Cystic Fibrosis Transmembrane Conductance Regulator Attaches Tumor Suppressor PTEN to the Membrane and Promotes Anti Pseudomonas aeruginosa Immunity.

The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl-/- mice, which lack the NH2-amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy.

The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma.

Insights into cancer genetics can lead to therapeutic opportunities. By cross-referencing chromosomal changes with an unbiased genetic screen we identify the ephrin receptor A7 (EPHA7) as a tumor suppressor in follicular lymphoma (FL). EPHA7 is a target of 6q deletions and inactivated in 72% of FLs. Knockdown of EPHA7 drives lymphoma development in a murine FL model. In analogy to its physiological function in brain development, a soluble splice variant of EPHA7 (EPHA7(TR)) interferes with another Eph-receptor and blocks oncogenic signals in lymphoma cells. Consistent with this drug-like activity, administration of the purified EPHA7(TR) protein produces antitumor effects against xenografted human lymphomas. Further, by fusing EPHA7(TR) to the anti-CD20 antibody (rituximab) we can directly target this tumor suppressor to lymphomas in vivo. Our study attests to the power of combining descriptive tumor genomics with functional screens and reveals EPHA7(TR) as tumor suppressor with immediate therapeutic potential.

Cadherin-17 as a target for the immunoPET of adenocarcinoma.

PURPOSE: Cadherin-17 (CDH17) is a calcium-dependent cell adhesion protein that is overexpressed in several adenocarcinomas, including gastric, colorectal, and pancreatic adenocarcinoma. High levels of CDH17 have been linked to metastatic disease and poor prognoses in patients with these malignancies, fueling interest in the protein as a target for diagnostics and therapeutics. Herein, we report the synthesis, in vitro validation, and in vivo evaluation of a CDH17-targeted 89Zr-labeled immunoPET probe. METHODS: The CDH17-targeting mAb D2101 was modified with an isothiocyanate-bearing derivative of desferrioxamine (DFO) to produce a chelator-bearing immunoconjugate - DFO-D2101 - and flow cytometry and surface plasmon resonance (SPR) were used to interrogate its antigen-binding properties. The immunoconjugate was then radiolabeled with zirconium-89 (t1/2 ~ 3.3 days), and the serum stability and immunoreactive fraction of [89Zr]Zr-DFO-D2101 were determined. Finally, [89Zr]Zr-DFO-D2101's performance was evaluated in a trio of murine models of pancreatic ductal adenocarcinoma (PDAC): subcutaneous, orthotopic, and patient-derived xenografts (PDX). PET images were acquired over the course of 5 days, and terminal biodistribution data were collected after the final imaging time point. RESULTS: DFO-D2101 was produced with a degree of labeling of ~ 1.1 DFO/mAb. Flow cytometry with CDH17-expressing AsPC-1 cells demonstrated that the immunoconjugate binds to its target in a manner similar to its parent mAb, while SPR with recombinant CDH17 revealed that D2101 and DFO-D2101 exhibit nearly identical KD values: 8.2 × 10-9 and 6.7 × 10-9 M, respectively. [89Zr]Zr-DFO-D2101 was produced with a specific activity of 185 MBq/mg (5.0 mCi/mg), remained >80% stable in human serum over the course of 5 days, and boasted an immunoreactive fraction of >0.85. In all three murine models of PDAC, the radioimmunoconjugate yielded high contrast images, with high activity concentrations in tumor tissue and low uptake in non-target organs. Tumoral activity concentrations reached as high as >60 %ID/g in two of the cohorts bearing PDXs. CONCLUSION: Taken together, these data underscore that [89Zr]Zr-DFO-D2101 is a highly promising probe for the non-invasive visualization of CDH17 expression in PDAC. We contend that this radioimmunoconjugate could have a significant impact on the clinical management of patients with both PDAC and gastrointestinal adenocarcinoma, most likely as a theranostic imaging tool in support of CDH17-targeted therapies.

UDP-glucose pyrophosphorylase 2, a regulator of glycogen synthesis and glycosylation, is critical for pancreatic cancer growth.

