Kisspeptin regulation of genes involved in cell invasion and angiogenesis in first trimester human trophoblast cells

The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A

Normosmic Congenital Hypogonadotropic Hypogonadism Due to TAC3/TACR3 Mutations: Characterization of Neuroendocrine Phenotypes and Novel Mutations

CONTEXT: TAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway. OBJECTIVE: To evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations. RESULTS: From a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%). We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants) found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn) probably caused by the misfolding of the mutated NK3R protein. We found a statistically significant (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio. CONCLUSION: The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations

GPR54 (KISS1R) Transactivates EGFR to Promote Breast Cancer Cell Invasiveness

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness

Pathogenesis of adolescent idiopathic scoliosis in girls - a double neuro-osseous theory involving disharmony between two nervous systems, somatic and autonomic expressed in the spine and trunk: possible dependency on sympathetic nervous system and hormones with implications for medical therapy

Anthropometric data from three groups of adolescent girls - preoperative adolescent idiopathic scoliosis (AIS), screened for scoliosis and normals were analysed by comparing skeletal data between higher and lower body mass index subsets. Unexpected findings for each of skeletal maturation, asymmetries and overgrowth are not explained by prevailing theories of AIS pathogenesis. A speculative pathogenetic theory for girls is formulated after surveying evidence including: (1) the thoracospinal concept for right thoracic AIS in girls; (2) the new neuroskeletal biology relating the sympathetic nervous system to bone formation/resorption and bone growth; (3) white adipose tissue storing triglycerides and the adiposity hormone leptin which functions as satiety hormone and sentinel of energy balance to the hypothalamus for long-term adiposity; and (4) central leptin resistance in obesity and possibly in healthy females. The new theory states that AIS in girls results from developmental disharmony expressed in spine and trunk between autonomic and somatic nervous systems. The autonomic component of this double neuro-osseous theory for AIS pathogenesis in girls involves selectively increased sensitivity of the hypothalamus to circulating leptin (genetically-determined up-regulation possibly involving inhibitory or sensitizing intracellular molecules, such as SOC3, PTP-1B and SH2B1 respectively), with asymmetry as an adverse response (hormesis); this asymmetry is routed bilaterally via the sympathetic nervous system to the growing axial skeleton where it may initiate the scoliosis deformity (leptin-hypothalamic-sympathetic nervous system concept = LHS concept). In some younger preoperative AIS girls, the hypothalamic up-regulation to circulating leptin also involves the somatotropic (growth hormone/IGF) axis which exaggerates the sympathetically-induced asymmetric skeletal effects and contributes to curve progression, a concept with therapeutic implications. In the somatic nervous system, dysfunction of a postural mechanism involving the CNS body schema fails to control, or may induce, the spinal deformity of AIS in girls (escalator concept). Biomechanical factors affecting ribs and/or vertebrae and spinal cord during growth may localize AIS to the thoracic spine and contribute to sagittal spinal shape alterations. The developmental disharmony in spine and trunk is compounded by any osteopenia, biomechanical spinal growth modulation, disc degeneration and platelet calmodulin dysfunction. Methods for testing the theory are outlined. Implications are discussed for neuroendocrine dysfunctions, osteopontin, sympathoactivation, medical therapy, Rett and Prader-Willi syndromes, infantile idiopathic scoliosis, and human evolution. AIS pathogenesis in girls is predicated on two putative normal mechanisms involved in trunk growth, each acquired in evolution and unique to humans

Kisspeptin signaling is required for the luteinizing hormone response in anestrous ewes following the introduction of males

The introduction of a novel male stimulates the hypothalamic-pituitary-gonadal axis of female sheep during seasonal anestrus, leading to the resumption of follicle maturation and ovulation. How this pheromone cue activates pulsatile secretion of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) is unknown. We hypothesised that pheromones activate kisspeptin neurons, the product of which is critical for the stimulation of GnRH neurons and fertility. During the non-breeding season, female sheep were exposed to novel males and blood samples collected for analysis of plasma LH profiles. Females without exposure to males served as controls. In addition, one hour before male exposure, a kisspeptin antagonist (P-271) or vehicle was infused into the lateral ventricle and continued for the entire period of male exposure. Introduction of a male led to elevated mean LH levels, due to increased LH pulse amplitude and pulse frequency in females, when compared to females not exposed to a male. Infusion of P-271 abolished this effect of male exposure. Brains were collected after the male effect stimulus and we observed an increase in the percentage of kisspeptin neurons co-expressing Fos, by immunohistochemistry. In addition, the per-cell expression of Kiss1 mRNA was increased in the rostral and mid (but not the caudal) arcuate nucleus (ARC) after male exposure in both aCSF and P-271 treated ewes, but the per-cell content of neurokinin B mRNA was decreased. There was also a generalized increase in Fos positive cells in the rostral and mid ARC as well as the ventromedial hypothalamus of females exposed to males. We conclude that introduction of male sheep to seasonally anestrous female sheep activates kisspeptin neurons and other cells in the hypothalamus, leading to increased GnRH/LH secretion

Kisspeptin antagonists

Kisspeptin is now known to be an important regulator of the hypothalamic-­pituitary-gonadal axis and is the target of a range of regulators, such as steroid hormone feedback, nutritional and metabolic regulation. Kisspeptin binds to its cognate receptor, KISS1R (also called GPR54), on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion—thus gating down-stream events supporting reproduction. The development of peripherally active kisspeptin antagonists could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer. The following chapter discusses the advances made in the search for both peptide and small molecule kisspeptin antagonists and their use in delineating the role of kisspeptin within the reproductive system. To date, four peptide antagonists and one small molecule antagonist have been designed