gene expression was evaluated in clones of the acute lymphoblastic leukemic cell line CEM that were sensitive or resistant to apoptosis evoked by the glucocorticoid, dexamethasone (Dex). Founding clones CEM-C7 (glucocort...
Gene expression was evaluated in clones of the acute lymphoblastic leukemic cell line CEM that were sensitive or resistant to apoptosis evoked by the glucocorticoid, dexamethasone (Dex). Founding clones CEM-C7 (glucocorticoid sensitive) and CEM-C1 (glucocorticoid resistant) were subcloned to maximize uniformity of each population studied. Among subclones of C1, our original pseudodiploid clone of glucocorticoid-resistant cells, we found a high proportion of hyperploid clones. Most C1 subclones were glucocorticoid resistant but two C1 subclones were found to be revertants to glucocorticoid sensitivity. Glucocorticoid receptor content of the C1 subclones varied almost 5-fold but higher quantity of receptors did not guarantee steroid sensitivity. Gene expression analysis was carried out on microchips containing representations for approximately 12,600 human genes. When a group of four subclones from C1 (three glucocorticoid-resistant and one glucocorticoid-sensitive revertant) were compared with the glucocorticoid-sensitive subclone CEM-C7-14 for basal gene expression, the four C1 subclones clustered closely and far from C7-14. Thus, basal gene expression in the C1 subclones differed for a large number of genes from that in the C7 subclone. Reversion to glucocorticoid sensitivity did not cause a major shift in basal gene expression to a more C7-like state. Three clones (one revertant glucocorticoid sensitive from C1 subclone, one C7 sensitive subclone, and one C1 glucocorticoid-resistant subclone) were compared for the genes regulated by treatment for 20 hours with 10(-6)M Dex. This interval brings the cells to a point just before the onset of apoptosis. We tested the hypothesis that a distinctive set of genes would be regulated in the glucocorticoid-sensitive clones. This proved to be so. In three experiments, at our chosen levels of discrimination, 39 genes were consistently induced > or = 2.5-fold and 21 genes were consistently reduced > or = 2-fold in glucocorticoid-sensitive clones but not in the glucocorticoid-resistant clone. The glucocorticoid-resistant clone showed induction or reduction of 88 genes different from those regulated in the glucocorticoid-sensitive clones. These data support our hypothesis and further show that the glucocorticoid-resistant clone is capable of responding to steroid but with a different set of genes. We propose that a general metabolic switch accounts for the alteration.
Genomic analysis of glucocorticoid-regulated promoters in murine T-lymphoma cells.
Chen L, Finnerty C, Gustafson WC … +6 more , Bush CR, Chi P, Guo H, Luxon B, Fields AP, Thompson EA
Recent Prog Horm Res · 2003 · PMID 12795418 · Publisher ↗
We have undertaken a high-throughput analysis to identify targets of glucocorticoid regulation in P1798 murine T-lymphoma cells. G1/S-arrested cultures were treated for 8 hours with 0.1 microM dexamethasone (dex) in the...
We have undertaken a high-throughput analysis to identify targets of glucocorticoid regulation in P1798 murine T-lymphoma cells. G1/S-arrested cultures were treated for 8 hours with 0.1 microM dexamethasone (dex) in the presence and absence of 1 microg/ml cycloheximide. Untreated cultures and cultures exposed to cycloheximide alone were prepared as controls. RNA was isolated and gene expression analyzed using Affymetrix MG-U74A oligonucleotide arrays (Gene Chips). Three independent experiments were performed. The data were analyzed using a variety of statistical and analytical approaches in order to identify primary transcriptional targets of the glucocorticoid receptor. We identified 44 genes that increase by > 2-fold in both dex-treated and dex + cycloheximide-treated cultures (relative to control and cycloheximide-treated cultures) in three replicate experiments. Statistical analysis of control data indicate that the probability that a given probeset would, as a result of random error, increase > 2-fold both in the presence and absence of cycloheximide in two independent experiments is approximately 7 x 10(-9). We have retrieved from the Celera mouse genomic sequence 8 kb of promoter sequence, spanning 4 kb either side of the 5'-end of the cDNA from eight of the induced genes. These sequences were analyzed for potential glucocorticoid receptor binding sites. Five of these genes contain the sequence ACAnnnTGTnCT within 4 kb of the presumptive transcriptional start site. Eight control genes were selected at random and analyzed for the sequence ACAnnnTGTnCT. Two control genes had such sequences within 4 kb of the transcriptional start site.