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs.

Androgen-induced insulin resistance is ameliorated by deletion of hepatic androgen receptor in females.

Androgen excess is one of the most common endocrine disorders of reproductive-aged women, affecting up to 20% of this population. Women with elevated androgens often exhibit hyperinsulinemia and insulin resistance. The mechanisms of how elevated androgens affect metabolic function are not clear. Hyperandrogenemia in a dihydrotestosterone (DHT)-treated female mouse model induces whole body insulin resistance possibly through activation of the hepatic androgen receptor (AR). We investigated the role of hepatocyte AR in hyperandrogenemia-induced metabolic dysfunction by using several approaches to delete hepatic AR via animal-, cell-, and clinical-based methodologies. We conditionally disrupted hepatocyte AR in female mice developmentally (LivARKO) or acutely by tail vein injection of an adeno-associated virus with a liver-specific promoter for Cre expression in ARfl/fl mice (adLivARKO). We observed normal metabolic function in littermate female Control (ARfl/fl ) and LivARKO (ARfl/fl ; Cre+/- ) mice. Following chronic DHT treatment, female Control mice treated with DHT (Con-DHT) developed impaired glucose tolerance, pyruvate tolerance, and insulin tolerance, not observed in LivARKO mice treated with DHT (LivARKO-DHT). Furthermore, during an euglycemic hyperinsulinemic clamp, the glucose infusion rate was improved in LivARKO-DHT mice compared to Con-DHT mice. Liver from LivARKO, and primary hepatocytes derived from LivARKO, and adLivARKO mice were protected from DHT-induced insulin resistance and increased gluconeogenesis. These data support a paradigm in which elevated androgens in females disrupt metabolic function via hepatic AR and insulin sensitivity was restored by deletion of hepatic AR.

Deficiency of arcuate nucleus kisspeptin results in postpubertal central hypogonadism.

Kisspeptin (encoded by Kiss1), a neuropeptide critically involved in neuroendocrine regulation of reproduction, is primarily synthesized in two hypothalamic nuclei: the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). AVPV kisspeptin is thought to regulate the estrogen-induced positive feedback control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), and the preovulatory LH surge in females. In contrast, ARC kisspeptin neurons, which largely coexpress neurokinin B and dynorphin A (collectively named KNDy neurons), are thought to mediate estrogen-induced negative feedback control of GnRH/LH and be the major regulators of pulsatile GnRH/LH release. However, definitive data to delineate the specific roles of AVPV versus ARC kisspeptin neurons in the control of GnRH/LH release is lacking. Therefore, we generated a novel mouse model targeting deletion of Kiss1 to the ARC nucleus (Pdyn-Cre/Kiss1fl/fl KO) to determine the functional differences between ARC and AVPV kisspeptin neurons on the reproductive axis. The efficacy of the knockout was confirmed at both the mRNA and protein levels. Adult female Pdyn-Cre/Kiss1fl/fl KO mice exhibited persistent diestrus and significantly fewer LH pulses when compared with controls, resulting in arrested folliculogenesis, hypogonadism, and infertility. Pdyn-Cre/Kiss1fl/fl KO males also exhibited disrupted LH pulsatility, hypogonadism, and variable, defective spermatogenesis, and subfertility. The timing of pubertal onset in males and females was equivalent to controls. These findings add to the current body of evidence for the critical role of kisspeptin in ARC KNDy neurons in GnRH/LH pulsatility in both sexes, while directly establishing ARC kisspeptin's role in regulating estrous cyclicity in female mice, and gametogenesis in both sexes, and culminating in disrupted fertility. The Pdyn-Cre/Kiss1fl/fl KO mice present a novel mammalian model of postpubertal central hypogonadism.NEW & NOTEWORTHY We demonstrate through a novel, conditional knockout mouse model of arcuate nucleus (ARC)-specific kisspeptin in the KNDy neuron that ARC kisspeptin is critical for estrous cyclicity in female mice and GnRH/LH pulsatility in both sexes. Our study reveals that ARC kisspeptin is essential for normal gametogenesis, and the loss of ARC kisspeptin results in significant hypogonadism, impacting fertility status. Our findings further confirm that normal puberty occurs despite a loss of ARC kisspeptin.

