Connections between prolactin and ovarian cancer.

Ovarian cancer (OC) is characterized by a high morbidity and mortality, highlighting a great need for a better understanding of biological mechanisms that affect OC progression and improving its early detection methods. This study investigates effects of prolactin (PRL) on ovarian cancer cells, analyzes PRL receptors (PRLR) in tissue micro arrays and relates PRLR expression to survival of ovarian cancer. A database, composed of transcript profiles from OC, was searched for PRLR expression and results were put in relation to survival. Expression of PRLR in OC tissue sections and OC cell lines SKOV3, OV2008 and OVSAHO was assessed using immunohistochemistry, western blots and quantitative real-time PCR. The biological function of PRLR was evaluated by proliferation, colony formation and wound healing assays. Levels of PRLR mRNA are related to survival; in epithelial OC a high PRLR mRNA expression is related to a shorter survival. Analysis of a tissue micro array consisting of 84 OC showed that 72% were positive for PRLR immuno-staining. PRLR staining tended to be higher in OC of high grade tumors compared to lower grades. PRLR mRNA and protein can further be detected in OC cell lines. Moreover, in vitro treatment with PRL significantly activated the JAK/STAT pathway. PRLR expression is associated with OC survivals. PRL and its receptor may play an onco-modulatory role and promote tumor aggressiveness in OC. Alternatively, increased PRLR levels may form a base for the development of PRLR antagonist or PRLR antagonist-drug conjugate to increase selective uptake of anti-cancer drugs.

Effects of prolactin on ventricular myocyte shortening and calcium transport in the streptozotocin-induced diabetic rat.

The physiological role of prolactin (PRL) in the heart, and in particular the diabetic heart, are largely unknown. The effects of PRL on ventricular myocyte shortening and Ca2+ transport in the streptozotocin (STZ) - induced diabetic and in age-matched control rats were investigated. PRL receptor protein, myocyte shortening, intracellular [Ca2+], L-type Ca2+ current were measured by Western blot, cell imaging, fluorescence photometry and whole-cell patch-clamp techniques, respectively. Compared to normal Tyrode solution (NT), PRL (50 ng/ml) significantly (p < 0.05) increased the amplitude of shortening in myocytes from control (7.43 ± 0.38 vs. 9.68 ± 0.46 %) and diabetic (6.57 ± 0.24 vs. 8.91 ± 0.44 %) heart (n = 44-49 cells). Compared to NT, PRL (50 ng/ml) significantly increased the amplitude of Ca2+ transients in myocytes from control (0.084 ± 0.004 vs. 0.115 ± 0.007 Fura-2 ratio units) and diabetic (0.087 ± 0.007 vs. 0.112 ± 0.006 Fura-2 ratio units) heart (n = 36-50 cells). PRL did not significantly alter the amplitude of caffeine-evoked Ca2+ transients however, PRL significantly increased the fractional release of Ca2+ in myocytes from control (21 %) and diabetic (14 %) and heart. The rate of Ca2+ transient recovery following PRL treatment was significantly increased in myocytes from diabetic and control heart. Amplitude of L-type Ca2+ current was not significantly altered by diabetes or by PRL. PRL increased the amplitude of shortening and Ca2+ transients in myocytes from control and diabetic heart. Increased fractional release of sarcoplasmic reticulum Ca2+ may partly underlie the positive inotropic effects of PRL in ventricular myocytes from control and STZ-induced diabetic rat.

Human Cytomegalovirus Infection Induces High Expression of Prolactin and Prolactin Receptors in Ovarian Cancer.