Role of defective apoptosis in type 1 diabetes and other autoimmune diseases.
Hayashi T, Faustman DL
Recent Prog Horm Res · 2003 · PMID 12795417 · Publisher ↗
Lymphocyte development, selection, and education are strictly controlled to prevent autoimmunity, with potentially autoreactive cells being removed by apoptosis. Dysregulation of apoptosis is a central defect in diverse...
Lymphocyte development, selection, and education are strictly controlled to prevent autoimmunity, with potentially autoreactive cells being removed by apoptosis. Dysregulation of apoptosis is a central defect in diverse murine autoimmune diseases. In murine models of autoimmune lupus, for example, mutations in the death receptor Fas (CD95) or in its ligand, FasL (CD95L), have been identified and shown to render lymphoid cells resistant to apoptosis. In contrast, select lymphoid subpopulations of mice with autoimmune diabetes manifest an increased susceptibility to apoptosis as a result of impaired activation of the transcription factor nuclear factor-kappa B (NF-kappaB), which normally protects cells against tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. The genetic basis of this defect in NF-kappaB activation is a mutation in the promoter-enhancer region of a gene that encodes an essential subunit (LMP2) of the proteasome. Although no specific genetic defects have been identified in most common forms of human autoimmune disease, functional assays consistently demonstrate heightened apoptosis attributable to multiple death signaling pathways.
Identification of a nuclear factor kappa B-dependent gene network.
Tian B, Brasier AR
Recent Prog Horm Res · 2003 · PMID 12795416 · Publisher ↗
Nuclear factor-kappa B (NF-kappaB) is a highly inducible transcription factor that plays an important role in the hepatic acute-phase response, innate/adaptive immunity, and cellular survival through the induction of gen...
Nuclear factor-kappa B (NF-kappaB) is a highly inducible transcription factor that plays an important role in the hepatic acute-phase response, innate/adaptive immunity, and cellular survival through the induction of genetic networks. The major transcriptional-activating species Rel A-NF-kappaB is a cytoplasmic complex whose nuclear translocation is controlled by its association with a family of inhibitory proteins, termed IkappaBs. Activation of NF-kappaB results in the targeted proteolysis of IkappaB, releasing NF-kappaB to enter the nucleus and bind to specific sequences in target promoters. Because the genomic actions of NF-kappaB are influenced by the stimulus applied and the promoter context/chromatin structure in which it binds, the spectrum of NF-kappaB-regulated genes has not been elucidated. We have begun to address this question, exploiting a tightly regulated cellular system expressing a nondegradable IkappaBalpha mutant that completely inhibits NF-kappaB action. High-density oligonucleotide microarrays were used to identify genetic responses in response to complex biological stimuli (viral replication) in the presence and absence of NF-kappaB. Using statistical and informatics tools, we identified two groups of NF-kappaB-dependent genes with distinct expression profiles: 1) a group with high constitutive expression whose expression levels fall in response to viral exposure and constitutive mRNA expression increases from NF-kappaB blockade, and 2) a group where constitutive expression was very low (or undetectable) and, after stimulation, expression levels strongly increased. In this group, NF-kappaB blockade inhibited the viral induction of genes. This latter cluster includes chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins (e.g., complement components, growth factor regulators). These data reveal complexity in the genetic response to NF-kappaB and serve as a foundation for further informatics analysis to identify genetic features common to up- and downregulated NF-kappaB-dependent promoters.