Conditional knockout of kisspeptin signaling in brown adipose tissue increases metabolic rate and body temperature and lowers body weight.

The peptide kisspeptin and its receptor, Kiss1r, act centrally to stimulate reproduction. Evidence indicates that kisspeptin signaling is also important for body weight (BW) and metabolism. We recently reported that Kiss1r KO mice develop obesity, along with reduced metabolism and energy expenditure, independent of estradiol levels. Outside the brain, Kiss1r is expressed in several metabolic tissues, including brown adipose tissue (BAT), but it is unknown which specific tissue is responsible for the metabolic phenotype in Kiss1r KOs. We first determined that global Kiss1r KO mice have significant alterations in body temperature and BAT thermogenic gene expression, perhaps contributing to their obesity. Next, to test whether kisspeptin signaling specifically in BAT influences BW, metabolism, or body temperature, we used Cre/lox technology to generate conditional Kiss1r knockout exclusively in BAT (BAT-Kiss1r KO). Unlike global Kiss1r KOs, BAT-Kiss1r KOs (lacking Kiss1r in just BAT) were not hypogonadal, as expected. Surprisingly, however, BAT-Kiss1r KOs of both sexes displayed significantly lower BW and adiposity than controls. This novel BAT-Kiss1r KO phenotype was of greater magnitude in females and was associated with improved glucose tolerance, increased metabolism, energy expenditure, and locomotor activity, along with increased body temperature and BAT gene expression, specifically Cox8b. Our findings suggest that the previously observed obesity and decreased metabolism in global Kiss1r KOs reflect impaired kisspeptin signaling in non-BAT tissues. However, the novel finding of increased metabolism and body temperature and lower BW in BAT-Kiss1r KOs reveal a previously unidentified role for endogenous kisspeptin signaling in BAT in modulating metabolic and thermogenic physiology.

Late-onset renal hypertrophy and dysfunction in mice lacking CTRP1.

Local and systemic factors that influence renal structure and function in aging are not well understood. The secretory protein C1q/TNF-related protein 1 (CTRP1) regulates systemic metabolism and cardiovascular function. We provide evidence here that CTRP1 also modulates renal physiology in an age- and sex-dependent manner. In mice lacking CTRP1, we observed significantly increased kidney weight and glomerular hypertrophy in aged male but not female or young mice. Although glomerular filtration rate, plasma renin and aldosterone levels, and renal response to water restriction did not differ between genotypes, CTRP1-deficient male mice had elevated blood pressure. Echocardiogram and pulse wave velocity measurements indicated normal heart function and vascular stiffness in CTRP1-deficient animals, and increased blood pressure was not due to greater salt retention. Paradoxically, CTRP1-deficient mice had elevated urinary sodium and potassium excretion, partially resulting from reduced expression of genes involved in renal sodium and potassium reabsorption. Despite renal hypertrophy, markers of inflammation, fibrosis, and oxidative stress were reduced in CTRP1-deficient mice. RNA sequencing revealed alterations and enrichments of genes in metabolic processes in CTRP1-deficient animals. These results highlight novel contributions of CTRP1 to aging-associated changes in renal physiology.

Tanycyte ablation in the arcuate nucleus and median eminence increases obesity susceptibility by increasing body fat content in male mice.

Tanycytes are radial glial cells located in the mediobasal hypothalamus. Recent studies have proposed that tanycytes play an important role in hypothalamic control of energy homeostasis, although this has not been directly tested. Here, we report the phenotype of mice in which tanycytes of the arcuate nucleus and median eminence were conditionally ablated in adult mice. Although the cerebrospinal fluid-hypothalamic barrier was rendered more permeable following tanycyte ablation, neither the blood-hypothalamic barrier nor leptin-induced pSTAT3 activation in hypothalamic parenchyma were affected. We observed a significant increase in visceral fat distribution accompanying insulin insensitivity in male mice, without significant effect on either body weight or food intake. A high-fat diet tended to accelerate overall body weight gain in tanycyte-ablated mice, but the development of visceral adiposity and insulin insensitivity was comparable to wildtype. Thermoneutral housing exacerbated fat accumulation and produced a shift away from fat oxidation in tanycyte-ablated mice. These results clarify the extent to which tanycytes regulate energy balance, and demonstrate a role for tanycytes in regulating fat metabolism.