One of the potential biomarkers for ovarian cancer patients is high serum level of prolactin (PRL), which is a growth factor that may promote tumor cell growth. The prolactin receptor (PRLR) and human cytomegalovirus (HCMV) proteins are frequently detected in ovarian tumor tissue specimens, but the potential impact of HCMV infection on the PRL system have so far not been investigated. In this study, HCMV's effects on PRL and PRLR expression were assessed in infected ovarian cancer cells (SKOV3) by PCR and Western blot techniques. The levels of both PRL and PRLR transcripts as well as the corresponding proteins were highly increased in HCMV-infected SKOV3 cells. Tissue specimens obtained from 10 patients with ovarian cancer demonstrated high expression of PRLR, HCMV-IE, and pp65 proteins. Extensive expression of PRLR was detected in all examined ovarian tumor tissue specimens except for one from a patient who had focal expression of PRLR and this patient was HCMV-negative in her tumor. In conclusion, PRL and PRLR were induced to high levels in HCMV-infected ovarian cancer cells and PRLR expression was extensively detected in HCMV-infected ovarian tissue specimens. Highly induced PRL and PRLR by HCMV infection may be of relevance for the oncomodulatory role of this virus in ovarian cancer.

Association between prolactin receptor expression and proliferation in the endometrium of obese women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is associated with increased risk of endometrial cancer. There is growing evidence that prolactin and its receptor (PRLR) are involved in the development of cancer. We assessed endometrial expression of PRLR mRNA, and immunostaining of PRLR and the proliferation marker Ki67 on different cycle days in obese (OB-PCOS) and normal-weight women with PCOS and body mass index-matched controls. The OB-PCOS group underwent a 3 months lifestyle intervention. Prior to intervention, obese women with PCOS and controls had lower endometrial levels of PRLR mRNA in proliferative endometrium than the normal-weight groups (p < .05). After intervention, six OB-PCOS women had confirmed ovulation, while 12 remained anovulatory. Both these subgroups displayed higher immunostaining of PRLR in endometrial stroma, and in the anovulatory subgroup also increased Ki67, on cycle days 21-23 compared with controls (p < .05). In obese controls, the PRLR mRNA expression was decreased in secretory endometrium compared with proliferative endometrium (p = .004). A corresponding change within the cycle was not found in OB-PCOS women. Immunostaining of PRLR in the secretory phase correlated positively with Ki67 (p < .05) in the endometrium. These observations suggest that short-term lifestyle intervention can restore ovulation but not normalize PRLR expression in the endometrium of obese women with PCOS. Trial registration: ISRCTN, ISRCTN18400086, https://doi.org/10.1186/ISRCTN18400086.

An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation.

Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (KD = 2.3±0.2 nM) and mouse serum albumin (KD = 0.38±0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21±3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

The Risk of Offspring Psychiatric Disorders in the Setting of Maternal Obesity and Diabetes.

BACKGROUND: Prenatal exposure to metabolic disturbances is associated with increased risk of offspring neurodevelopmental impairment and autism spectrum disorder, while little is known about the joint effect of maternal obesity and diabetes. With this study, we aim to assess the joint effect of maternal obesity and diabetes on the risk for offspring psychiatric and mild neurodevelopmental disorders. METHODS: Nationwide registries were used to link data of all live births in Finland between 2004 and 2014 (n = 649 043). Cox proportional hazards modeling adjusting for potential confounders was applied to estimate the effect of maternal obesity, pregestational diabetes mellitus (PGDM), and gestational diabetes mellitus, as well as their joint effects, on the outcomes of offspring psychiatric and mild neurodevelopmental diagnoses and offspring prescription of psychotropic drugs. RESULTS: Among mothers without diabetes, severely obese mothers had 67% to 88% increased risk of having a child with mild neurodevelopmental disorders (hazard risk ratio [HR] = 1.69; 95% confidence interval [CI] = 1.54-1.86), attention-deficit/hyperactivity disorder or conduct disorder (HR = 1.88; 95% CI = 1.58-2.23), and psychotic, mood, and stress-related disorders (HR = 1.67; 95% CI = 1.31-2.13) compared with mothers with a normal BMI. PGDM implied a further risk increase for all groups of psychiatric diagnoses with onset in childhood or adolescence in mothers with severe obesity. Marked effects were found particularly for autism spectrum disorder (HR = 6.49; 95% CI = 3.08-13.69), attention-deficit/hyperactivity disorder and conduct disorder (HR = 6.03; 95% CI = 3.23-11.24), and mixed disorders of conduct and emotions (HR = 4.29; 95% CI = 2.14-8.60). Gestational diabetes mellitus did not increase the risk highly for these offspring disorders. CONCLUSIONS: Maternal PGDM combined with severe maternal obesity markedly increases the risk of several children's psychiatric and mild neurodevelopmental disorders.