Statistical approach to DNA chip analysis.
Svrakic NM, Nesic O, Dasu MR … +2 more , Herndon D, Perez-Polo JR
Recent Prog Horm Res · 2003 · PMID 12795415 · Publisher ↗
Statistical methods for analyzing data from DNA microarray experiments are reviewed. Specifically, we discuss common experimental setups, methods for data reduction and clustering, and analysis of time-course experiments...
Statistical methods for analyzing data from DNA microarray experiments are reviewed. Specifically, we discuss common experimental setups, methods for data reduction and clustering, and analysis of time-course experiments. While early microarray studies focused mainly on the basic methodological and technical aspects of DNA arrays, emphasis has shifted to biological, medical, and clinical applications. We mention several of these and present results from our recent research as illustrative examples. New developments in this ever-growing field are outlined.
Gene expression profiling for prediction of clinical characteristics of breast cancer.
Huang E, West M, Nevins JR
Recent Prog Horm Res · 2003 · PMID 12795414 · Publisher ↗
We have applied techniques of gene expression analysis to the analysis of human breast cancer by identifying metagene models with the capacity to discriminate breast tumors based on estrogen receptor (ER) status as well...
We have applied techniques of gene expression analysis to the analysis of human breast cancer by identifying metagene models with the capacity to discriminate breast tumors based on estrogen receptor (ER) status as well as the propensity for lymph node metastasis. We assess the utility and validity of these models in predicting status of tumors in cross-validation determinations. The practical value of such approaches relies on the ability not only to assess relative probabilities of clinical outcomes for future samples but also to provide an honest assessment of the uncertainties associated with such predictive classifications, based on the selection of gene subsets for each validation analysis. This latter point is of critical importance to the ability of applying these methodologies to clinical assessment of tumor phenotype. It is also clear from ER predictions that these analyses identify genes known to be involved in ER function but also identify new candidate genes involved in ER function. We believe these gene expression phenotypes have the potential to characterize the complex genetic alterations that typify the neoplastic state in a way that truly reflects the complexity of the regulatory pathways that are affected.
The use of DNA microarrays to assess clinical samples: the transition from bedside to bench to bedside.
Copland JA, Davies PJ, Shipley GL … +3 more , Wood CG, Luxon BA, Urban RJ
Recent Prog Horm Res · 2003 · PMID 12795413 · Publisher ↗
The advent of gene array technology brings the ability to classify disease states to the molecular level by examining changes in all mRNAs expressed in cells or tissues. Comparing changes in gene expression patterns betw...
The advent of gene array technology brings the ability to classify disease states to the molecular level by examining changes in all mRNAs expressed in cells or tissues. Comparing changes in gene expression patterns between normal and diseased cells and/or tissues has elucidated unique subsets of genes identifiable to a specific disease. Already, new subclassifications of specific cancers have been discovered, belying that genomic profiling can uniquely distinguish a specific disease state and tissue of origin. This technology bestows the ability to examine global changes occurring in a cell or tissue(s), thereby allowing the elucidation of alterations in dysregulated biological, biochemical, and molecular events leading to disease states such as diabetes, hypertension, infertility, obesity, osteoporosis, and atherosclerosis. Furthermore, genomic profiling will lead to new molecular targets for the development of drug therapeutics. Futuristically, one could envision personalized patient therapies based upon identification of specific aberrant signaling pathways that can be targeted for drug therapy.
A functional proteomics approach to signal transduction.
Graves PR, Haystead TA
Recent Prog Horm Res · 2003 · PMID 12795412 · Publisher ↗
The purpose of this review is to highlight how proteomics techniques can be used to answer specific questions related to signal transduction in a wide variety of systems. In our laboratory, we utilize proteomic technolog...