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

Multiplex Quantification Identifies Novel Exercise-regulated Myokines/Cytokines in Plasma and in Glycolytic and Oxidative Skeletal Muscle.

Exercise is known to confer major health benefits, but the underlying mechanisms are not well understood. The systemic effects of exercise on multi-organ systems are thought to be partly because of myokines/cytokines secreted by skeletal muscle. The extent to which exercise alters cytokine expression and secretion in different muscle fiber types has not been systematically examined. Here, we assessed changes in 66 mouse cytokines in serum, and in glycolytic (plantaris) and oxidative (soleus) muscles, in response to sprint, endurance, or chronic wheel running. Both acute and short-term exercise significantly altered a large fraction of cytokines in both serum and muscle, twenty-three of which are considered novel exercise-regulated myokines. Most of the secreted cytokine receptors profiled were also altered by physical activity, suggesting an exercise-regulated mechanism that modulates the generation of soluble receptors found in circulation. A greater overlap in cytokine profile was seen between endurance and chronic wheel running. Between fiber types, both acute and chronic exercise induced significantly more cytokine changes in oxidative compared with glycolytic muscle. Further, changes in a subset of circulating cytokines were not matched by their changes in muscle, but instead reflected altered expression in liver and adipose tissues. Last, exercise-induced changes in cytokine mRNA and protein were only minimally correlated in soleus and plantaris. In sum, our results indicate that exercise regulates many cytokines whose pleiotropic actions may be linked to positive health outcomes. These data provide a framework to further understand potential crosstalk between skeletal muscle and other organ compartments.

Helping Scholars Overcome Socioeconomic Barriers to Medical and Biomedical Careers: Creating a Pipeline Initiative.

Problem: To achieve their potential in medical and biomedical careers, students (scholars) from under-resourced backgrounds must build sophisticated skills and develop confidence and professionalism. To flourish in an advanced educational system that may be unfamiliar, these scholars also need networks of mentors and role models. These challenges can affect scholars at multiple stages of their education. Intervention: To meet these challenges, we created a broad and innovative biomedical research-focused pipeline program: the Johns Hopkins Initiative for Careers in Science in Medicine (CSM Initiative). This initiative targets three levels: high school, undergraduate, and post-baccalaureate/pre-doctoral (graduate and medical). We provide training in essential academic, research, professional, and social skills to meet the unique challenges of our scholars from under-resourced backgrounds. Scholars also build relationships with mentors who provide career guidance and support. We present an overview of the training and assessment at each level of this initiative. Context: The initiative took place at an institution located in the greater Baltimore area and that is endowed with exceptional doctoral and postdoctoral trainees, staff, and faculty including clinicians, physician-scientists, and scientists who served as key role models and mentors. Our pipeline program draws from local high school students and a local and national pool of undergraduates and post-baccalaureates preparing for medical or graduate school. Impact: Our goals for the high school scholars are significant improvement in academic skills, increased confidence, and matriculation into higher education systems. Currently, at least 83% of high school scholars have matriculated into four-year college programs and 73% have chosen science, technology, engineering, math, and medicine (STEMM)-related majors. Among undergraduate participants, 42% have matriculated thus far into medical or biomedical graduate programs and this number is expected to rise as more scholars graduate from college and either enter graduate training or pursue STEMM careers. Another 25% have returned to our post-baccalaureate program. Among post-baccalaureate scholars, 71% have now matriculated into doctoral-level graduate biomedical programs (medical or graduate school) and the remaining 29% are pursuing careers in STEMM-related fields such as biomedical research with some still aiming at graduate-level education. Our long-term goal is to see a large majority of our scholars become successful professionals in medicine, biomedical research, allied healthcare, or other STEMM fields. Analysis of the early phases of the CSM initiative demonstrates such outcomes are attainable. Lessons Learned: This program provides experiences in which scholars develop and practice core competencies essential for developing their self-identity as scientists and professionals. The most important lesson learned is that mentorship teams must be highly dynamic, flexible, thoughtful, and personal in responding to the wide range of challenges and obstacles that scholars from under-resourced backgrounds must overcome to achieve career success.

