A new glucocorticoid receptor species: relation to induction of tryptophan dioxygenase by glucocorticoids

When rats were treated with a high dose (greater than or equal to 20 micrograms/100 g BW) of dexamethasone, their liver cytosol showed a predominant peak of specific binding protein of glucocorticoid eluting with 0.13-0.14 M NaCl on diethylaminoethyl cellulose chromatography. On the other hand, when rats were treated with a low dose (approximately 2 micrograms/100 g BW) of dexamethasone, the cytosol showed only the same peak as that of untreated rats, which is widely thought to be that of the typical glucocorticoid receptor. The appearance of the new binding peak depended both on the dose of hormone and the time (from 30 min to 20 h) after dexamethasone treatment; it disappeared 40 h after treatment. A peak similar to that in the cytosol also appeared in the nuclear fraction after treatment with a high dose of hormone. The glucocorticoid-inducible enzymes, tryptophan dioxygenase [(TO) EC 1.13.11.11] and tyrosine aminotransferase (EC 2.6.1.5) were assayed as physiological markers of receptor function. TO induction correlated well with the appearance of the new binding peak in terms of dose- and time dependence on glucocorticoid, whereas tyrosine aminotransferase induction did not. The new peak of glucocorticoid-binding protein detected in the cytosol and nuclear fractions may thus represent the glucocorticoid receptor species involved in TO induction, physiological changes under stress, and pharmacological changes after therapy with high doses of glucocorticoid hormones.     

Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.     

Neonatal induction of a nuclear protein that binds to the c-fos enhancer.

The expression of the c-fos gene is transiently induced at birth in most organs in the mouse. To study the basis of this induction we searched for a nuclear factor that binds to the 5' regulatory region of the c-fos gene. Gel mobility shift assays with tissue extracts revealed fast (band I) and slow (band III) migrating bands, which represent factor binding to the c-fos enhancer, termed the serum response element (SRE). Neonatal extracts preferentially elicited band I, with low or undetectable levels of band III, whereas fetal and adult extracts generated predominantly band III, with reduced levels of band I. These results indicate that the SRE-binding activity changes during perinatal development and that the appearance of band I, which coincides with diminution of band III, correlates with neonatal c-fos induction. Methylation interference and competition analyses showed that the neonatal factor (band I) binds to the SRE at a site different from the adult factor (band III). DNA-binding activity of the adult factor, but not the neonatal factor, was sensitive to phosphatase treatment. Furthermore, the adult factor, but not the neonatal factor, shared antigenic specificity with the human serum response factor (SRF) that is expressed in cultured cells irrespective of c-fos gene induction. We conclude that band I in neonates represents a SRE-binding factor that is distinct from the SRF, which may be responsible for the neonatal induction of the c-fos gene. The band III factor was indistinguishable from the SRF in all criteria tested.     

Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis

Human neonates infected with herpes simplex virus 1 (HSV-1) develop one of three distinct patterns of infection: (i) infection limited to the skin, eye or mouth; (ii) infection of the CNS; or (iii) disseminated infection. The disseminated form usually involves the liver, adrenal gland, and lung, and resembles the clinical picture of bacterial sepsis. This spectrum of symptoms in HSV-1-infected neonates suggests that inflammatory cytokines play a significant role in the pathogenesis of the disease. Recent studies suggest that the Toll-like receptors (TLRs) may play an important role in the induction of inflammatory cytokines in response to viruses. TLRs are mammalian homologues of Toll, a Drosophila protein that is essential for host defense against infection. Engagement of TLRs by bacterial, viral, or fungal components leads to the production and release of cytokines and other antimicrobial products. Here, we demonstrate that TLR2 mediates the inflammatory cytokine response to HSV-1 by using both transfected cell lines and knockout mice. Studies of infected mice revealed that HSV-1 induced a blunted cytokine response in TLR2(-/-) mice. Brain levels of monocyte chemoattractant protein 1 chemokine were significantly lower in TLR2(-/-) mice than in either wild-type or TLR4(-/-) mice. TLR2(-/-) mice had reduced mortality compared with wild-type mice. The differences between TLR2(-/-) mice and both wild-type and TLR4(-/-) mice in the induction of monocyte chemoattractant protein 1, brain inflammation, or mortality could not be accounted for on the basis of virus levels. Thus, these studies suggest the TLR2-mediated cytokine response to HSV-1 is detrimental to the host.     

Soluble interleukin-2 receptor in Crohn's disease: relation of serum concentrations to disease activity.

Serum concentrations of soluble interleukin-2 receptor (sIL-2R) were measured as a marker of immune activation in a group of 30 patients with Crohn's disease. sIL-2R concentrations were determined by enzyme linked immunosorbent assay during periods of active and inactive disease and correlated with standard parameters of disease activity. Serum concentrations of sIL-2R were significantly raised in patients with active Crohn's disease compared with patients with inactive disease (p less than 0.001) and control subjects. There was a significant correlation between serum sIL-2R concentrations and disease activity as assessed by the Harvey-Bradshaw index (r = 0.42, p less than 0.01), platelet numbers (r = 0.49, p less than 0.01), and orosomucoid (r = 0.47, p less than 0.01), alpha 1 antitrypsin (r = 0.44, p less than 0.01), and C reactive protein concentrations (r = 0.48, p less than 0.001) but not with the erythrocyte sedimentation rate. Measurement of serum sIL-2R concentration is a simple and useful laboratory means of assessing disease activity. Raised concentrations in patients with active Crohn's disease is further evidence for in vivo immune activation occurring in this disease.     

