Dai Chida

Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis

ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus–pituitary–adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R–MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.

Functional role of acetylcholine and the expression of cholinergic receptors and components in osteoblasts

Recent studies have indicated that acetylcholine (ACh) plays a vital role in various tissues, while the role of ACh in bone metabolism remains unclear. Here we demonstrated that ACh induced cell proliferation and reduced alkaline phosphatase (ALP) activity via nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) in osteoblasts. We detected mRNA expression of several nAChRs and mAChRs. Furthermore, we showed that cholinergic components were up‐regulated and subunits/subtypes of acetylcholine receptors altered during osteoblast differentiation. To our knowledge, this is the first report demonstrating that osteoblasts express specific acetylcholine receptors and cholinergic components and that ACh plays a possible role in regulating the proliferation and differentiation of osteoblasts.

Characterization of a protein tyrosine phosphatase (RIP) expressed at a very early stage of differentiation in both mouse erythroleukemia and embryonal carcinoma cells

From our previous studies, several protein tyrosine phosphatases (PTPase) are implicated in the early events leading to in vitro differentiation of both mouse erythroleukemia (MEL) and embryonal carcinoma (F9) cells. Among the PTPases, recent experiments suggest that a new PTPase (RIP) plays a critical role in differentiation processes, particularly at their early stages. We isolated cDNA clones for RIP from a RNA preparation isolated from differentiating MEL cells, and determined the total 7932 bp base sequence for RIP cDNA. The cDNA codes for a putative 269.8 kDa (2450 amino acids) protein with a PTPase catalytic domain. We have demonstrated that the transcripts exist in multiple forms, and among mouse tissues they were found predominantly in kidney and, to a lesser extent, in lung, heart, brain and testis. The RIP gene was mapped between D5Mit90 and D5Mit25 on mouse chromosome 5.

RhoH Plays Critical Roles in FcεRI-Dependent Signal Transduction in Mast Cells

RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon FcεRI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, FcεRI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLCγ1, and PLCγ2 were impaired, however phosphorylation of the γ-subunit of FcεRI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates FcεRI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk.

Characterization of mice deficient in Melanocortin 2 receptor on a B6/Balbc mix background

We have previously reported that Melanocortin 2 receptor (MC2R(-/-)) deficient mice on B6 N5 generations exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata (zF) and lack of detectable levels of corticosterone, and reduced serum concentrations of aldosterone and epinephrine. All MC2R(-/-) mice on B6/N8 background die within 2 days after birth, while about half of the MC2R(-/-) mice on B6/Balbc mix background survived to adulthood. Both male and female MC2R(-/-) mice were fertile, suggesting that normal development and function of reproductive organs. MC2R(-/-) mice delivered from MC2R(-/-) dams failed to survive due to lung failure, suggesting that fetal or maternal corticosterone is essential for lung maturation. MC2R(-/-) mice failed to activate the hypothalamic-pituitary-adrenal axis in response to both immune and non-immune stimuli. MC2R(-/-) mice maintained glomerular structure and achieved electrolyte homeostasis by the activation of the renin-angiotensin-aldosterone system under low aldosterone and undetectable levels of corticosterone.

Increased fat:carbohydrate oxidation ratio in Il1ra−/− mice on a high-fat diet is associated with increased sympathetic tone

Aims/hypothesisProinflammatory cytokines, including IL-1, exert pleiotropic effects on the neuro–immuno–endocrine system. Previously, we showed that mice with knockout of the gene encoding IL-1 receptor antagonist (Il1ra−/−, also known as Il1rn−/−) have a lean phenotype. The present study was designed to analyse the mechanisms leading to this lean phenotype.MethodsIl1ra−/− mice were fed a high-fat diet following weaning. Energy expenditure, body temperature, heart rate, blood parameters, urinary catecholamines and adipose tissue were analysed.ResultsIl1ra−/− mice exhibited resistance to obesity induced by a high-fat diet; this resistance was associated with increased energy expenditure and a decreased respiratory quotient, indicating that the ratio of fat:carbohydrate metabolism in Il1ra−/− mice is greater than in controls. Activity level in Il1ra−/− mice was significantly decreased and body temperature was significantly increased, compared with wild-type (WT) mice. Inguinal white adipose tissues in Il1ra−/− mice express increased levels of Ucp1 and mitochondrial respiratory chain genes compared with WT mice. Histological analysis of adipose tissue in Il1ra−/− mice revealed that brown adipose tissue is hyperactive and inguinal white adipose tissue contains smaller cells, which exhibit the distinctive multilocular appearance of brown adipocytes. Urinary epinephrine and norepinephrine excretion in Il1ra−/− mice was significantly increased compared with WT mice, suggesting that Il1ra−/− mice have increased sympathetic tone. Consistent with this, heart rate in Il1ra−/− mice was also significantly increased.Conclusions/interpretationOur results show that Il1ra−/− mice have increased energy expenditure, fat:carbohydrate oxidation ratio, body temperature, heart rate and catecholamine production. All of these observations are consistent with an enhanced sympathetic tone.

