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BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism is heterogeneously distributed worldwide, with the highest and lowest frequencies of the T allele in Mexico and Africa, respectively, and a south-to-north gradient in Europe. Distribution of MTHFR 1298A-->C is less well known. It has been hypothesized that 677T frequency could result in part from gene-nutrient interactions. OBJECTIVE: The objective was to compare the association of 677T and 1298C alleles with plasma concentrations of homocysteine, folate, and vitamin B-12 in geographical areas with contrasting 677T allele frequencies. DESIGN: Healthy young adults (n = 1277) were recruited in Mexico City, the West African countries of Bénin and Togo, France, and Sicily (Italy). Homocysteine, folate, and vitamin B-12 were measured in plasma, and MTHFR polymorphisms were measured in genomic DNA. RESULTS: Mexico City and Sicily reported the highest and Bénin and Togo reported the lowest plasma concentrations of folate. Mexico City had the highest 677T allele prevalence and the lowest influence of 677TT genotype on homocysteine, whereas the opposite was observed in Africa. The prevalence of the 1298C allele was lowest in the Mexicans and Africans and highest in the French. The percentage of the 677T genotype was significantly associated with the folate concentrations in 677CC carriers in a univariate analysis (R = 0.976; 95% CI: 0.797, 0.996; P < 0.0002) and in a multiple regression model that included homocysteine, vitamin B-12, and age (P = 0.0002). CONCLUSION: Our data agree with the hypothesis of a gene-nutrient interaction between MTHFR 677C-->T polymorphism and folate status that may confer a selective advantage of TT-homozygous genotype when dietary intake of folate is adequate, at least in the areas studied. Copyright © 2006 American Society for Clinical Nutrition [ABSTRACT FROM AUTHOR]

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5–50 µM range (EC50=4.3 µM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 µM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[γ- S]=2-MeSATP≥ADP, where ATP[γ- S] is adenosine 5′- O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate (α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 µM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 µM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor. [ABSTRACT FROM AUTHOR]

Vascular anomalies (VAs) result from the defective development of the embryonic vascular system and feature dysplastic malformed vessels, which are not always apparent at birth. They do not regress over the patient's lifetime; they usually have commensurate growth during childhood and may worsen over time if not treated. VAs may cause chronic painful swelling, bleeding, functional deficits or vital structure obstruction. These patients' quality of life is usually impaired because of the chronicity and recurrence of the disease. We report on six cases of complicated VAs, refractory to current treatments, treated with rapamycin, an mTor inhibitor recently used in VAs., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Context: The ghrelin receptor GHS-R1a is highly expressed in human somatotroph adenomas and exhibits unusually high basal signaling activity. In humans, the suppression of this constitutive activity by mutation induces a short stature., Objective: Using a GHS-R1a inverse agonist, modified substance P (MSP), we explored the role of GHS-R1a constitutive activity in GH hypersecretion from somatotroph adenomas and as a putative therapeutic target., Design: The effects of MSP were assessed on GH secretion from 19 human somatotroph tumors in vitro. Moreover, these effects were compared with those of octreotide (somatostatin receptor subtype 2 [sst2] agonist) and with the combination of both drugs. Expression and localization of GHS-R1a and sst2 were studied., Results: For all tumors, MSP inhibited GH secretion in a dose-dependent manner from 13 to 64%. Moreover, MSP enhanced octreotide-induced GH inhibition. For five tumors, the effects of combined MSP plus octreotide treatment were significantly higher than the sum of effects of each drug alone. MSP increased the membrane localization of GHS-R1a and of microdomains colocalizing sst2-GHS-R1a, highlighting the cooperation between the two drugs., Conclusions: The GHS-R1a inverse agonist could open new therapeutic options for acromegalic patients, particularly patients partially sensitive to octreotide whose GH secretion is not completely controlled by the sst2 agonist.

Abundant evidences have shown that ghrelin, by its binding to GHS-R1a, plays an important role for fundamental physiological functions. Increasing attention is given to the GHS-R1a unusually high constitutive activity and its contribution to downstream signaling and physiological processes. Here, we review recent lines of evidences showing that the interaction between ligand-binding pocket TM domains and the ECL2 could be partially responsible for this high constitutive activity. Interestingly, GHSR-1a constitutive activity activates in turn the downstream PLC, PKC, and CRE signaling pathways and this activation is reversed by the inverse agonist [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)]-substance P (MSP). Noteworthy, GHSR-1a exhibits a C-terminal-dependent constitutive internalization. Non-sense GHS-R1a mutation (Ala204Glu), first discovered in Moroccan patients, supports the role of GHSR-1a constitutive activity in physiological impairments. Ala204Glu-point mutation, altering exclusively the GHSR-1a constitutive activity, was associated with familial short stature syndrome. Altogether, these findings suggest that GHS-R1a constitutive activity could contribute to GH secretion or body weight regulation. Consequently, future research on basic and clinical applications of GHS-R1a inverse agonists will be challenging and potentially rewarding.