The ubiquitin ligase Cullin5SOCS2 regulates NDR1/STK38 stability and NF-κB transactivation.

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2-/- mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway.

Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

Role of Prolactin Receptors in Lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.

Suppressor of cytokine signaling 2 (SOCS2) deletion protects against multiple low dose streptozotocin-induced type 1 diabetes in adult male mice.

BACKGROUND: Diabetes type 1 is characterized by the failure of beta cells to produce insulin. Suppressor of cytokine signaling (SOCS) proteins are important regulators of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. Previous studies have shown that GH can prevent the development of type I diabetes in mice and that SOCS2 deficiency mimics a state of increased GH sensitivity. METHODOLOGY: The elevated sensitivity of SOCS2-/- mice to GH and possibly to PRL was the rationale to analyze the effects of multiple low dose streptozotocin (MLDSTZ)-induced diabetes in SOCS2-/- mice. RESULTS: We show that 6-month-old SOCS2-/- mice, but not 2-month-old mice, were less sensitive to MLDSTZ-induced diabetes, compared to controls. MLDSTZ treatment induced glucose intolerance in both SOCS2+/+ and SOCS2-/- mice, as shown by glucose tolerance tests, with SOCS2+/+ mice showing a more marked intolerance, compared to SOCS2-/- mice. Furthermore, insulin tolerance tests showed that the SOCS2-/- mice have an improved hypoglycemic response to exogenous insulin, compared to SOCS2+/+ mice. Moreover, in isolated islets, lipotoxic effects on insulin release could partly be overcome by ligands, which bind to GH or PRL receptors. CONCLUSION: Knockdown of SOCS2 makes mice less sensitive to MLDSTZ. These results are consistent with the proposal that elimination of SOCS2 in pancreatic islets creates a state of ß-cell hypersensitivity to GH/PRL that mimics events in pregnancy, and which is protective against MLDSTZ-induced type I diabetes in mice. SOCS2-dependent control of ß-cell survival may be of relevance to islet regeneration and survival in transplantation.

Effect of a macronutrient preload on blood glucose level and pregnancy outcome in gestational diabetes.

AIM: To investigate the effect of a macro-nutrient preload (Inzone Vitality) on blood glucose levels and pregnancy outcomes of gestational diabetes. The preload method involves the ingestion of a smaller amount of a macronutrient composition half an hour before regular meals. The hypothesis was that preload treatment will reduce postprandial glycemia in gestational diabetes. METHODS: Sixty-six diagnosed cases of gestational diabetes were randomly selected from gynecology and obstetrics outpatient clinic at Xinqiao Hospital in Chongqing. The patients were divided into an intervention group (33 cases) and a control group (33 cases), according to odd-even numbers of the random cases. The intervention group was treated with a macro-nutrient preload given 0.5 h before regular meals and the control group was given a comparative treatment consisting of a milk powder with similar energy content. The two groups were studied until delivery and the measured parameters included fasting blood glucose (FBG), 2-hour postprandial blood glucose (2h-PBG), delivery mode and neonatal birth weight. RESULTS: The two groups showed no differences in FBG or 2h-PBG before the nutritional intervention. FBG and 2h-PBG after intervention and before delivery were significantly lower in the intervention group, treated with the macro nutrient preload compared to the control group (P < 0.01). Changes in FBG and 2h-PBG before and after the intervention were investigated and the difference in the intervention group was significantly greater than corresponding values in the control group (P < 0.05, P < 0.01). The neonatal birth weight and delivery mode was not significantly different (P > 0.05). CONCLUSION: A macro-nutrient composition, used as a preload, is effective in controlling FBG and PBG of gestational diabetes.

Effects of a Macro-Nutrient Preload on Type 2 Diabetic Patients.

