Conditions associated with increased growth hormone and prolactin sensitivity

Growth Hormone (GH) and Prolactin (PRL) are critical regulators of body growth and metabolism. Secretion and actions of GH and PRL are regulated at several levels and by different factors. The biological actions of these hormones are initiated by their binding to the respective membrane bound receptors of GH and PRL (GHR and PRLR). Several hormone systems are characterized by changes in target tissue sensitivity. Key factors in hormone sensitivity include the number of particular receptors and the duration of receptor activated intracellular signals. A common theme concerning this is e.g. that tyrosine phosphorylated intracellular proteins become inactivated by tyrosine phosphatases or by proteasomal breakdown. In this thesis a particular focus is put on two different proteins, Suppressors of Cytokine Signaling2 (SOCS2) and Tuberous Sclerosis Complex2 (TSC2) that uniquely impinge on JAK-STAT activation and on mTOR activation. Study I. We explored the influence of SOCS2 on glucose metabolism by using a mouse model of diabetes induced by multiple low dose streptozotocin (MLDSTZ). Pancreatic islets from untreated SOCS2-/- mice appeared larger than in wild-type (WT) controls, which could explain the augmented serum insulin levels observed in SOCS2-/- mice. Pancreatic islets, derived from SOCS2-/- mice showed increased GHR and PRLR staining, which suggest a higher sensitivity to GH/PRL-STAT5 signals in SOCS2-/- than in WT-derived β-cells. Our results suggest that SOCS2 ablation can partly overcome β-cell destruction induced by MLDSTZ. Study II and III. In these studies we aimed to investigate the relevance of PRL in two different human tumors, i.e. lymphangioleiomyomatosis (LAM) and glioblastoma (GBM), by analyzing features of the PRLR and the effect of a novel PRLR antagonist (PRLRA) in such tumors. Reduction of TSC2 (the disease causing gene in LAM) increased PRLR levels in LAM cells and PRL stimulated LAM cell proliferation; an effect that could be blocked with the PRLRA. In GBM, PRLR was detectable in cultured GBM cells as well as in tissue sections from patients with GBM. In cell culture GBM studies, PRL treatment increased STAT5 phosphorylation as well as cell invasion and both effects could be blocked by the PRLRA. In summary, our studies indicate that the tissue sensitivity to GH/PRL is regulated by SOCS2 and TSC2 proteins. Since both SOCS2 and TSC2 have links to different disorders, an increased GH/PRL sensitivity in such conditions could play a functional role. To block an increased PRL sensitivity we have developed a novel PRLRA and demonstrated its efficacy in cell cultures

The ubiquitin ligase Cullin5^SOCS2 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

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

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 β-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 β-cell survival may be of relevance to islet regeneration and survival in transplantation.The Spanish Ministry of Science and Innovation, with the European Regional Development Fund, supported this research by grants-in-aid to L.F.-P. (SAF2012– 37344). L.F.-P. was also supported by grants-in-aid from ACIISI (PI2010/0110) and Alfredo Martin-Reyes Foundation (Arehucas)-FICIC. The Danish Council for Independent Research and the Novo Nordisk Foundation supports A.F-M. G.N. is supported by a grant from Swedish Heart and Lung Foundation. A.A is supported by a fellowship from Sultan Qaboos University, Muscat, Oman. M.M-G. is a recipient of predoctoral fellowship from ULPGCMEC (AP2001–3499) and Fundación Universitaria de Las Palmas (INNOVA).Peer Reviewe

Addition of exogenous Prl stimulates cell proliferation in a manner which can be blocked by PrlRA.

<p>LAM/TSC cells were cultured in different concentrations of serum (0%, 2%, 10%). Human Prl was added to sub-confluent cells at a concentration of 200 ng/ml, with or without PrlRA (20, 50, 200 ng/ml). After 72 hours, cells were subject to the crystal violet assay. The X-axis depicts serum concentration and the Y-axis shows relative cell proliferation, as determined by absorbance at 600 nm. Each data point represents a triplicate assay. All values are +/- standard deviation, P-value <0.05.</p

Human LAM cells express PrlR, and prolactin stimulates phosphorylation of STAT3 and Erk.

<p>LAM/TSC cells were cultured in serum-free medium overnight. The cells were then exposed to different doses of PrlRA (20, 50, 200 ng/ml) for 15 minutes, and subsequently the cells were exposed to 200 ng/ml Prl for 60 minutes or PBS as a control. Then protein extracts were prepared for Western blot analysis, and probed with antibodies for P-STAT3, STAT3, P-Erk and Erk. (A) Western blot to analyze PrlR in LAM/TSC control cells; an antibody against human PrlR detected a protein band of 89–90 kD. (B) Western blot, using antibodies directed against P-STAT3, STAT3, P-Erk and Erk in LAM/TSC cells following treatment as described in Material and methods section. (C) Densitometric quantification of Western blot signals, in which the Y-axis depicts the ratio between phosphorylated STAT3 and Erk to the total protein.</p

Immunofluorescence of CRL-2620 using PrlR and GHR antibodies.

<p>Cells were transfected with TSC2 or control siRNAs. After over-night incubation, the cells were washed 3 times with PBS and incubated with antibodies for PrlR and GHR. The figures show that PrlR immunostaining was markedly increased following TSC2 knock-down. A strong immunofluorescence signal was observed at intracellular locations. In the negative control panel, cells were incubated with mouse IgG and rabbit IgG antibodies. Fluorescence images were acquired at a magnification of 63x.</p

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&rsquo;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

Detection of PrlR in human LAM cells using immunofluorescence.

<p>Staining LAM/TSC cells using different PrlR antibodies detect a significant immunofluorescence signal in LAM/TSC cells. The antibodies employed were U5 (Thermoscientific) and A12B1 (Invitrogen).</p