The Role of Glucocorticoids in Pregnancy, Parturition, Lactation, and Nurturing in Melanocortin Receptor 2-Deficient Mice

Maternal glucocorticoids are critical for fetal development, but overexpression can be deleterious. Previously we established a mouse line deficient in melanocortin receptor 2 (MC2R). MC2R(-/-) mice have undetectable levels of corticosterone despite high levels of ACTH and defects resembling those in patients with familial glucocorticoid deficiency. Here we analyzed the role of glucocorticoids in pregnancy, parturition, lactation, and nurturing in MC2R(-/-) mice. MC2R(-/-) mice were fertile and produced normal litters when crossed with MC2R(+/+) mice. However, MC2R(-/-) females crossed with MC2R(-/-) males had no live births, and approximately 20% of the embryos at d 18.5 of pregnancy were of normal body size but were dead when born. MC2R(-/-) pregnant females crossed with MC2R(+/+) males had detectable serum corticosterone levels, suggesting the transplacental passage of corticosterone from fetus to mother. MC2R(+/-) pups delivered from MC2R(-/-) females crossed with MC2R(+/+) males mice thrived poorly with MC2R(-/-) mothers but grew to adulthood when transferred to foster mothers after birth, suggesting that MC2R(-/-) females are poor mothers or cannot nurse. MC2R(-/-) females had normal alveoli, but penetration of mammary epithelium into fat pads and expression of milk proteins were reduced. Myoepithelial cells, which force milk out of the alveoli, were fully developed and differentiated. Pup retrieval behavior was normal in MC2R(-/-) mice. Exogenous corticosterone rescued expression of milk proteins in MC2R(-/-) mothers, and the pups of treated mothers grew to adulthood. Our results reveal the importance of glucocorticoids for fetal survival late in pregnancy, mammary gland development, and milk protein gene expression.

Expression of the Protein Tyrosine Phosphatase β2 Gene in Mouse Erythroleukemia Cells Induces Terminal Erythroid Differentiation

We have cloned cDNA for protein tyrosine phosphatase β2, which had been implicated in erythroid differentiation of mouse erythroleukemia cells. Expression of cDNA constructs, in which β2 cDNA is placed under the control of mouse metallothionein-I promoter, by ZnCl2 converted a significant portion (20 to 38%) of the cells to erythroid-like cells, which is 25-50% of the erythroid differentiation efficiency observed by conventional erythroid-inducing agents. Furthermore, introduction and expression of altered protein tyrosine phosphatase β2 cDNA constructs designed to produce the enzyme lacking the phosphatase activity inhibited erythroid differentiation by 100-20%, depending upon the concentration of erythroid-inducing agents employed. These results strongly suggest that protein tyrosine phosphatase β2 is involved in triggering erythroid differentiation in mouse erythroleukemia cells.

Functional Hypothalamic Amenorrhea Due to Increased CRH Tone in Melanocortin Receptor 2-Deficient Mice

Exposure to chronic stressors results in dysregulation of the hypothalamic-pituitary-adrenal axis and a disruption in reproduction. CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis induces the secretion of ACTH from the pituitary, which stimulates adrenal steroidogenesis via the specific cell-surface melanocortin 2 receptor (MC2R). Previously, we demonstrated that MC2R(-/-) mice had undetectable levels of corticosterone despite high ACTH levels. Here, we evaluated the reproductive functions of female MC2R(-/-) mice and analyzed the mechanism of the disrupted cyclicity of these mice. The expression of CRH in the paraventricular nucleus was significantly increased in MC2R(-/-) mice under nonstressed conditions. Although MC2R(-/-) females were fertile, they showed a prolonged estrous cycle. After hormonal stimulation, MC2R(-/-) females produced nearly-normal numbers of eggs, but slightly less than MC2R(+/-) females, and showed near-normal ovarian histology. During diestrus, the number of GnRH-positive cells in the medial preoptic area was significantly reduced in MC2R(-/-) females. CRH type 1 receptor antagonist restored estrous cyclicity in MC2R(-/-) females. Kisspeptin-positive areas in the arcuate nucleus were comparable, whereas kisspeptin-positive areas in the anteroventral periventricular nucleus in MC2R(-/-) females were significantly reduced compared with MC2R(+/-) females, suggesting that arcuate nucleus kisspeptin is not involved, but anteroventral periventricular nucleus kisspeptin may be involved, in the maintenance of estrous cyclicity. Our findings show that high levels of hypothalamic CRH disturb estrous cyclicity in the female animals and that the MC2R(-/-) female is a unique animal model of functional hypothalamic amenorrhea.

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice.

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.