The effect of in vivo growth hormone treatment on blood gene expression in adults with growth hormone deficiency reveals potential biomarkers to monitor growth hormone therapy.

OBJECTIVE: Growth hormone (GH) replacement therapy is presently utilized in the treatment of adult GH deficiency (AGHD). Adult responses to GH treatment are highly variable and, apart from measurement of IGF-I, few tools are currently available for monitoring GH treatment progress. As GH receptors are expressed in certain blood cell types, changes in gene expression in peripheral blood can reflect perturbations induced as a result of GH therapy. DESIGN/PATIENTS: We have carried out a pilot study to identify GH-responsive genes in blood, and have assessed the utility of GH-responsive genes in monitoring GH therapy in AGHD. Blood was collected from ten women diagnosed with AGHD syndrome both before and 4 weeks after initiation of GH substitutive therapy. RNA was extracted from peripheral blood mononuclear cells (PBMCs) and changes in response to GH were detected using microarray-based gene analysis. RESULTS: All patients responded to GH replacement therapy, with serum levels of IGF-I increasing by an average of 307% (P = 0.0003) while IGFBP-3 increased by an average of 182% (P = 0.0002). Serum levels of triglycerides, LDL-C, HDL-C, APOA1 or APOB did not change after 1 month of GH treatment. By contrast, we detected an increase in Lp(a) serum levels (P = 0.0149). Using a stringent selection cutoff of P

Steroid binding sites in liver membranes: interplay between glucocorticoids, sex steroids, and pituitary hormones.

Steroid hormones activate target cells through specific receptors that discriminate among ligands based upon recognition of distinct structural features. For most known steroids, membrane and nuclear receptors co-exist in many target cells. However, while the structure of the nuclear receptors and their function as transcriptional activators of specific target genes is generally well understood, the identity of the membrane receptors remains elusive. Using pharmacological and biochemical approaches, we are beginning to characterize receptors for glucocorticoids and anabolic-androgenic steroids in male rat liver membranes. Male rat liver endoplasmic reticulum contains two steroid binding sites which are functionally related and associated with a 90-134 kDa oligomeric protein: (1) the low-affinity glucocorticoid binding site (LAGS), composed at least in part of two peptides (37 and 53 kDa) that bind glucocorticoids and (2) the stanozolol binding protein (STBP), composed at least in part of three peptides (22, 31, and 55 kDa) that bind the synthetic androgen stanozolol. These steroid binding proteins have many properties different from those of classical nuclear receptors, with the salient differences being a failure to recognize "classical" ligands for nuclear receptors together with marked differences in biochemical properties and physiological regulation. The mechanism of interaction of glucocorticoids with the LAGS can be clearly distinguished from that with STBP. Moreover, STBP shows an extremely narrow pharmacological profile, being selective for ST and its analog, danazol, among more than 100 steroids and non-steroidal compounds that were assayed, including those that are able to displace glucocorticoids from the LAGS. The level of LAGS activity undergoes dramatic variations following changes from the physiological serum levels of thyroid hormones, glucocorticoids, GH, vitamin A, and E2. However, neither thyroid hormones nor GH have a critical role on STBP activity. The STBP is functionally related to LAGS. We have suggested a novel mechanism for STBP whereby membrane-associated glucocorticoid binding activity is targeted by stanozolol (and 16beta-hydroxylated stanozolol): stanozolol modulates glucocorticoid activity in the liver through negative allosteric modulation of the LAGS resulting in an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

Gene expression profile of the aging process in rat liver: normalizing effects of growth hormone replacement.

The mechanisms that control life span and age-related phenotypes are not well understood. It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. To test this hypothesis, and to improve our understanding of the cellular and molecular mechanisms behind the aging process, we have analyzed age-induced changes in gene expression patterns through the application of DNA chip technology. In the present study, the aging process was analyzed in rat liver in the presence or absence of GH replacement. Out of 3,000 genes printed on the microarrays, approximately 1,000 were detected in the rat liver. Among these, 47 unique transcripts were affected by the aging process in male rat livers. The largest groups of age-regulated transcripts encoded proteins involved in intermediary metabolism, mitochondrial respiration, and drug metabolism. Approximately 40% of the differentially expressed gene products were normalized after GH treatment. The majority of those transcripts have previously not been shown to be under GH control. The list of gene products that showed normalized expression levels in GH-treated old rats may shed further insight on the action and mechanism behind the positive effects of GH on, for example, fuel metabolism and body composition observed in different animal and human studies.