The purpose of this review is to highlight how proteomics techniques can be used to answer specific questions related to signal transduction in a wide variety of systems. In our laboratory, we utilize proteomic technologies to elucidate signal transduction pathways involved in smooth muscle contraction and relaxation, cell growth and tumorigenesis, and the pathogenesis of malaria. We see the real application of this technology as a tool to enhance the power of existing approaches such as classical yeast and mouse genetics, tissue culture, protein expression systems, and site-directed mutagenesis. Our basic approach is to examine only those proteins that differ by some variable from the control sample. In this way, the number of proteins to be processed by electrophoresis, Edman degradation, or mass spectrometry is greatly reduced. In addition, since only those proteins that change in response to a given biological treatment are analyzed, the experimental outcome provides information about specific signaling pathways. Examples of typical experiments in our laboratory are measurement of changes in protein phosphorylation in response to treatment of cells with growth factors or specific drugs, characterization of proteins associated with a bait protein in a "pull-down" experiment, or measurement of changes in protein expression. Frequently, in these experiments, it is necessary to define complex protein mixtures. To achieve this goal, we utilize a variety of techniques to isolate specific types of proteins or "subproteomes" for further analysis. In this review, we discuss strategies used in our laboratory for studying signaling pathways, including subproteome isolation, proteome mining, and analysis of the phosphoproteome.
Regulation of the mitotic and meiotic cell cycles in the male germ line.
Wolgemuth DJ, Laurion E, Lele KM
Recent Prog Horm Res · 2002 · PMID 12017557 · Publisher ↗
Mammalian gametogenesis provides a unique system in which to study cell-cycle regulation. Furthermore, understanding the genetic program controlling the mitotic and meiotic divisions of the germ line will provide insight...
Mammalian gametogenesis provides a unique system in which to study cell-cycle regulation. Furthermore, understanding the genetic program controlling the mitotic and meiotic divisions of the germ line will provide insight into understanding infertility and new directions for contraception. Male and female germ cells have stages of cell-cycle regulation in common, including a mitotic proliferative stage, entry into meiosis, completion of a reductive division, and entry into a quiescent state awaiting signals at fertilization. However, the timing of these events - and, indeed, even the stage of development at which these events occurs - differs in the two sexes. The genes involved in controlling these specialized mitotic and meiotic cycles of mammalian germ cell differentiation are only now being identified. They include a complex array of kinases, phosphatases, regulatory proteins (e.g., cyclins), and an equally complex array of substrates, including components of the nuclear and cytoplasmic structures involved in cell division. This chapter provides an overview of our current understanding of cell-cycle regulation in mammalian mitotic cells and the importance of restriction points. A summary of observations regarding the expression of various cell-cycle regulatory genes in mouse gametes is provided, along with comments on interesting differences between mitotic and meiotic cells. Finally, the role of the novel A-type cyclin, cyclin A1, during male meiosis is discussed in depth.
Prolactin modulation of immune and inflammatory responses.
Yu-Lee LY
Recent Prog Horm Res · 2002 · PMID 12017556 · Publisher ↗
Prolactin (PRL), a pituitary peptide hormone, is known to regulate diverse physiological functions via its effects on cellular processes such as proliferation, differentiation, and cell survival. All these activities are...
Prolactin (PRL), a pituitary peptide hormone, is known to regulate diverse physiological functions via its effects on cellular processes such as proliferation, differentiation, and cell survival. All these activities are mediated by the PRL receptor (PRL-R), a member of the hematopoietin cytokine receptor superfamily. To understand PRL-dependent mitogenic signaling in T cells, we cloned PRL. PRL-R, one mediator of PRL signaling, signal transducer and activator of transcription (Stat) 5b, and a panel of PRL-inducible immediate early-response genes from T cells. We are employing one of these PRL-inducible genes, the transcription factor interferon regulatory factor-1 (IRF-1), a multifunctional immune regulator gene, as a tool to understand how PRL modulates T-cell proliferative responses. In investigating regulatory events along the PRL-R/Janus activating kinase (JAK)/Stat/IRF-1 signaling pathway, we show that Stat factors can activate as well as inhibit IRF-1 promoter activity and that cross talk between Stat and nuclear factor (NF)kappaB signaling pathways also regulates IRF-1 expression. In understanding how signaling pathways cross talk at the IRF-1 promoter, we obtained insights into how PRL can modulate immune and inflammatory responses. These findings have much broader implications, not only for cells in the immune system but also for other PRL-responsive cells and tissues.