Impairments in the reproductive axis of female mice lacking estrogen receptor ß in GnRH neurons.

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ERα, and ERß. Lack of functional ERα and ERß genes in vivo significantly affects reproductive function; however, the target tissues and signaling pathways in the hypothalamus are not clearly defined. Here, we describe the generation and reproductive characterization of a complete-ERß KO (CERßKO) and a GnRH neuron-specific ERßKO (GERßKO) mouse models. Both ERßKO mouse models displayed a delay in vaginal opening and first estrus. Hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression levels in both ERßKO mice were similar to control mice; however female CERßKO and GERßKO mice had lower basal and surge serum gonadotropin levels. Although a GnRH stimulation test in both female ERßKO models showed preserved gonadotropic function in the same animals, a kisspeptin stimulation test revealed an attenuated response by GnRH neurons, suggesting a role for ERß in normal GnRH neuron function. No alteration in estrogen-negative feedback was observed in either ERßKO mouse models after ovariectomy and estrogen replacement. Further, abnormal development of ovarian follicles with low serum estradiol levels and impairment of fertility were observed in both ERßKO mouse models. In male ERßKO mice, no differences in the timing of pubertal onset or serum luteinizing hormone and follicle-stimulating hormone levels were observed as compared with controls. Taken together, these data provide in vivo evidence for a role of ERß in GnRH neurons in modulating puberty and reproduction, specifically through kisspeptin responsiveness in the female hypothalamic-pituitary-gonadal axis.

The regulation of reproductive neuroendocrine function by insulin and insulin-like growth factor-1 (IGF-1).

The mammalian reproductive hormone axis regulates gonadal steroid hormone levels and gonadal function essential for reproduction. The neuroendocrine control of the axis integrates signals from a wide array of inputs. The regulatory pathways important for mediating these inputs have been the subject of numerous studies. One class of proteins that have been shown to mediate metabolic and growth signals to the CNS includes Insulin and IGF-1. These proteins are structurally related and can exert endocrine and growth factor like action via related receptor tyrosine kinases. The role that insulin and IGF-1 play in controlling the hypothalamus and pituitary and their role in regulating puberty and nutritional control of reproduction has been studied extensively. This review summarizes the in vitro and in vivo models that have been used to study these neuroendocrine structures and the influence of these growth factors on neuroendocrine control of reproduction.

Dynamic and extensive metabolic state-dependent regulation of cytokine expression and circulating levels.

Cytokines play diverse and critical roles in innate and acquired immunity, and several function within the central nervous system and in peripheral tissues to modulate energy metabolism. The extent to which changes in energy balance impact the expression and circulating levels of cytokines (many of which have pleiotropic functions) has not been systematically examined. To investigate metabolism-related changes in cytokine profiles, we used a multiplex approach to assess changes in 71 circulating mouse cytokines in response to acute (fasting and refeeding) and chronic (high-fat feeding) alterations in whole body metabolism. Refeeding significantly decreased serum levels of IL-22, IL-1α, soluble (s)IL-2Rα, and soluble vascular endothelial growth factor receptor 3 (VEGFR3), but markedly increased granulocyte colony-stimulating factor (G-CSF), IL-1ß, chemokine (C-C motif) ligand (CCL2), sIL-1RI, lipocalin-2, pentraxin-3, tissue inhibitor of metalloproteinase (TIMP-1), and serum amyloid protein (SAP) relative to the fasted state. Interestingly, only a few of these changes paralleled the alterations in expression of their corresponding mRNAs. Functional studies demonstrated that central delivery of G-CSF increased, whereas IL-22 decreased, food intake. Changes in food intake were not accompanied by acute alterations in orexigenic (Npy and Agrp) and anorexigenic (Pomc and Cart) neuropeptide gene expression in the hypothalamus. In the context of chronic high-fat feeding, circulating levels of chemokine (C-X-C) ligand (CXCL1), serum amyloid protein A3 (SAA3), TIMP-1, α1-acid glycoprotein (AGP), and A2M were increased, whereas IL-12p40, CCL4, sCD30, soluble receptor for advanced glycation end products (sRAGE), CCL12, CCL20, CX3CL1, IL-16, IL-22, and haptoglobin were decreased relative to mice fed a control low-fat diet. These results demonstrate that both short- and long-term changes in whole body metabolism extensively alter cytokine expression and circulating levels, thus providing a foundation and framework for further investigations to ascertain the metabolic roles for these molecules in physiological and pathological states.