Polymorphisms of the beta2-adrenergic receptor gene (ADRB2) in relation to cardiovascular risk factors in men

OBJECTIVE: To investigate the effect of polymorphisms in codon 16 (Arg16Gly) and codon 27 (Gln27Glu) of the beta2-adrenergic receptor gene (ADRB2) on anthropometric, endocrine, metabolic and haemodynamic variables. DESIGN: A cross-sectional study. SUBJECTS: A subgroup of 284 Swedish men from a population sample of 1040 at the age of 51 years. MAIN OUTCOME MEASURES: Genotype examination of ADRB2 polymorphisms in codon 16 and codon 27 with polymerase chain reaction and restriction fragment length polymorphism. Anthropometric measurements included body mass index, waist-to-hip ratio and abdominal sagittal diameter. Endocrine measurements included blood levels of testosterone, insulin-like growth factor I, and leptin plus salivary cortisol. Overnight fasting values of serum insulin, blood glucose, triglycerides, total, low and high density lipoprotein cholesterol, as well as blood pressure and resting heart rate, were also determined. RESULTS: Polymorphisms were frequent in both codon 16 and codon 27. The Arg16Gly genotype showed significant relationships to elevated central distribution of body fat and to systolic blood pressure, whilst the Glu27Glu genotype was associated with elevated leptin and triglyceride levels but not to other measurements, including obesity variables. CONCLUSIONS: We conclude that only a few cardiovascular risk factors are associated with DNA sequence variation in the ADRB2 in Swedish men.     

Distinct melanocortin 2 receptor accessory protein domains are required for melanocortin 2 receptor interaction and promotion of receptor trafficking

Melanocortin 2 receptor (MC2R) is the receptor for the pituitary hormone ACTH. When activated, MC2R stimulates cAMP production and adrenal steroidogenesis. The functional expression of the receptor requires melanocortin 2 receptor accessory protein (MRAP), a single-transmembrane domain protein involved in the trafficking of MC2R from the endoplasmic reticulum to the cell surface. Mutations in both MC2R and MRAP cause the inherited disease familial glucocorticoid deficiency. At present, little is known regarding the mechanism of MRAP in MC2R functional expression. Here we report the characterization of MRAP in the trafficking of MC2R to the cell surface and the formation of a functional receptor. We identify the transmembrane domain of MRAP as the MC2R interaction domain and a conserved N-terminal tyrosine-rich domain of MRAP that is required for trafficking MC2R to the cell surface.     

Altered control of gastric acid secretion in gastrin-cholecystokinin double mutant mice

BACKGROUND & AIMS: Three pathways control gastric acid secretion: the gastrin-enterochromaffin-like (ECL) cell axis, the vagus-parietal cell axis, and the cholecystokinin (CCK)-D cell axis. Mice lacking gastrin or both gastrin and CCK were examined to determine the role of the hormones. METHODS: Acid was measured after pylorus ligation, and biopsies from gastrin knockout (KO), gastrin-CCK double-KO, and wild-type (WT) mice were collected for biochemical, immunocytochemical, and electron-microscopic examination. RESULTS: The ECL cells were inactive in both groups of mutant mice but the cell number was unaffected. Both parietal cell number and level of H(+)/K(+)-ATPase messenger RNA (mRNA) were reduced in the mutant strains, but gastrin-CCK double-KO mice displayed more active parietal cells and larger acid output than the gastrin KO mice. The acid response to histamine in double-KO mice was unchanged whereas that to gastrin was diminished, but it could be restored by infusion of gastrin. Oxyntic D-cell density was the same in both mutant strains, but the D cells were more active in the gastrin KO than in the double-KO mice. CCK infusion in gastrin-CCK double-KO mice raised the somatostatin mRNA level and inhibited acid secretion to the level seen in gastrin KO mice. Vagotomy and atropine abolished acid secretion in all 3 groups of mice. CONCLUSIONS: Lack of gastrin impairs the gastrin-ECL axis, whereas lack of gastrin and CCK impairs both hormonal pathways. In the gastrin-CCK double-KO mice, acid secretion is only controlled by cholinergic vagal stimulation, which normalizes the acid output.     