AIMS: Macro-nutrient preloads given 30 min before regular meals may improve metabolism. The aim was to investigate how type 2 diabetic patients react to a preload consisting of a blend of macro-nutrients with a low-glycemic index (Inzone Preload(®)). METHODS: In a before-after study design, 30 subjects with type 2 diabetes mellitus (T2DM) were enrolled in a 12-week program. All subjects were given Inzone Preload (43% proteins, 29% carbohydrates, 10% lipids, and 9% fibers, 71 kcal), 30 min before each meal during 12 weeks. Fasting glucose and postprandial 2 h glucose were monitored every second week. Body weight (BW) and waist circumference were measured each month. Fasting plasma glucose, glycosylated hemoglobin, serum lipids, fasting insulin, C-reactive protein, and homeostasis model assessment were evaluated before and after the intervention. Subjective appetite was monitored using visual analogue scales after the Inzone Preload. RESULTS: The dietary intervention significantly influenced several metabolic parameters compared to base line. Inzone Preload treatment reduced mean postprandial plasma glucose levels (12.2 ± 1.2 vs. 10.5 ± 2.0 mmol/L), HbA1c (7.4 ± 0.3 vs. 7.1 ± 0.2%), mean total cholesterol (4.8 ± 0.9 vs. 4.3 ± 0.8 mmol/L), low-density lipoprotein cholesterol (2.8 ± 0.6 vs. 2.5 ± 0.4 mmol/L), and CRP (1.5 ± 1.4 vs. 0.7 ± 0.7 mg/L). BW loss of more than 3% was seen in 13 participants (43%). Feelings of satiety were significantly higher after Inzone Preload than after habitual breakfast (p < 0.05). No significant changes in fasting blood glucose, high-density lipoprotein and total triacylglycerol, HOMA-IR, and HOMA-ß were observed. CONCLUSION: A macro-nutrient preload treatment reduces postprandial glucose, inflammatory markers, and serum lipids in patients with T2DM. Approximately half of the study group also displayed reduced BW.

Metabolomics: a tool for the diagnosis of GH deficiency and for monitoring GH replacement?

The diagnostic value of insulin-like growth factor 1 (IGF1) for GH deficiency (GHD) in adults is not optimal. Molecular profiling could be used for biomarker discovery. The aim of this pilot study was to compare the serum metabolome between GHD patients and healthy controls, and identification of potential markers for diagnosis and/or for individual GH dosing. A total of ten patients with GHD, median age of 55 years and BMI of 27 kg/m(2), were compared with ten healthy age- and gender-matched controls. The serum metabolic profiles were generated using gas chromatography-coupled mass spectroscopy on fasting samples taken in the morning from the controls and at baseline and during 6 months of GH replacement in the patients with GHD. The difference in low-molecular weight compounds (LMC) distinguished the healthy controls from GHD patients. Among 285 measured metabolites, 13 were identified as being most important in differentiating GHD patients from controls. Of these, 11 could not be structurally annotated but many were classified as lipids. The difference in the LMC pattern persisted despite normalisation of IGF1 following GH replacement. GH replacement increased the levels of specific fatty acid compounds and decreased the levels of certain amino acids. No metabolite changed in response to GH treatment, to the same extent as IGF1. The measurement of 285 metabolites resulted in a unique pattern in GHD, but changes in the metabolite patterns during GH treatment were limited. The utility of metabolomics to find new markers in GHD and GH replacement remains to be further elucidated.

SOCS2 mediates the cross talk between androgen and growth hormone signaling in prostate cancer.

UNLABELLED: Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa. SUMMARY: Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

Influence of neonatal hypothyroidism on hepatic gene expression and lipid metabolism in adulthood.