Endoplasmic reticulum stress prolongs GH-induced Janus kinase (JAK2)/signal transducer and activator of transcription (STAT5) signaling pathway.

The desensitization of the GH-induced Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) signaling pathway plays a crucial role in GH regulation of hepatic genes. Previous studies have demonstrated that the inactivation of the GH-induced JAK2/STAT5 pathway is regulated by protein translation and suppressors of cytokine signaling (SOCS). In this study we sought to explore the relationships between endoplasmic reticulum stress, GH-induced JAK2/STAT5 activity and SOCS expression. 1,2-bis(o-Aminophenoxy)ethane-N,N,N,N-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM), used to provoke endoplasmic reticulum stress, caused a drastic inhibition of protein translation that correlated with the phosphorylation of the eukaryotic translation initiation factor 2alpha. Both GH and BAPTA-AM caused a rapid induction of the transcription factor C/EBP homology protein (CHOP) and an additive effect was observed with combined treatment, which suggests a regulatory role of GH on endoplasmic reticulum stress. Endoplasmic reticulum stress did not interfere with the rapid GH activation of STAT5 DNA binding activity. However, BAPTA-AM prolonged the DNA binding activity of STAT5 without affecting STAT5 or JAK2 protein levels. GH-induced phosphorylation of JAK2 and STAT5 DNA binding activity were prolonged in the presence of BAPTA-AM, suggesting that endoplasmic reticulum stress prevents the inactivation of STAT5 DNA binding activity by modulating the rate of JAK2/STAT5 dephosphorylation. Like BAPTA-AM, the endoplasmic reticulum stressors dithiothreitol and A23187 also prolonged the GH-induced STAT5 DNA binding activity. We were not able to correlate BAPTA-AM effects to the GH-dependent expression of SOCS proteins or SOCS mRNA, suggesting that endoplasmic reticulum stress modulates the rate of JAK2/STAT5 dephosphorylation through mechanisms other than inhibition of SOCS expression. This study indicates that cellular stress may modulate transcription through the JAK/STAT pathway.

In vivo quantitative phosphoproteomic profiling identifies novel regulators of castration-resistant prostate cancer growth.

Prostate cancer remains a leading cause of cancer-related mortality worldwide owing to our inability to treat effectively castration-resistant tumors. To understand the signaling mechanisms sustaining castration-resistant growth, we implemented a mass spectrometry-based quantitative proteomic approach and use it to compare protein phosphorylation in orthotopic xenograft tumors grown in either intact or castrated mice. This investigation identified changes in phosphorylation of signaling proteins such as MEK, LYN, PRAS40, YAP1 and PAK2, indicating the concomitant activation of several oncogenic pathways in castration-resistant tumors, a notion that was confirmed by tumor transcriptome analysis. Further analysis demonstrated that the activation of mTORC1, PAK2 and the increased levels of YAP1 in castration-resistant tumors can be explained by the loss of androgen inhibitory actions. The analysis of clinical samples demonstrated elevated levels of PAK2 and YAP1 in castration-resistant tumors, whereas knockdown experiments in androgen-independent cells demonstrated that both YAP1 and PAK2 regulate cell colony formation and cell invasion activity. PAK2 also influenced cell proliferation and mitotic timing. Interestingly, these phenotypic changes occur in the absence of obvious alterations in the activity of AKT, MAPK or mTORC1 pathways, suggesting that PAK2 and YAP1 may represent novel targets for the treatment of castration-resistant prostate cancer. Pharmacologic inhibitors of PAK2 (PF-3758309) and YAP1 (Verteporfin) were able to inhibit the growth of androgen-independent PC3 xenografts. This work demonstrates the power of applying high-resolution mass spectrometry in the proteomic profiling of tumors grown in vivo for the identification of novel and clinically relevant regulatory proteins.

Differential cloning of growth hormone-regulated hepatic transcripts in the aged rat.

