Regulating Mechanisms of Gonadal and Pituitary Tumorigenesis in Mice Producing Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are structurally and functionally similar glycoprotein hormones acting through the same luteinizing hormone chorionic gonadotropin receptor (LHCGR). The functions of LH in reproduction and hCG in pregnancy are well known. Recently, the expression of LHCGR has been found in many nongonadal tissues and cancers, and this has raised the question of whether LH/hCG could affect the function or tumorigenesis of these nongonadal tissues. We have also previously generated an hCG expressing mouse model presenting nongonadal phenotypes. Using this model it is possible to improve our understanding of nongonadal action of highly elevated LH/hCG. In the current study, we analyzed the effect of moderately and highly elevated hCG levels on male reproductive development and function. The main finding was the appearance of fetal Leydig cell (FLC) adenomas in prepubertal males. However, the development and differentiation of FLCs were not significantly affected. We also show that the function of hCG is different in FLCs and in adult Leydig cells (ALC), because in the latter cells hCG was not able to induce tumorigenesis. In FLCs, LHCGR is not desensitized or downregulated upon ligand binding. In this study, we found that the testicular expression of two G protein-coupled receptor kinases responsible for receptor desensitization or downregulation is increased in adult testis. Results suggest that the lack of LHCGR desensitization or downregulation in FLCs protect testosterone (Te) synthesis, but also predispose FLCs for LH/hCG induced adenomas. However, all the hCG induced nongonadal changes observed in male mice were possible to explain by the elevated Te level found in these males. Our findings indicate that the direct nongonadal effects of elevated LH/hCG in males are not pathophysiologically significant. In female mice, we showed that an elevated hCG level was able to induce gonadal tumorigenesis. hCG also induced the formation of pituitary adenomas (PA), but the mechanism was indirect. Furthermore, we found two new potential risk factors and a novel hormonally induced mechanism for PAs. Increased progesterone (P) levels in the presence of physiological estradiol (E2) levels induced the formation of PAs in female mice. E2 and P induced the expression and nuclear localization of a known cell-cycle regulator, cyclin D1. A calorie restricted diet was also able to prevent the formation of PAs, suggesting that obesity is able to promote the formation of PAs. Hormone replacement therapy after gonadectomy and hormone antagonist therapy showed that the nongonadal phenotypes observed in hCG expressing female mice were due to ovarian hyperstimulation. A slight adrenal phenotype was evident even after gonadectomy in hCG expressing females, but E2 and P replacement was able to induce a similar phenotype in WT females without elevated LH/hCG action. In conclusion, we showed that the direct effects of elevated hCG/LH action are limited only to the gonads of both sexes. The nongonadal phenotypes observed in hCG expressing mice were due to the indirect, gonadal hormone mediated effects of elevated hCG. Therefore, the gonads are the only physiologically significant direct targets of LHCGR signalling.Siirretty Doriast

Extragonadal LH/hCG action-Not yet time to rewrite textbooks

Gonadotropins are indispensable in both sexes in the regulation of gonadal sex steroid production and gametogenesis. In addition to their well-established classical actions, numerous recent publications have indicated the presence and function of luteinizing hormone/chorionic gonadotropin receptors (LH/hCG-R) in a variety of extragonadal tissues. However, the physiological significance of such effects has remained unclear. We have generated two genetically modified mouse models, one with excessive production of hCG and the other with targeted disruption of LH/hCG-R gene, and used them to address the functions of LH and hCG. Numerous gonadal and extragonadal phenotypes were found in the models with the two extremes of LH/hCG action. However, when the extragonadal effects were scrutinized in greater detail, they all appeared to arise through modification of gonadal function, either through enhanced or inhibited response to LH/hCG stimulation. Hence, further evidence is needed before the extragonadal LH/hCG-R expression can be considered functionally significant.Fil: Pakarainen, Tomi. University of Turku; FinlandiaFil: Ahtiainen, Petteri. University of Turku; FinlandiaFil: Zhang, Fu Ping. University of Helsinki; FinlandiaFil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Poutanen, Matti. University of Turku; FinlandiaFil: Huhtaniemi, Ilpo. University of Turku; Finlandia. IMperial College London; Reino Unid

Enhanced LH action in transgenic female mice expressing hCGβ-subunit induces pituitary prolactinomas; the role of high progesterone levels