The Ras/Raf/MEK/ERK is a conserved signalling pathway involved in the control of fundamental cellular processes. Despite extensive research, how this pathway can process a myriad of diverse extracellular inputs into substrate specificity to determine biological outcomes is not fully understood. It has been established that the ERK1/2 pathway is an integrative point in the control of the pituitary function exerted by various extracellular signals. In addition we previously established that the GTPases Ras and Rap1 play a key role in the regulation of ERK1/2-dependent prolactin transcription by EGF or the cAMP-dependent neuropeptide VIP. In this report, using the FRET-based biosensor of ERK activity (EKAR) in the pituitary GH4C1 cell line, we show that both EGF and VIP tightly control the spatiotemporal dynamic of activated ERK with different magnitude and duration. Importantly, we provide the first evidence of a differential control of cytoplasmic and nuclear pools of activated ERK by the GTPases Ras and Rap1. Ras is required for nuclear magnitude and duration of EGF-dependent ERK activation, whereas it is required for both VIP-activated cytoplasmic and nuclear ERK pools. Rap1 is exclusively involved in VIP-activated ERK nuclear pool. Moreover, consistent with the control of the nuclear pool of activated ERK by the GTPases, we observe the same differential role of Ras and Rap1 on ERK nuclear translocation triggered by EGF or VIP. Together these findings identify Ras and Rap1 as determinant partners in shaping nuclear and cytoplasmic ERK kinetics in response to EGF and VIP, which in turn should control pituitary secretion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The treatment of growth hormone (GH)- and prolactin (PRL)-secreting tumors resistant to current therapeutic molecules (somatostatin and dopamine analogues) remains challenging. To target these tumors specifically, we chose to inactivate a gene coding for a crucial factor in cell proliferation and hormonal regulation, specifically expressed in pituitary, by using a dominant-negative form of this gene involved in human pituitary deficiencies: transcription factor Pit-1 (POU1F1) mutated on arginine 271 to tryptophan (R271W). After lentiviral transfer, the effect of R271W was studied in vitro on human tumoral somatotroph and lactotroph cells and on the murine mammosomatotroph cell line GH4C1 and in vivo on GH4C1 subcutaneous xenografts in nude mice. R271W induced a decrease in GH and PRL hypersecretion by controlling the transcription of the corresponding hormones. This mutant decreased cell viability by an apoptotic mechanism and in vivo blocked the tumoral growth and GH secretion of xenografts obtained after transplantation of GH4C1 expressing mutant R271W. The strategy of using a dominant-negative form of a main factor controlling cell proliferation and hormonal secretion, and exclusively expressed in pituitary, seems promising for the gene therapy of human pituitary tumors and may be translated to other types of tumors maintaining some differentiation features.

Deoxynivalenol (DON), one of the most abundant trichothecenes found on cereals, has been implicated in mycotoxicoses in both humans and farm animals. Low-dose toxicity is characterized by reduced weight gain, diminished nutritional efficiency, and immunologic effects. The levels and patterns of human food commodity contamination justify that DON consumption constitutes a public health issue. DON stability during processing and cooking explains its large presence in human food. We characterized here DON intoxication by showing that the toxin concomitantly affects feeding behavior, body temperature, and locomotor activity after both per os and central administration. Using c-Fos expression mapping, we identified the neuronal structures activated in response to DON and observed that the pattern of neuronal populations activated by the toxin resembled those induced by inflammatory signals. By real-time PCR, we report the first evidences for a DON-induced central inflammation, attested by the strong upregulation of interleukin-1β, interleukin-6, tumor necrosis factor-α, cyclooxygenase-2, and microsomal prostaglandin synthase-1 (mPGES-1) messenger RNA. However, silencing prostaglandins E2 signaling pathways using mPGES-1 knockout mice, which are resistant to cytokine-induced sickness behavior, did not modify the responses to the toxin. These results reveal that, despite strong similarities, behavioral changes observed after DON intoxication differ from classical sickness behavior evoked by inflammatory cytokines.