It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. This study was performed to elucidate age-related changes in GH-dependent effects at the level of gene expression. Through the application of complementary DNA representational difference analysis (RDA) we have identified gene products that are reduced during aging in rat liver. The expression of these genes was restored upon GH treatment. Results from reverse Northern and ribonuclease protection analysis confirmed that the RDA products were truly differentially expressed. In addition to well characterized GH-regulated genes, including CYP2C12, CYP2C13, and alpha2u-globulin, we demonstrate the differential expression of at least 11 genes previously not known to be under GH control. Several hepatic transcripts encoding enzymes and receptors involved in the metabolism of protein, carbohydrates, and lipids were identified. Other RDA products consisted of transcripts encoding proteins involved in ATP synthesis, detoxification of reactive oxygen species, or immune responses. This list of GH-regulated genes in the old rat may shed further light on the action and mechanism behind the positive effects of GH on, for example, body composition and the immune system that have been observed in different animal and human studies.

Growth hormone regulation of SOCS-2, SOCS-3, and CIS messenger ribonucleic acid expression in the rat.

The SOCS (suppressors of cytokine signaling) proteins have been suggested to function as inhibitors of cytokine receptor signaling. We have analyzed SOCS-2, SOCS-3, and CIS expression in relation to GH actions in the rat. SOCS-2, SOCS-3, and CIS transcripts were detected in various GH responsive tissues, including liver, muscle, and fat. In addition to the finding that different tissues express different levels of SOCS-2, SOCS-3, and CIS messenger RNA (mRNA), the steady-state levels of these SOCS transcripts were dependent on the endocrine status of the animal. SOCS-3 expression was 5-fold higher in fat from old compared with younger rats. Hypophysectomy reduced the levels of SOCS-2 and CIS mRNA in liver, muscle, and fat, whereas SOCS-3 expression was unchanged. Using primary cultures of rat hepatocytes, GH was shown to increase SOCS-2, SOCS-3, and CIS mRNA levels with different kinetics. SOCS-3 was rapidly and transiently induced, whereas SOCS-2 and CIS were increased in a slower fashion. Glucocorticoids blocked GH-induced SOCS-3 expression in cultured hepatocytes, whereas SOCS-2 and CIS expression was potentiated. Our data fit well with a concept of SOCS proteins acting as modulators of GH signal transduction.

Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat.

Complementary DNA microarrays containing 3000 different rat genes were used to study the consequences of severe hormonal deficiency (hypophysectomy) on the gene expression patterns in heart, liver, and kidney. Hybridization signals were seen from a majority of the arrayed complementary DNAs; nonetheless, tissue-specific expression patterns could be delineated. Hypophysectomy affected the expression of genes involved in a variety of cellular functions. Between 16-29% of the detected transcripts from each tissue changed expression level as a reaction to this condition. Chronic treatment of hypophysectomized animals with human GH also caused significant changes in gene expression patterns. The study confirms previous knowledge concerning certain gene expression changes in the above-mentioned situations and provides new information regarding hypophysectomy and chronic human GH effects in the rat. Furthermore, we have identified several new genes that respond to GH treatment. Our results represent a first step toward a more global understanding of gene expression changes in states of hormonal deficiency.

Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C.

Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, Stat5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. However, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity were prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing protein synthesis. Furthermore, inhibition of protein synthesis potentiated GH-induced transcriptional activity in BRL-4 cells transiently transfected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-induced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 and mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Transactivation of SPIGLE1 by GH was potentiated by mepacrine and D609 but not by the phospholipase A2 inhibitor AACOCF3. Thus, a regulatory circuit of GH-induced transcription through the Jak2/Stat5-signaling pathway includes a prompt GH-induced activation of Jak2/Stat5 followed by a negative regulatory response; ongoing protein synthesis and intracellular signaling pathways, where phospholipase C activity is involved, play a critical role to desensitize the GH-activated Jak2/Stat5-signaling pathway.

In vitro interaction between STAT 5 and JAK 2; dependence upon phosphorylation status of STAT 5 and JAK 2.