Anabolic steroids.
Kuhn CM
Recent Prog Horm Res · 2002 · PMID 12017555 · Publisher ↗
The term "anabolic steroids" refers to testosterone derivatives that are used either clinically or by athletes for their anabolic properties. However, scientists have questioned the anabolic effects of testosterone and i...
The term "anabolic steroids" refers to testosterone derivatives that are used either clinically or by athletes for their anabolic properties. However, scientists have questioned the anabolic effects of testosterone and its derivatives in normal men for decades. Most scientists concluded that anabolic steroids do not increase muscle size or strength in people with normal gonadal function and have discounted positive results as unduly influenced by positive expectations of athletes, inferior experimental design, or poor data analysis. There has been a tremendous disconnect between the conviction of athletes that these drugs are effective and the conviction of scientists that they aren't. In part, this disconnect results from the completely different dose regimens used by scientists to document the correction of deficiency states and by athletes striving to optimize athletic performance. Recently, careful scientific study of suprapharmacologic doses in clinical settings - including aging, human immunodeficiency virus, and other disease states - supports the efficacy of these regimens. However, the mechanism by which these doses act remains unclear. "Anabolism" is defined as any state in which nitrogen is differentially retained in lean body mass, either through stimulation of protein synthesis and/or decreased breakdown of protein anywhere in the body. Testosterone, the main gonadal steroid in males, has marked anabolic effects in addition to its effects on reproduction that are easily observed in developing boys and when hypogonadal men receive testosterone as replacement therapy. However, its efficacy in normal men, as during its use in athletes or in clinical situations in which men are eugonadal, has been debated. A growing literature suggests that use of suprapharmacologic doses can, indeed, be anabolic in certain situations; however, the clear identification of these situations and the mechanism by which anabolic effects occur are unclear. Furthermore, the pharmacology of "anabolism" is in its infancy: no drugs currently available are "purely" anabolic but all possess androgenic properties as well. The present review briefly recapitulates the historic literature about the androgenic/anabolic steroids and describes literature supporting the anabolic activity of these drugs in normal people, focusing on the use of suprapharmacologic doses by athletes and clinicians to achieve anabolic effects in normal humans. We will present the emerging literature that is beginning to explore more specific mechanisms that might mediate the effects of suprapharmacologic regimens. The terms anabolic/androgenic steroids will be used throughout to reflect the combined actions of all drugs that are currently available.
Sex steroids and bone.
Manolagas SC, Kousteni S, Jilka RL
Recent Prog Horm Res · 2002 · PMID 12017554 · Publisher ↗
The adult skeleton is periodically remodeled by temporary anatomic structures that comprise juxtaposed osteoclast and osteoblast teams and replace old bone with new. Estrogens and androgens slow the rate of bone remodeli...