Thyroid hormones are required for normal growth and development in mammals. Congenital-neonatal hypothyroidism (CH) has a profound impact on physiology, but its specific influence in liver is less understood. Here, we studied how CH influences the liver gene expression program in adulthood. Pregnant rats were given the antithyroid drug methimazole (MMI) from GD12 until PND30 to induce CH in male offspring. Growth defects due to CH were evident as reductions in body weight and tail length from the second week of life. Once the MMI treatment was discontinued, the feed efficiency increased in CH, and this was accompanied by significant catch-up growth. On PND80, significant reductions in body mass, tail length, and circulating IGF-I levels remained in CH rats. Conversely, the mRNA levels of known GH target genes were significantly upregulated. The serum levels of thyroid hormones, cholesterol, and triglycerides showed no significant differences. In contrast, CH rats showed significant changes in the expression of hepatic genes involved in lipid metabolism, including an increased transcription of PPARα and a reduced expression of genes involved in fatty acid and cholesterol uptake, cellular sterol efflux, triglyceride assembly, bile acid synthesis, and lipogenesis. These changes were associated with a decrease of intrahepatic lipids. Finally, CH rats responded to the onset of hypothyroidism in adulthood with a reduction of serum fatty acids and hepatic cholesteryl esters and to T3 replacement with an enhanced activation of malic enzyme. In summary, we provide in vivo evidence that neonatal hypothyroidism influences the hepatic transcriptional program and tissue sensitivity to hormone treatment in adulthood. This highlights the critical role that a euthyroid state during development plays on normal liver physiology in adulthood.

SOCS2 deletion protects against hepatic steatosis but worsens insulin resistance in high-fat-diet-fed mice.

Hepatic steatosis is a prominent feature in patients with growth hormone (GH) deficiency. The ubiquitin ligase SOCS2 attenuates hepatic GH signaling by inhibiting the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) axis. Here, we investigated the role of SOCS2 in the development of diet-induced hepatic steatosis and insulin resistance. SOCS2-knockout (SOCS2(-/-)) mice and wild-type littermates were fed for 4 mo with control or high-fat diet, followed by assessment of insulin sensitivity, hepatic lipid content, and expression of inflammatory cytokines. SOCS2(-/-) mice exhibited increased hepatic TG secretion by 77.6% (P<0.001) as compared with wild-type control mice and were protected from high-fat-diet (HFD)-induced hepatic steatosis, showing 49.3% (P<0.01) reduction in liver TG levels compared to HFD-fed wild-type littermates. In contrast, we found that HFD-triggered attenuation of systemic insulin sensitivity was more marked in SOCS2(-/-) mice. Livers from the HFD-fed SOCS2(-/-) mice showed increased NF-κB activity as well as elevated expression of genes for the inflammatory cytokines IFN-γ and IL-6. An inhibitory role of SOCS2 on Toll-like receptor 4 signaling was demonstrated in macrophages obtained from the SOCS2(-/-) and wild-type mice. This study identified SOCS2 as an important regulator of hepatic homeostasis under conditions of high-fat dietary stress.

Effects of oestrogen and testosterone therapy on serum metabolites in postmenopausal women.

OBJECTIVES: Metabolite profiles of body fluids or tissue extracts can be regarded as important indicators of physiological or pathological states. Whether hormone-specific alterations of the serum metabolome can be identified using this technique has not been tested yet. The aim of this study was to investigate metabolic responses during hormone therapy in postmenopausal women by a nontargeted metabolomics approach. METHODS: Sixty naturally postmenopausal women were randomly assigned to treatment with testosterone undecanoate 40 mg every second day; estradiol valerate 2 mg daily; or the combination of both. Serum metabolites were determined by gas chromatography-mass spectrometry (GC-MS) before and after 3 months of treatment. Metabolites affected by the treatment were identified and correlated with changes in insulin sensitivity and lipid profiles. RESULTS: Treatment-dependent and hormone-specific effects on serum metabolites were observed, ranging between 69% reduction and 184% increase, but the metabolites that best explained the differences could not be structurally identified. Effects on annotated metabolites were less associated with clinical parameters as compared to established serum markers for adverse lipid and carbohydrate metabolism, such as cholesterol and triglycerides. However, cystine, lysine and tyrosine were shown to change in correlation with insulin sensitivity and high-density lipoprotein cholesterol levels in response to testosterone, indicating that those responses were somehow related to each other. CONCLUSIONS: Oestrogen- and androgen-specific alterations in the serum metabolome could be identified using GC-MS, reflecting hormone-specific effects on whole body metabolism. New knowledge regarding steroid-mediated metabolic responses within different tissues might be obtained using a similar approach on tissue extracts.

LPS regulates SOCS2 transcription in a type I interferon dependent autocrine-paracrine loop.