A working model for haematopoietic cytokine signal transduction has been hypothesised as follows. Binding of cytokines to specific receptor molecules leads to phosphorylation and activation of receptor associated members of the Janus kinase family. This is followed by tyrosine phosphorylation of the associated receptor and members of the STAT (signal transducer and activator of transcription) family of DNA-binding transcription factors. Phosphorylation is accompanied by STAT dimerisation, nuclear transport and activation of gene transcription. Activation of gene transcription is mediated by the binding of STAT dimers to palindromic STAT response elements. A number of areas of confusion remain; not least the mechanism by which multiple cytokines signal via a limited number of STATs. A role has been suggested for phosphorylated receptor tyrosine residues as STAT docking sites on activated receptor-JAK complexes. According to this model the amino acid sequence context of key tyrosine residues confers receptor specificity upon STAT activation. There is some controversy as to whether this model applies to STAT 5. The heterologous expression of STAT 5 in Sf 9 insect cells using the baculovirus expression system is described here. Protein of the correct molecular weight was expressed and found to be phosphorylated on tyrosine residues and to bind to a STAT response DNA element. This binding was dependent upon the phosphorylation status of the STAT protein. DNA binding could be abolished in vitro by treatment with a phosphotyrosine phosphatase and restored in vitro by treatment with activated recombinant JAK 2. The protein was purified to near homogeneity using a simple ion exchange/gel filtration chromatography procedure. The interaction between purified recombinant STAT 5 and JAK 2, either expressed by baculovirus or endogenously expressed in Buffalo rat liver cells, was studied. In both cases STAT 5 in its non-phosphorylated form was found to form a stable complex with activated JAK 2. Non-activated JAK 2 and phosphorylated STAT 5 were unable to participate in complex formation. The results presented provide a mechanistic basis for the activation of STAT 5 by a wide range of cytokines capable of activating JAK 2.

Mitogen-activated protein kinase kinase inhibition decreases growth hormone stimulated transcription mediated by STAT5.

We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).

Estrogens regulate the hepatic effects of growth hormone, a hormonal interplay with multiple fates.

The liver responds to estrogens and growth hormone (GH) which are critical regulators of body growth, gender-related hepatic functions, and intermediate metabolism. The effects of estrogens on liver can be direct, through the direct actions of hepatic ER, or indirect, which include the crosstalk with endocrine, metabolic, and sex-differentiated functions of GH. Most previous studies have been focused on the influence of estrogens on pituitary GH secretion, which has a great impact on hepatic transcriptional regulation. However, there is strong evidence that estrogens can influence the GH-regulated endocrine and metabolic functions in the human liver by acting at the level of GHR-STAT5 signaling pathway. This crosstalk is relevant because the widespread exposition of estrogen or estrogen-related compounds in human. Therefore, GH or estrogen signaling deficiency as well as the influence of estrogens on GH biology can cause a dramatic impact in liver physiology during mammalian development and in adulthood. In this review, we will summarize the current status of the influence of estrogen on GH actions in liver. A better understanding of estrogen-GH interplay in liver will lead to improved therapy of children with growth disorders and of adults with GH deficiency.

Determination of the structural changes by FT-IR, Raman, and CP/MAS 13C NMR spectroscopy on retrograded starch of maize tortillas.

The nixtamalization, production and storage of tortillas in refrigeration cause several changes on the starch structure, resulting in an increased crystallinity and therefore a higher content of resistant starch. The IR analysis for resistant starch (RS) showed a band at 1047cm-1 associated to the retrogradation process; this band was due to the weakening of the intermolecular H-bonds. These associated together to form ordered regions. The Raman analysis shows a characteristic band at 856cm-1 corresponding to C-C skeletal modes of glucose of α-1,4 glycosidic linkage starches, and a band at 480cm-1 attributed to skeletal vibrations of the pyranose ring in the glucose unit of starches. These changes may be related to the polymerization degree of the starch molecules, as well as to the retrogradation of amylose and amylopectin. The spectrum of 13C CP-MAS/NMR for RS3 supports the results obtained by IR and Raman. Lipidic and proteic groups were observed which may be in the form of complexes with amylose. One can proclaim that the existence of the salt form is induced and stabilized by the interactions dominating the V amylose structure in the solid state.

Ezrin mediates c-Myc actions in prostate cancer cell invasion.