The adult skeleton is periodically remodeled by temporary anatomic structures that comprise juxtaposed osteoclast and osteoblast teams and replace old bone with new. Estrogens and androgens slow the rate of bone remodeling and protect against bone loss. Conversely, loss of estrogen leads to increased rate of remodeling and tilts the balance between bone resorption and formation in favor of the former. Studies from our group during the last 10 years have elucidated that estrogens and androgens decrease the number of remodeling cycles by attenuating the birth rate of osteoclasts and osteoblasts from their respective progenitors. These effects result, in part, from the transcriptional regulation of genes responsible for osteoclastogenesis and mesenchymal cell replication and/or differentiation and are exerted through interactions of the ligand-activated receptors with other transcription factors. However, increased remodeling alone cannot explain why loss of sex steroids tilts the balance of resorption and formation in favor of the former. Estrogens and androgens also exert effects on the lifespan of mature bone cells: pro-apoptotic effects on osteoclasts but anti-apoptotic effects on osteoblasts and osteocytes. These latter effects stem from a heretofore unexpected function of the classical "nuclear" sex steroid receptors outside the nucleus and result from activation of a Src/Shc/extracellular signal-regulated kinase signal transduction pathway probably within preassembled scaffolds called caveolae. Strikingly, estrogen receptor (ER) alpha or beta or the androgen receptor can transmit anti-apoptotic signals with similar efficiency, irrespective of whether the ligand is an estrogen or an androgen. More importantly, these nongenotropic, sex-nonspecific actions are mediated by the ligand-binding domain of the receptor and can be functionally dissociated from transcriptional activity with synthetic ligands. Taken together, these lines of evidence strongly suggest that, in sex steroid deficiency, loss of transcriptional effects may be responsible for the increased osteoclastogenesis and osteoblastogenesis and thereby the increased rate of bone remodeling. Loss of nongenotropic anti-apoptotic effects on mature osteoblasts and osteocytes, in combination with an opposite effect on the lifespan of mature osteoclasts, may be responsible for the imbalance between formation and resorption and the progressive loss of bone mass and strength. Elucidation of the dual function of sex steroid receptors has important pathophysiologic and pharmacologic implications. Specifically, synthetic ligands of the ER that can evoke the nongenotropic but not the genotropic signal may be bone anabolic agents, as opposed to natural estrogens or selective estrogen receptor modulators that are antiresorptive agents. The same ligands may also circumvent the side effects associated with conventional hormone replacement therapy.
Molecular basis of mammalian gamete binding.
Miller DJ, Shi X, Burkin H
Recent Prog Horm Res · 2002 · PMID 12017553 · Publisher ↗
Despite the importance of fertilization for controlling human reproduction, regulating animal production, and promoting preservation of endangered species, the molecular basis underlying gamete binding and fertilization...
Despite the importance of fertilization for controlling human reproduction, regulating animal production, and promoting preservation of endangered species, the molecular basis underlying gamete binding and fertilization has been perplexing. More progress has been made in the mouse than in other mammals and, recently, targeted deletion of specific genes in the mouse has yielded intriguing results. This review will emphasize research performed by our laboratory and others done primarily with mouse gametes but will include some interesting observations from other mammals. Studies of murine fertilization indicate that oligosaccharides on the egg coat glycoprotein ZP3 bind sperm. The precise oligosaccharides that bind sperm are the subject of considerable debate. ZP3 also induces exocytosis of the sperm acrosome, allowing sperm to penetrate through the egg coat (zona pellucida). A number of candidate ZP3 receptors have been proposed and studies of beta1,4galactosyltransferase-I (GalT-I) are reviewed here in the most detail. Sperm from mice with a targeted deletion of GalT-I still are able to bind the zona pellucida but are unable to acrosome react and penetrate through the zona. Therefore, the unique role of GalT-I appears to be in signal transduction. GalT-I forms a complex with heterotrimeric G proteins and activates signaling, leading to exocytosis in sperm and in heterologous cells expressing GalT-I. Other signaling steps triggered by GalT-I are under active investigation; this receptor forms a complex with a protein kinase anchoring protein. After exocytosis of the acrosome, sperm penetrate the zona pellucida and fuse with the oocyte plasma membrane using ADAM family members on sperm and integrins on oocytes. These proteins, along with the tetraspanins on oocytes, may form a complex web at gamete fusion. Targeted deletion of specific genes in this putative complex has provided important information about their redundancy. After the oocyte is fertilized, the binding site for GalT-I is lost from ZP3, preventing additional sperm from binding to the zona pellucida. New technical advances and creative ideas offer the opportunity to make important advances and to solve the conundrum of fertilization.
About
- Frequency
- Sun
- Papers found
- 200
- RSS feed
- Subscribe