Recent studies suggest that SOCS2 is involved in the regulation of TLR signaling. In this study, we found that the expression of SOCS2 is regulated in human monocyte-derived DC by ligands stimulating TLR2, 3, 4, 5, 8 and 9 signaling. SOCS2 induction by LPS was dependent on the type I IFN regulated transcription factors IRF1 and IRF3 as shown by using silencing RNAs for IRFs. Blocking endogenous type I IFN signaling, by neutralizing antibodies to the receptor IFNAR2, abolished SOCS2 mRNA expression after TLR4 stimulation. Transcription factors STAT3, 5 and 6 displayed putative binding sites in the promoter regions of the human SOCS2 gene. Subsequent silencing experiments further supported that STAT3 and STAT5 are involved in LPS induced SOCS2 regulation. In mice we show that SOCS2 mRNA induction is 45% lower in bone marrow derived macrophages derived from MyD88(-/-) mice, and do not increase in BMMs from IRF3(-/-) mice after BCG infection. In conclusion, our results suggest that TLR4 signaling indirectly increases SOCS2 in late phase mainly via the production of endogenous type I IFN, and that subsequent IFN receptor signaling activates SOCS2 via STAT3 and STAT5.

Androgen-independent effects of Serenoa repens extract (Prostasan®) on prostatic epithelial cell proliferation and inflammation.

Extracts from Serenoa repens are widely used for the treatment of benign prostatic hyperplasia (BPH) and traditionally for prostatitis. In the present study we evaluated the biological effects of Serenoa repens extract (Prostasan®) on prostate cells beyond its known antiandrogenic actions. Prostasan® inhibited epidermal growth factor (EGF) and lipopolysaccharide (LPS) induced proliferation of the prostatic epithelial, androgen independent cell line PC-3. At effective concentrations of 50 µg/mL, Prostasan® partly displaced EGF from EGF receptor (EGFR) but fully blocked EGF-induced cell proliferation of PC-3 cells. Similarly, Prostasan® inhibited LPS-induced proliferation of PC-3 cells without affecting LPS activation of the NFĸB pathway via toll-like receptor-4 (TLR-4). Additionally, Prostasan® reduced the constitutive secretion of monocyte chemotactic protein-1 (MCP-1), the LPS-induced secretion of IL-12 and inhibited MCP-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in the presence of LPS on PC-3 cells. Taken together, our results suggest that S. repens extracts, in addition to other reported effects on BPH development and prostatitis, inhibits EGF-dependent growth and proinflammatory responses of the prostate epithelial cells.

The SOCS2 ubiquitin ligase complex regulates growth hormone receptor levels.

Growth Hormone is essential for the regulation of growth and the homeostatic control of intermediary metabolism. GH actions are mediated by the Growth Hormone Receptor; a member of the cytokine receptor super family that signals chiefly through the JAK2/STAT5 pathway. Target tissue responsiveness to GH is under regulatory control to avoid excessive and off-target effects upon GHR activation. The suppressor of cytokine signalling 2 (SOCS) is a key regulator of GHR sensitivity. This is clearly shown in mice where the SOCS2 gene has been inactivated, which show 30-40% increase in body length, a phenotype that is dependent on endogenous GH secretion. SOCS2 is a GH-stimulated, STAT5b-regulated gene that acts in a negative feedback loop to downregulate GHR signalling. Since the biochemical basis for these actions is poorly understood, we studied the molecular function of SOCS2. We demonstrated that SOCS2 is part of a multimeric complex with intrinsic ubiquitin ligase activity. Mutational analysis shows that the interaction with Elongin B/C controls SOCS2 protein turnover and affects its molecular activity. Increased GHR levels were observed in livers from SOCS2⁻/⁻ mice and in the absence of SOCS2 in in vitro experiments. We showed that SOCS2 regulates cellular GHR levels through direct ubiquitination and in a proteasomally dependent manner. We also confirmed the importance of the SOCS-box for the proper function of SOCS2. Finally, we identified two phosphotyrosine residues in the GHR to be responsible for the interaction with SOCS2, but only Y487 to account for the effects of SOCS2. The demonstration that SOCS2 is an ubiquitin ligase for the GHR unveils the molecular basis for its physiological actions.