The etiology of pituitary adenomas remains largely unknown, with the exception of involvement of estrogens in the formation of prolactinomas. We have examined the molecular pathogenesis of prolactin-producing pituitary adenomas in transgenic female mice expressing the human choriongonadotropin (hCG) β-subunit. The LH/CG bioactivity is elevated in the mice, with consequent highly stimulated ovarian progesterone (P4) production, in the face of normal estrogen secretion. Curiously, despite normal estrogen levels, large prolactinomas developed in these mice, and we provide here several lines of evidence that the elevated P4 levels are involved in the growth of these estrogen-dependent tumors. The antiprogestin mifepristone inhibited tumor growth, and combined postgonadectomy estradiol/P4 treatment was more effective than estrogen alone in inducing tumor growth. Evidence for direct growth-promoting effect of P4 was obtained from cultures of primary mouse pituitary cells and rat somatomammotroph GH3 cells. The mouse tumors and cultured cells revealed stimulation of the cyclin D1/cyclin-dependent kinase 4/retinoblastoma protein/transcription factor E2F1 pathway in the growth response to P4. If extrapolated to humans, and given the importance of endogenous P4 and synthetic progestins in female reproductive functions and their pharmacotherapy, it is relevant to revisit the potential role of these hormones in the origin and growth of prolactinomas

A nationwide study on parathyroid carcinoma

Background: Parathyroid carcinoma (PC) is rare and diagnostically challenging. Reported outcomes are rather poor and the incidence might be increasing.Material and methods: We performed a nationwide study on all cases (n=32) diagnosed in 2000-2011 in Finland, and compared clinical and histopathological characteristics and outcome to atypical parathyroid (APA; n=28) and parathyroid adenomas (PA; n=72). The incidence in years 1955-1999 was compared to that in 2000-2013.Results: Preoperatively, calcium and parathyroid hormone concentrations were higher in PC compared to APA and PA (1.76, 1.56 and 1.44mmol/l, pPeer reviewe

TSH elevations as the first laboratory evidence for pseudohypoparathyroidism type Ib (PHP-Ib).

Hypocalcemia and hyperphosphatemia because of resistance toward parathyroid hormone (PTH) in the proximal renal tubules are the most prominent abnormalities in patients affected by pseudohypoparathyroidism type Ib (PHP-Ib). In this rare disorder, which is caused by GNAS methylation changes, resistance can occur toward other hormones, such as thyroid-stimulating hormone (TSH), that mediate their actions through G protein-coupled receptors. However, these additional laboratory abnormalities are usually not recognized until PTH-resistant hypocalcemia becomes clinically apparent. We now describe four pediatric patients, first diagnosed with subclinical or overt hypothyroidism between the ages of 0.2 and 15 years, who developed overt PTH-resistance 3 to 20 years later. Although anti-thyroperoxidase (anti-TPO) antibodies provided a plausible explanation for hypothyroidism in one of these patients, this and two other patients revealed broad epigenetic GNAS abnormalities, which included loss of methylation (LOM) at exons AS, XL, and A/B, and gain of methylation at exon NESP55; ie, findings consistent with PHP-Ib. LOM at GNAS exon A/B alone led in the fourth patient to the identification of a maternally inherited 3-kb STX16 deletion, a well-established cause of autosomal dominant PHP-Ib. Although GNAS methylation changes were not detected in additional pediatric and adult patients with subclinical hypothyroidism (23 pediatric and 39 adult cases), hypothyroidism can obviously be the initial finding in PHP-Ib patients. One should therefore consider measuring PTH, along with calcium and phosphate, in patients with unexplained hypothyroidism for extended periods of time to avoid hypocalcemia and associated clinical complications