Nonfunctioning pituitary adenomas (NFPA; gonadotroph derived), even not inducing hormonal hypersecretion, cause significant morbidity by compression neighboring structures. No effective and specific medical methods are available so far for treating these tumors. The pituitary homeobox 2 (PITX2) gene is a member of the bicoid-like homeobox transcription factor family, which is involved in the Wnt/Dvl/β-catenin pathway. PITX2 is overexpressed in NFPA. PITX2 mutations are known to be responsible for Axenfield Rieger syndrome, a genetic disorder in which pituitary abnormalities have been detected. The R91P mutant identified in Axenfeld Rieger syndrome is a dominant-negative factor, which is able to block the expression of several pituitary genes activated by PITX2. To better understand the role of Pitx2 on gonadotroph tumorigenesis and to explore new approach for inhibiting tumoral growth, the R91P mutant was transferred via a lentiviral vector in tumoral gonadotroph cells of two kinds: the αT3-1 cell line and human adenoma cells. R91P mutant and small interfering RNA directed against Pitx2 both decreased the viability of αT3-1 cells via an apoptotic mechanism involving the activation of executioner caspase. Similar effects of the R91P mutant were observed on human gonadotroph cells in primary culture. Therefore, Pitx2 overexpression may play an antiapoptotic role during NFPA tumorigenesis.

Physiological regulations of energy balance and body weight imply highly adaptive mechanisms which match caloric intake to caloric expenditure. In the central nervous system, the regulation of appetite relies on complex neurocircuitry which disturbance may alter energy balance and result in anorexia or obesity. Deoxynivalenol (DON), a trichothecene, is one of the most abundant mycotoxins found on contaminated cereals and its stability during processing and cooking explains its widespread presence in human food. DON has been implicated in acute and chronic illnesses in both humans and farm animals including weight loss. Here, we provide the first demonstration that DON reduced feeding behavior and modified satiation and satiety by interfering with central neuronal networks dedicated to food intake regulation. Moreover, our results strongly suggest that during intoxication, DON reaches the brain where it modifies anorexigenic balance. In view of the widespread human exposure to DON, the present results may lead to reconsider the potential consequences of chronic DON consumption on human eating disorders.

For decades, the struggle against inflammation and related disorders has constituted an important field in medical practice, with strategies mainly aimed at inhibiting compounds produced through the arachidonic acid pathway. Thus, specific COX-2 inhibitors or "coxibs", were recently designed, that play an increasing but controversial role in reducing inflammatory phenomenon. Lately, several patents have been generated which target the specific inhibition of the microsomal Prostanglandin E synthase-1 (mPGES-1). This enzyme, which was cloned and characterized at the end of the nineties, catalyzes under inflammatory stimuli the last step of PGE2 synthesis. A corpus of data is now available illustrating the pivotal role played by this enzyme in numerous symptoms linked to inflammation such as fever, anorexia or pain. The present review highlights the current state of knowledge of the involvement of mPGES-1 in sickness behaviour and in other inflammation-related disorders and summarizes the recent patents related to mPGES-1 and its specific inhibition.

Previous evidence has shown that prostaglandins play a key role in the development of sickness behavior observed during inflammatory states. In particular, prostaglandin E2 (PGE2) is produced in the brain by a variety of inflammatory signals such as endotoxins or cytokines. Its injection has been also shown to induce symptoms of sickness behavior. The role of cyclooxygenase enzymes (COX), the rate-limiting enzymes converting arachidonic acid into prostaglandins, in sickness behavior has been extensively studied, and it has been demonstrated that strategies aiming at inhibiting these enzymes limit anorexia, body weight loss and fever in animals with inflammatory diseases. However, inhibiting COX activity may lead to negative gastric or cardiovascular effects, since COX enzymes play a role in the synthesis of others prostanoids with various and sometimes contrasting properties. Recently, prostaglandin E synthases (PGES), which specifically catalyze the final step of PGE2 biosynthesis, were characterized. Among these enzymes, the microsomal prostaglandin E synthase-1 (mPGES-1) was of a particular interest since it was shown to be up-regulated by inflammatory signals in a variety of cell types. Moreover, mPGES-1 was shown to be crucial for correct immune-to-brain communication and induction of fever and anorexia by pro-inflammatory agents. This review takes stock of previous knowledge and recent advances in understanding the role of prostaglandins and of their specific synthesizing enzymes in the molecular mechanisms underlying sickness behavior. The review concludes with a short summary of key questions that remain to be addressed and points out therapeutic developments in this research field.