The forced overexpression of c-Myc in mouse prostate and in normal human prostate epithelial cells results in tumor transformation with an invasive phenotype. How c-Myc regulates cell invasion is poorly understood. In this study, we have investigated the interplay of c-Myc and androgens in the regulation of prostate cancer cell invasion. We found that c-Myc induces cell invasion and anchorage-independent growth by regulating ezrin protein expression in the presence of androgens. The activity of the ezrin promoter is controlled by androgens through c-Myc, which binds to a phylogenetically conserved E-Box located in the proximal promoter region. Besides, we also show that ezrin is an important regulator of c-Myc protein levels. These effects are achieved through androgen-induced changes in ezrin phosphorylation, which results in the regulation of downstream signals. These downstream signals involve the modulation of Akt and GSK-3beta activity resulting in increased c-Myc protein synthesis and inhibition of its degradation. In summary, we have shown a key role for ezrin as a mediator of c-Myc-induced tumorigenesis in prostate cancer cells.

Role of pituitary hormones on 17alpha-ethinylestradiol-induced cholestasis in rat.

Estrogens cause intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal hormone replacement therapy. 17alpha-Ethinylestradiol (EE) is a synthetic estrogen widely used to cause experimental cholestasis in rodents with the aim of examining molecular mechanisms involved in this disease. EE actions on the liver are thought to be mediated by estrogen receptor alpha (ERalpha) and pituitary hormones. We tested this hypothesis by analyzing metabolic changes induced by EE in livers from hypophysectomized (HYPOX) and hypothyroid rats. Microarray studies revealed that the number of genes regulated by EE was increased almost 4-fold in HYPOX rat livers compared with intact males. Little overlap was apparent between the effects of EE in intact and HYPOX rats, demonstrating that pituitary hormones play a critical role in the hepatic effects of EE. Consistently, hypophysectomy protects the liver against induction by EE of serum bilirubin and alkaline phosphatase, two markers of cholestasis and hepatotoxicity and modulates the effects of EE on several genes involved in bile acid homeostasis (e.g., FXR, SHP, BSEP, and Cyp8b1). Finally, we demonstrate a novel mechanism of action of EE through binding and negative regulation of glucocorticoid receptor-mediated transcription. In summary, pituitary- and ERalpha-independent mechanisms contribute to development of EE-induced changes in liver transcriptome. Such mechanisms may be relevant when this model of EE-induced cholestasis is evaluated. The observation that the pharmacological effects of estrogen in liver differ in the absence or presence of the pituitary could be clinically relevant, because different drugs that block actions of pituitary hormones are now available.

IGF-II regulates metastatic properties of choriocarcinoma cells through the activation of the insulin receptor.

Choriocarcinoma is a highly malignant tumor that can arise from trophoblasts of any type of gestational event but most often from complete hydatidiform mole. IGF-II plays a fundamental role in placental development and may play a role in gestational trophoblastic diseases. Several studies have shown that IGF-II is expressed at high levels in hydatidiform moles and choriocarcinoma tissues; however, conflicting data exist on how IGF-II regulates the behaviour of choriocarcinoma cells. The purpose of this study was to determine the contribution of the receptors for IGF-I and insulin to the actions of IGF-II on the regulation of choriocarcinoma cells metastasis. An Immuno Radio Metric Assay was used to analyse the circulating and tissue levels of IGF-I and IGF-II in 24 cases of hydatidiform mole, two cases of choriocarcinoma and eight cases of spontaneous abortion at the same gestational age. The JEG-3 choriocarcinoma cell line was used to investigate the role of IGF-II in the regulation of cell invasion. We found that mole and choriocarcinoma tissue express high levels of IGF-II compared to first trimester placenta. Both IGF-I and IGF-II regulate choriocarcinoma cell invasion in a dose dependent manner but through a different mechanism. IGF-II effects involve the activation of the InsR while IGF-I uses the IGF-IR. The positive effects of IGF-II on invasion are the result of enhanced cell adhesion and chemotaxis (specifically towards collagen IV). The actions of IGF-II but not those of IGF-I were sensitive to inhibition by the insulin receptor inhibitor HNMPA(AM)3. Our results demonstrate that the insulin receptor regulates choriocarcinoma cell invasion.