Differential effects of exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation and survival

The prostate gland is regulated by multiple hormones and growth factors that may also affect prostate tumorigenesis. Growth hormone (GH) contributes to prostate development and function, but the direct effects of GH on prostate cancer cells are not well understood. The expression of endogenous GH in prostate cancer cell lines has also been observed, suggesting the potential for an effect of autocrine GH. In the present study, we measure the levels of GH and GH receptor (GHR) mRNA in multiple prostate cancer and normal prostate‐derived cell lines, and compare the effects of exogenous and autocrine GH on LNCaP prostate cancer cell proliferation and apoptosis, and the associated signal transduction pathways. We found that GHR and GH expression were higher in the prostate cancer cell lines, and that exogenous GH increased LNCaP cell proliferation, but had no effect on apoptosis. In contrast, autocrine GH overexpression reduced LNCaP cell proliferation and increased apoptosis. The distinct actions of exogenous and autocrine GH were accompanied by differences in the involvement of GHR‐associated signal transduction pathways, and were paralleled by an alteration in the subcellular localization of GHR, in which autocrine GH appeared to sequester GHR in the Golgi and endoplasmic reticulum. This alteration of GHR trafficking may underlie a distinct mode of GH‐mediated signaling associated with the effect of autocrine GH. These findings clarify the potential effects of GH on prostate cancer cell function, and indicate that the activity of autocrine GH may be distinct from that of endocrine GH in prostate cancer cells. J. Cell. Biochem. 114: 1322–1335, 2013. © 2012 Wiley Periodicals, Inc.     

Growth hormone activity in mitochondria depends on GH receptor Box 1 and involves caveolar pathway targeting

Growth hormone (GH) binding to its receptor (GHR) initiates GH-dependent signal transduction and internalization pathways to generate the biological effects. The precise role and way of action of GH on mitochondrial function are not yet fully understood. We show here that GH can stimulate cellular oxygen consumption in CHO cells transfected with cDNA coding for the full-length GHR. By using different GHR cDNA constructs, we succeeded in determining the different parts of the GHR implicated in the mitochondrial response to GH. Polarography and two-photon excitation fluorescence microscopy analysis showed that the Box 1 of the GHR intracellular domain was required for an activation of the mitochondrial respiration in response to a GH exposure. However, confocal laser scanning microscopy demonstrated that cells lacking the GHR Box 1 could efficiently internalize the hormone. We demonstrated that internalization mediated either by clathrin-coated pits or by caveolae was able to regulate GH mitochondrial effect: these two pathways are both essential to obtain the GH stimulatory action on mitochondrial function. Moreover, electron microscopic and biochemical approaches allowed us to identify the caveolar pathway as essential for targeting GH and GHR to mitochondria.     

Growth hormone stimulates the tyrosine phosphorylation of 42- and 45-kDa ERK-related proteins.

Growth hormone (GH) influences a number of tissue-specific biological activities in diverse cell types. However, little is known about the biochemical pathway by which the signal initiated by GH binding to its cell-surface receptor is transduced. The GH receptor has been reported to be phosphorylated on tyrosine in 3T3-F442A cells, a cell line in which GH promotes differentiation and inhibits mitogen-stimulated growth; however, it is not known whether tyrosine phosphorylation plays a role in GH signal transduction. We report that GH treatment of 3T3-F442A cells resulted in the rapid tyrosine phosphorylation of at least four proteins. These included 42- (pp42) and 45-kDa (pp45) proteins immunologically related to ERK1 (extracellular signal-regulated kinase 1), a member of a family of serine/threonine protein kinases that are phosphorylated on tyrosine in response to mitogens. Prolonged phorbol ester pretreatment attenuated the tyrosine phosphorylation of pp42 and pp45 in platelet-derived growth factor-treated cells, but not in GH-treated cells. Maximal GH-stimulated tyrosine phosphorylation of pp42 and pp45 coincided with peak levels of a 42-kDa renaturable MBP kinase activity in lysates of GH-treated cells resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The observation that multiple cellular proteins are rapidly phosphorylated on tyrosine in response to physiological concentrations of GH suggests that tyrosine phosphorylation plays a role in GH signal transduction. Moreover, the stimulation of tyrosine phosphorylation of ERK-related proteins by GH suggests that mitogens and nonmitogens may employ common phosphotyrosyl proteins in the activation of ultimately distinct cellular programs.     

Tyrosine phosphorylation: a signal for the activation of the phagocyte respiratory burst

The respiratory burst of the phagocytic cell is a unique function which provides the cell with a series of reactive oxygen intermediates which it can use to kill ingested microorganisms. The complex signal transduction pathways responsible for regulating the respiratory burst are yet to be fully elucidated. Protein tyrosine kinases are now recognised as being critical components in the regulatory pathways controlling many cellular functions. In this report we review the evidence implicating tyrosine phosphorylation as an important signal for the activation of the phagocyte respiratory burst.     