Background/aim: Protein kinase C (PKC) is a family of isoenzymes playing a key role in the regulation of gonadotrope cell functions. Specific PKC isoforms are activated and downregulated differentially by gonadotropin-releasing hormone (GnRH) and the phorbol ester TPA. In the present study, focusing mainly on PKC epsilon, the mechanisms underlying the proteasome-dependent downregulation of GnRH-activated PKC epsilon and TPA-sensitive PKC alpha and epsilon isoenzymes were investigated in alphaT3-1 gonadotrope cells., Methods/results: In pull-down assays involving the use of glutathione-agarose affinity beads conjugated with a GST-fusion protein containing ubiquitin-associated domains of Rad23 that bind very likely to K48-linked polyubiquitinated proteins, TPA induced rapid (within 15 min) and sustained (up to 4 h) PKC alpha and PKC epsilon polyubiquitination. However, GnRH selectively elicited receptor-dependent polyubiquitination of PKC epsilon, but not that of PKC alpha. The GnRH-evoked PKC epsilon polyubiquitination was a strong, fast process (taking place as early as 10 min) which decreased progressively with time (but was still detectable after 4 h of treatment). In addition, no apparent association between PKC epsilon and the lysosomal compartment was observed upon performing double-labeling immunofluorescence and confocal microscopy, after either 10 min or 1 hour of stimulation by GnRH or the phorbol ester., Conclusion: In alphaT3-1 gonadotrope cells, polyubiquitination is therefore the event triggering GnRH-evoked PKC epsilon desensitization as well as TPA-induced PKC alpha and PKC epsilon downregulations; it precedes the respective isoenzyme's degradation by the proteasome complex.

In human somatotroph adenomas, growth hormone (GH) hypersecretion can be inhibited by somatostatin analogues such as octreotide. Unfortunately, serum GH levels reach normal values in only 60% of treated patients. The decreased sensitivity to octreotide is strongly related to a lower expression of somatostatin receptor sst2. In this present study, the sst2 gene was transferred by an adenoviral vector (Ad-sst2) in human somatotroph (n = 7) and lactotroph (n = 2) adenomas in vitro. Sst2 mRNA levels and sst2 immunostaining dramatically increased after infection. Ten days after infection at 20 multiplicity of infection (MOI), sst2 gene transfer decreased cell viability from 19% to 90% by caspase-dependent apoptosis. At low viral doses (5 MOI), Ad-sst2 decreased GH or prolactin (PRL) basal secretion and mRNA expression. Somatotroph tumors were classified in three groups according to their octreotide sensitivity. Four days after infection by 5 MOI Ad-sst2, the maximal GH suppression by octreotide increased from 31% to 57% in the octreotide partially resistant group and from 0% to 27% in the resistant ones. In the octreotide-sensitive group, EC(50) values significantly decreased from 1.3 x 10(-11) to 6.6 x 10(-13) mol/L without improving maximal GH suppression. Finally, lactotroph tumors, nonresponding to octreotide in basal conditions, became octreotide sensitive with a maximal PRL suppression of 43% at 10(-8) mol/L. Therefore, sst2 reexpression is able to improve octreotide sensitivity. Sst2 gene transfer may open new therapeutic strategies in treatment combined with somatostatin analogues.

The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.

The anterior pituitary-specific transcription factor Pit-1 was initially identified and cloned as a transactivator of the prolactin (PRL) and GH genes and later as a regulator of the TSHb gene. It was found to be a major developmental regulator, because natural Pit-1 gene mutations cause a dwarf phenotype in mice and cause combined pituitary hormone deficiency associated with pituitary hypoplasia in humans. To further investigate the growth-promoting effects of Pit-1, we used a strategy based on the use of dominant-negative Pit-1 mutants as an alternative means of inactivating endogenous Pit-1 functions. R271W, a Pit-1 mutant identified in one allele in patients with severe combined pituitary hormone deficiency, and Pit-1Delta1-123, a deletion mutant in which only the DNA binding domain of Pit-1 is conserved, were generated, and their ability to abolish the effects of the endogenous native Pit-1 in the differentiated proliferating somatolactotrope GH4C1 cell line was investigated. Enforced expression of the dominant-negative mutants in GH4C1 cells using recombinant lentiviral vectors decreased the levels of expression of known Pit-1 target genes such as PRL and GH, abolished the hormone release, and reduced cell viability by decreasing the growth rate and inducing apoptosis via a caspase-independent pathway. These results show for the first time that the growth-promoting effects of Pit-1 are at least partly due to the fact that this transcription factor prevents apoptotic cell death.