Adiponectin and adiponectin receptor gene variants in relation to type 2 diabetes and insulin resistance-related phenotypes

BACKGROUND: Alterations in adiponectin-mediated pathways are known to be associated with glucose intolerance, insulin resistance (IR), obesity, and type 2 diabetes (T2D) mellitus. Genetic variations in adiponectin (ADIPOQ) and adiponectin 1 and 2 receptor (ADIPOR1 and ADIPOR2) could have effects on IR-related phenotypes and T2D. Here we examine whether the polymorphic markers rs2241766 (ADIPOQ), rs22753738 (ADIPOR1), rs11061971 and rs16928751 (both in ADIPOR2) are implicated in susceptibility to T2D in a Russian population. METHODS: The polymorphic markers were genotyped in 129 T2D patients, and 117 non-diabetic controls, by polymerase chain reaction (PCR) restriction fragment length polymorphism approach. In the subjects, biochemical characteristics including serum insulin, plasma glucose and serum lipids/lipoproteins were measured and compared for correlation with the genetic variations studied. RESULTS: Allele T of rs11061971 and allele A of rs16928751 showed association with higher risk of diabetes providing odds ratios (OR) of 2.05 (p = 0.0025) and 1.88 (p = 0.018), respectively. Haplotype A-G consisting of allele A of rs11061971 and allele G of rs16928751 was associated with reduced risk of T2D (OR = 0.59, p_c = 0.0224). Compared to other variants, diabetic patients double homozygous for A/A of rs16928751 and G/G of rs16928751 had decreased homeostasis model assessment-insulin resistance (p_c = 0.0375) and serum triglycerides (p_c = 0.0285). CONCLUSIONS: The variants of ADIPOR2 confer susceptibility to T2D and are associated with some IR-related phenotypes in the Russian study population.     

CRIPT与人甘丙肽2型受体的相互作用

目的 通过寻找与人甘丙肽2型受体(hGalR2)C端相互作用的蛋白,以进一步探讨hGalR2转运和信号传导机制.方法 利用酵母双杂交实验寻找可以与hGalR2 C端相互作用的蛋白,并通过酵母双转验证和免疫共沉淀实验验证受体和目标蛋白之间的相互作用.结果 酵母双杂交方法结合免疫共沉淀实验发现和证实hGalR2与蛋白Cysteine-rich PDZ-binding protein(CRIPT)之间存在相互作用.结论 CRIPT可以与hGalR2结合而发生相互作用并可能因此参与hGalR2的转运或信号传导.     

Epistatic interaction between variations in the angiotensin I converting enzyme and angiotensin II type 1 receptor genes in relation to extent of coronary atherosclerosis

OBJECTIVE: To test the hypothesis that gene-gene interaction of the renin-angiotensin system is associated with an effect on the extent of coronary atherosclerosis. SETTING AND RESULTS: A cohort of 1162 patients with coronary artery disease were genotyped for genetic polymorphisms in the renin-angiotensin system. Patients carrying the D allele of the angiotensin I converting enzyme (ACE) gene had greater coronary extent scores (defined as the number of coronary segments with 5% to 75% stenosis) than those not carrying this allele (p = 0.006 in non-parametric analysis and p = 0.019 in parametric analysis). This association remained significant after adjusting for age, body mass index, hypertension, and diabetes, which were also significantly associated with coronary extent scores. There was a significant interaction (p = 0.033) between genotypes of ACE and angiotensin II type 1 receptor (AGTR1). The association between the ACE gene D allele and increased coronary extent scores was significant (p = 0.008 in non-parametric and p = 0.027 in parametric analysis) in those carrying the +1166 C allele of the AGTR1 gene, but was absent in those not carrying the AGTR1 gene +1166 C allele. CONCLUSION: These findings suggest that variation in the ACE and AGTR1 genes and their interaction may not only contribute to susceptibility of coronary artery disease as previously found but also modify the disease process, thus contributing to interindividual differences in severity of the disease.     

Gonadotropin induction of c-fos and c-myc expression and deoxyribonucleic acid synthesis in rat granulosa cells

Although gonadotropins stimulate ovarian granulosa cells to proliferate and differentiate into steroidogenic cells, little is known about the molecular mechanisms by which gonadotropins induce these fundamentally different responses. In this study the acute effects of PMSG on protooncogene expression, DNA synthesis, and steroid secretion were examined. The levels of c-fos, c-myc, and beta-actin mRNA were measured in total RNA samples from granulosa cells by quantitative polymerase chain reaction. PMSG increased the mRNA levels of c-fos, c-myc, and beta-actin within 15 min. Fos and myc proteins were localized within granulosa cells by immunocytochemistry. Less than 10% of granulosa cells stained for c-fos or c-myc proteins in the control samples. In contrast, approximately 40% of the cells stained for these protooncogene proteins 30 min after PMSG injection (P less than 0.05). These values declined to about 10% of the cells 60 min after PMSG injection. DNA synthesis, as estimated by [3H]thymidine incorporation, increased 30 and 60 min after PMSG (P less than 0.05). 17 beta-Estradiol and progesterone synthesis did not change within 60 min of PMSG injection. These data demonstrate that 1) c-fos and c-myc are expressed in ovarian granulosa cells; 2) the expression of the genes encoding c-fos, c-myc, and beta-actin is rapidly increased by gonadotropin; and 3) the increase in the corresponding products of the c-fos and the c-myc genes precedes an increase in DNA synthesis and steroid production. These data suggest that the expression of c-fos and c-myc may be a part of the molecular mechanism through which gonadotropins regulate granulosa cell function.