KRAS: Biology, Inhibition, and Mechanisms of Inhibitor Resistance.

KRAS is a small GTPase that is among the most commonly mutated oncogenes in cancer. Here, we discuss KRAS biology, therapeutic avenues to target it, and mechanisms of resistance that tumors employ in response to KRAS inhibition. Several strategies are under investigation for inhibiting oncogenic KRAS, including small molecule compounds targeting specific KRAS mutations, pan-KRAS inhibitors, PROTACs, siRNAs, PNAs, and mutant KRAS-specific immunostimulatory strategies. A central challenge to therapeutic effectiveness is the frequent development of resistance to these treatments. Direct resistance mechanisms can involve KRAS mutations that reduce drug efficacy or copy number alterations that increase the expression of mutant KRAS. Indirect resistance mechanisms arise from mutations that can rescue mutant KRAS-dependent cells either by reactivating the same signaling or via alternative pathways. Further, non-mutational forms of resistance can take the form of epigenetic marks, transcriptional reprogramming, or alterations within the tumor microenvironment. As the possible strategies to inhibit KRAS expand, understanding the nuances of resistance mechanisms is paramount to the development of both enhanced therapeutics and innovative drug combinations.

Loss of epithelial oestrogen receptor α inhibits oestrogen-stimulated prostate proliferation and squamous metaplasia via in vivo tissue selective knockout models.

Squamous metaplasia (SQM) is a specific phenotype in response to oestrogen in the prostate and oestrogen receptor (ER) α is required to mediate this response. Previous studies utilizing tissue recombination with seminal vesicle (SV) mesenchyme and prostatic ductal tips from wild type and ERαKO mice suggested that both epithelial and stromal ERα are necessary for SQM. However, tissue recombination is conducted in the renal capsule of immune-deficient mice, in which the microenvironment is different from normal prostate microenvironment in the intact mice. Furthermore, whether the requirement of stromal ERα in the SV for developing SQM is the same as in the prostate is unknown. Therefore, there is a clear need to evaluate the respective roles of ERα in prostate epithelial versus stromal compartments in the intact mouse. Here we generated a mouse model that has selectively lost ERα in either stromal (FSP-ERαKO) or epithelial prostate cells (pes-ERαKO) to determine the requirements of ERα for oestrogen-stimulated prostate proliferation and SQM. Our results indicated that FSP-ERαKO prostates develop full and uniform SQM, which suggests that loss of the majority (~65%) of stromal ERα will not influence oestrogen-mediated SQM. In contrast, loss of epithelial ERα inhibits oestrogen-mediated prostate growth and SQM evidenced by decreasing cytokertin 10 positive squamous cell stratification and differentiation, by reduced ERα protein expression in SQM compared to wild type mice ERα, and by the presence of normal proliferative activities in the oestrogen-treated pes-ERαKO prostates. These in vivo results suggest that epithelial ERα is required for oestrogen-mediated proliferative response and could be an appropriate target for preventing aberrant oestrogen signalling in the prostate.

Timing and completion of puberty in female mice depend on estrogen receptor alpha-signaling in kisspeptin neurons.