Multiple sites of tumorigenesis in transgenic mice overproducing hCG

We have produced transgenic (TG) mice expressing under the ubiquitin C promoter either the glycoprotein hormone common alpha-subunit (C(alpha)) or human chorionic gonadotropin (hCG) beta-subunit. C(alpha) overexpression alone had no phenotypic effect, but the hCG(beta) expressing females, presenting with moderately elevated levels of bioactive LH/hCG, due to dimerization of the TG hCG(beta) with endogenous C(alpha), developed multiple gonadal and extragonadal neoplasias. Crosses of the C(alpha) and hCG(beta) mice (hCG(alpha)beta) had >1000-fold elevated hCG levels, due to ubiquitous transgene expression, and presented with more aggressive tumour formation. The ovaries displayed initially strong luteinisation of all somatic cell types, leading to formation of luteomas, and subsequently to germ cell tumours (teratomas). The pituitary glands of TG females were massively enlarged, up to >100 mg, developing macroprolactinomas with very high prolactin (PRL) production. This endocrine response probably induced breast cancers in the mice. In contrast to the females, similar high levels of hCG in male mice had only marginal effects in adulthood, with slight Leydig cell hyperplasia and atrophy in the seminiferous epithelium. However, clear Leydig cell adenomas were observed in postnatal mice, apparently originating from fetal Leydig cells. In conclusion, these studies demonstrate marked tumorigenic effects of supraphysiological hCG levels in female mice, but clear resistance to similar changes in males. The extragonadal tumours were induced by hCG stimulated aberrant ovarian endocrine function, rather than by direct hCG action, because gonadectomy prevented all extragonadal phenotypes despite persistent hCG elevation. The phenotypes of the TG mice apparently represent exaggerated responses to hCG/LH and/or gonadal steroids. It remains to be explored to what extent they simulate respective responses in humans to pathophysiological elevation of the same hormones.Fil: Huhtaniemi, Ilpo. University of Turku; FinlandiaFil: Rulli, Susana Beatriz. Imperial College London; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Petteri, Ahtiainen. University of Turku; FinlandiaFil: Poutanen, Matti. University of Turku; Finlandi

Mechanisms of multiple forms of tumorigenesis in LH/hCG overexpressing transgenic mice

The two gonadotrophins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced by the pituitary gland as heterodimers composed of a common a-subunit and the hormone- specific b-subunit. They have a key role in ovarian and testicular functions by acting through binding to their specific seven-transmembrane domain G-protein coupled receptors (Burger et al., 2004). In the gonads, LH receptors are located in testicular Leydig cells and in ovarian theca, granulosa and luteal cells (Ascoli et al., 2002). FSH receptors are expressed in testicular Sertoli cells and in granulosa cells of the ovary (Simoni et al., 1997). Although primarily expressed in gonads, LH receptors are also found in numerous extragonadal tissues (Rao, 2001; Filicori et al., 2005). However, their physiological significance is still unclear (Pakarainen et al., 2007). The placental analogue of LH, human chorionic gonadotrophin (hCG) interacts with the same LH/hCG receptor as LH, and functions as an LH agonist with a longer half-life and higher biopotency than its pituitary counterpart (Jameson and Hollenberg,1993).Fil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ahtiainen, Petteri. University of Turku; FinlandiaFil: Poutanen, Matti. University of Turku; FinlandiaFil: Hutaniemi, Ilpo T.. University of Turku; Finlandia. Imperial College. London Institute Of Medical Sciences.

Genetically modified mouse models in studies of luteinising hormone action

Numerous genetically modified mouse models have recently been developed for the study of the pituitary-gonadal interactions. They include spontaneous or engineered knockouts (KO) of the gonadotrophin-releasing hormone (GnRH) and its receptor, the gonadotrophin common-alpha(Calpha), luteinising hormone (LH) beta and follicle-stimulating hormone (FSH) beta subunits, and the two gonadotrophin receptors (R), LHR and FSHR. In addition, there are also transgenic (TG) mice overexpressing gonadotrophin subunits and producing supraphysiological levels of these hormones. These models have offered relevant phenocopies for similar mutations in humans and to a great extent expanded our knowledge on normal and pathological functions of the hypothalamic-pituitary-gonadal (HPG) axis. The purpose of this article is to review some of our recent findings on two such mouse models, the LHR KO mouse (LuRKO), and the hCG overexpressing TG mouse (hCG+).Fil: Huhtaniemi, Ilpo. Imperial College London; Reino Unido. University of Turku; FinlandiaFil: Ahtiainen, Petteri. University of Turku; FinlandiaFil: Pakarainen, Tomi. University of Turku; FinlandiaFil: Rulli, Susana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Zhang, Fu Ping. University of Helsinki; FinlandiaFil: Poutanen, Mattu. University of Turku; Finlandi