生长激素受体及其介导的信号转导

生长激素受体(growth hormone receptor,GHR)是细胞因子／造血因子受体超级家族的一员。它通过二聚体的形式和生长激素(growth hormone,GH)相结合,然后诱发Janus激酶2(Janus kinase 2,JAK2)等细胞因子酪氨酸磷酸化并通过4条不同的途径将信号传入细胞内从而产生一系列的生理效应。现在了解GHR的结构特征、组织分布的基础上,对其介导的信号转导途径作进一步的阐明。     

A microarray gene analysis of peripheral whole blood in normal adult male rats after long-term GH gene therapy

The main aims of this study were to determine the effects of GH gene abuse/misuse in normal animals and to discover genes that could be used as candidate biomarkers for the detection of GH gene therapy abuse/misuse in humans. We determined the global gene expression profile of peripheral whole blood from normal adult male rats after long-term GH gene therapy using CapitalBio 27 K Rat Genome Oligo Arrays. Sixty one genes were found to be differentially expressed in GH gene-treated rats 24 weeks after receiving GH gene therapy, at a two-fold higher or lower level compared to the empty vector group (p < 0.05). These genes were mainly associated with angiogenesis, oncogenesis, apoptosis, immune networks, signaling pathways, general metabolism, type I diabetes mellitus, carbon fixation, cell adhesion molecules, and cytokine-cytokine receptor interaction. The results imply that exogenous GH gene expression in normal subjects is likely to induce cellular changes in the metabolism, signal pathways and immunity. A real-time qRT-PCR analysis of a selection of the genes confirmed the microarray data. Eight differently expressed genes were selected as candidate biomarkers from among these 61 genes. These 8 showed five-fold higher or lower expression levels after the GH gene transduction (p < 0.05). They were then validated in real-time PCR experiments using 15 single-treated blood samples and 10 control blood samples. In summary, we detected the gene expression profiles of rat peripheral whole blood after long-term GH gene therapy and screened eight genes as candidate biomarkers based on the microarray data. This will contribute to an increased mechanistic understanding of the effects of chronic GH gene therapy abuse/misuse in normal subjects.     

植物GH3基因家族研究进展

生长素在植物的整个生长发育过程中都具有重要的作用,其早期响应基因可归为3类:Aux/IAAs、GH3s、SAURs.通过功能基因组学的研究,特别是对相关突变体的分子遗传学与分子生物学的研究,使我们对这些基因家族的作用机理的理解更为深入.以下综述了植物GH3基因的结构、功能及表达调控模式,重点介绍了由GH3介导的生长素信号途径与其他信号转导途径之间的互作和GH3基因与植物逆境胁迫适应的关系.     

Signal transduction by protein tyrosine nitration: competition or cooperation with tyrosine phosphorylation-dependent signaling events?

This review article is an attempt to stimulate a discussion on the significance of protein tyrosine nitration to cellular signaling and its relationships with protein tyrosine phosphorylation. Initially, it provides basic information on growth factor and oxidants as modulators/mediators of tyrosine phosphorylation-dependent signal transduction pathways. The effects of exogenous and endogenous tyrosine nitration on such pathways were examined by reviewing published and unpublished observations. From an initial perspective that tyrosine nitration was a toxic manifestation of nitric oxide, the concept evolved to a protein modification that could also function in cellular signaling events, possibly cooperating with tyrosine phosphorylation.     

Signal transduction events in mammalian cells in response to ionizing radiation

Ionizing radiations elicit a variety of biological effects in mammalian cells. In recent years altered signal transduction has been recognized as a key cellular response to ionizing radiation. Several oncogenes, the products of which are components of signal transduction pathways and which are over-expressed in many tumors, are specifically induced in cells exposed to radiation. It has also become evident that the oncogene ras and the serine/threonine protein kinase oncogenes raf and PKC confer radio-resistance to tumor cells. Modulation of these genes or their activity by natural compounds may offer a strategy to treat cancer by enhancing radiation-induced apoptosis of tumor cells.     

Focal adhesion kinase: protein interactions and cellular functions

Integrin-mediated cell adhesion to extracellular matrix (ECM) plays important roles in a variety of biological processes. Recent studies suggested that integrins mediate signal transduction across the plasma membrane via activating several intracellular signaling pathways. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been shown to be a major mediator of integrin signal transduction pathways. Upon activation by integrins, FAK undergoes autophosphorylation as well as associations with several other intracellular signaling molecules. These interactions in the signaling pathways have been shown to regulation a variety of cellular functions such as cell spreading, migration, cell proliferation, apoptosis and cell survival. Recent progress in the understanding of FAK interactions with other proteins in the regulation of these cellular functions will be discussed in this review.     