Puberty onset is initiated by activation of neurons that secrete gonadotropin-releasing hormone (GnRH). The timing and progression of puberty may depend upon temporal coordination of two opposing central mechanisms--a restraint of GnRH secretion before puberty onset, followed by enhanced stimulation of GnRH release to complete reproductive maturation during puberty. Neuronal estrogen receptor α (ERα) has been implicated in both controls; however, the underlying neural circuits are not well understood. Here we test whether these mechanisms are mediated by neurons that express kisspeptin, a neuropeptide that modulates GnRH neurosecretion. Strikingly, conditional ablation of ERα in kisspeptin neurons results in a dramatic advancement of puberty onset in female mice. Furthermore, subsequent pubertal maturation is arrested in these animals, as they fail to acquire normal ovulatory cyclicity. We show that the temporal coordination of juvenile restraint and subsequent pubertal activation is likely mediated by ERα in two separate kisspeptin neuronal populations in the hypothalamus.

The gonadotropin-releasing hormone cell-specific element is required for normal puberty and estrous cyclicity.

Appropriate tissue-specific gene expression of gonadotropin-releasing hormone (GnRH) is critical for pubertal development and maintenance of reproductive competence. In these studies, a common element in the mouse GnRH (mGnRH) promoter, between -2806 and -2078 bp, is shown to mediate differential regulation of hypothalamic and ovarian mGnRH expression. To further characterize this region, we generated a knock-out mouse (GREKO(-/-)) with a deletion of the mGnRH promoter fragment between -2806 and -2078 bp. GnRH mRNA expression in the brain of GREKO(-/-) was less than the expression in wild-type mice; however, immunohistochemical analysis revealed no difference between the numbers of GnRH neurons among groups. GnRH mRNA expression in the ovary was fivefold higher in GREKO(-/-). The immunohistochemical staining for GnRH in the ovary increased in surface epithelial and granulosa cells and also in the corpora lutea of GREKO(-/-) mice. The reproductive phenotype revealed that the mean day of vaginal opening was delayed, and additionally, there was a significant decrease in the length of proestrus and diestrus-metestrus phases of the estrous cycle, resulting in a shortened estrous cycle in GREKO(-/-) mice. This work supports the hypothesis that the region of the GnRH promoter contained between -2806 and -2078 bp acts as a cell-specific enhancer in the GnRH neuron and as a repressor in the ovary. Deletion of this region in vivo implicates the GnRH promoter in mediating pubertal development and periodic reproductive cycling, and forms the foundation to define the nuclear proteins important for puberty and estrous cycling in mammals.

Jak2 is necessary for neuroendocrine control of female reproduction.

Gonadotropin-releasing hormone (GnRH) neurons represent the final common output of signals from the brain that regulates reproductive function. A wide range of environmental factors impact GnRH neuron activity including disease, stress, nutrition, and seasonal cues, as well as gonadal steroid hormones. The CNS response is thought to be mediated, at least in part, through intermediate signaling molecules that affect GnRH neuronal activity. In vitro, GnRH neuronal cell lines respond to a variety of ligands that activate the Jak (Janus-activated kinase)/STAT (signal transducers and activators of transcription) intracellular signaling pathway. To determine its biological function in reproduction, we used Cre (cAMP response element)/LoxP technology to generate GnRH neuron-specific Jak2 conditional knock-out (Jak2 G(-/-)) mice. GnRH mRNA levels were reduced in Jak2 G(-/-) mice when compared with controls, while the number of GnRH neurons was equivalent, indicating a reduction in GnRH gene expression. Secretion of GnRH is also reduced as basal serum luteinizing hormone (LH) levels were significantly lower in female Jak2 G(-/-) mice while the pituitary responded normally to exogenous GnRH. Preovulatory LH surge levels were blunted in Jak2 G(-/-) mice, which was correlated with reduced GnRH neuronal activation as assessed by c-Fos. However, the activation of GnRH neurons following release from estrogen-negative feedback is retained. Female Jak2 G(-/-) mice exhibited significantly delayed puberty and first estrus, abnormal estrous cyclicity, and impaired fertility. These results demonstrate an essential role for Jak2 signaling in GnRH neurons for normal reproductive development and fertility in female mice.