Signal transduction pathway for anterior-posterior development inDrosophila

InDrosophila, the establishment of embryonic polarity along the anterior-posterior axis of the egg is determined by the activity of maternal gene products that accumulate during oogenesis. Amongst these are the Bicoid, the Nanos, and the terminal class gene products, some of which are oncoproteins involved in signal transduction for the formation of terminal structures in the embryo. Several signal transduction pathways have been described inDrosophila, and this review explores the potential of oncogene studies using one of those pathways — the terminal class signal transduction pathway — to better understand the cellular mechanisms of proto-oncogenes that mediate cellular responses in vertebrates including humans.     

Targets of TGF beta-related signaling in Caenorhabditis elegans.

With the characterization of the Smads 5 years ago, it became possible to trace the TGFβ signal transduction pathway from the plasma membrane to the nucleus. Since that time, many Smad interaction partners, cofactors and target genes have been identified using a variety of experimental approaches and model systems. Understanding how these partners generate tissue specificity and crosstalk between pathways is an ongoing pursuit for the field of TGFβ signal transduction. The nematode Caenorhabditis elegans provides a simple, genetically tractable model organism in which to address this goal. This review will examine progress towards the identification of cellular and molecular targets of TGFβ-related signaling in C. elegans.     

Tyrosine nitration: Localisation,quantification, consequences for protein function and signal transduction

The nitration of free tyrosine or protein tyrosine residues generates 3-nitrotyrosine the detection of which has been utilised as a footprint for the in vivo formation of peroxynitrite and other reactive nitrogen species. The detection of 3-nitrotyrosine by analytical and immunological techniques has established that tyrosine nitration occurs under physiological conditions and levels increase in most disease states. This review provides an updated, comprehensive and detailed summary of the tissue, cellular and specific protein localisation of 3-nitrotyrosine and its quantification. The potential consequences of nitration to protein function and the pathogenesis of disease are also examined together with the possible effects of protein nitration on signal transduction pathways and on the metabolism of proteins.     

Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis

Background  

Prolactinoma is the most frequent pituitary tumor in humans. The dopamine D₂ receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma.     

Novel antitumor peptide hormones and their effect on signal transduction.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.     

Therapeutic potential of natural product signal transduction agents

Modern drug discovery embraces a strategy of targeting cellular signal transduction pathways as a means of finding new therapeutic agents. Historically, natural products derived from microorganisms have played an important role as drug leads and clinical candidates under this paradigm. The future drug potential of natural products as signal transduction agents looks promising, as illustrated by two key examples. First, substantial advances have been made in the development of inhibitors based on immunophilin ligand polyketides, which target the TOR-mediated pathways and can modulate processes including cell proliferation and cell-cycle arrest. Second, the discovery of natural product inhibitors of the ubiquitin-proteasome proteolytic signal transduction pathway represents an emerging field. Given these examples, together with the diversity of as yet undiscovered agents, natural product signal transduction agents offer great potential for future drug discovery efforts.     

Annexins: putative linkers in dynamic membrane–cytoskeleton interactions in plant cells

Summary. The plasma membrane, the most external cellular structure, is at the forefront between the plant cell and its environment. Hence, it is naturally adapted to function in detection of external signals, their transduction throughout the cell, and finally, in cell reactions. Membrane lipids and the cytoskeleton, once regarded as simple and static structures, have recently been recognized as significant players in signal transduction. Proteins involved in signal detection and transduction are organised in specific domains at the plasma membrane. Their aggregation allows to bring together and orient the downstream and upstream members of signalling pathways. The cortical cytoskeleton provides a structural framework for rapid signal transduction from the cell periphery into the nucleus. It leads to intracellular reorganisation and wide-scale modulation of cellular metabolism which results in accumulation of newly synthesised proteins and/or secondary metabolites which, in turn, have to be distributed to the appropriate cell compartments. And again, in plant cells, the secretory vesicles that govern polar cellular transport are delivered to their target membranes by interaction with actin microfilaments. In search for factors that could govern subsequent steps of the cell response delineated above we focused on an evolutionary conserved protein family, the annexins, that bind in a calcium-dependent manner to membrane phospholipids. Annexins were proposed to regulate dynamic changes in membrane architecture and to organise the interface between secretory vesicles and the membrane. Certain proteins from this family were also identified as actin binding, making them ideal mediators in cell membrane and cytoskeleton interactions. Correspondence and reprints: Laboratory of Plant Pathogenesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland.