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Search for other papers by J Quirk in
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Search for other papers by P Brown in
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The homeobox repressor Hesx1, expressed throughout Rathke's pouch and required for normal pituitary development, has been implicated in anterior pituitary pathogenesis in man. Prolonged expression of Hesx1 delays the appearance of anterior pituitary terminal differentiation markers in mice, particularly the gonadotroph hormones. We tested if Hesx1 could modulate gonadotrophin gene expression directly, and found that Hesx1 repressed both common alpha subunit (alpha GSU) and luteinising hormone beta-subunit (LH beta) gene promoters. Repression mapped to the Pitx1 homeodomain protein transactivation site in the proximal alpha GSU promoter, but did not map to the equivalent site on LH beta. Hesx1 repression of the alpha GSU Pitx1 site was overridden by co-transfection of Pitx1. In contrast, Hesx1 antagonised Pitx1 transactivation of LH beta in a dose-dependent manner. This was due to monomeric binding of Hesx1 on alpha GSU and homodimerisation on LH beta. The homodimerisation site comprises the Pitx1 DNA binding site and a proximal binding site, and mutation of either inhibited homodimer formation. Conversion of the LH beta Pitx1 DNA binding site to an alpha GSU-type did not promote homodimer formation, arguing that Hesx1 has pronounced site selectivity. Furthermore, mutation of the proximal half of the homodimerisation site blocked Hesx1 antagonisation of Pitx1 transactivation. We conclude that Hesx1 monomers repress gene expression, and homodimers block specific transactivation sites.
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Search for other papers by B. L. Brown in
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Search for other papers by P. R. M. Dobson in
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ABSTRACT
Bradykinin stimulated prolactin secretion from monolayer cultures of rat anterior pituitary cells, the stimulation being greater from the cells of male rats. This stimulated secretion was accompanied by a rise in total inositol phosphate accumulation, suggesting that the action of bradykinin is mediated by phosphoinositide hydrolysis. The increase in inositol phosphate accumulation was biphasic; a further sharp rise occurred when the concentration of bradykinin exceeded 1 μmol/l. This may indicate that bradykinin acts on other cell types in the pituitary gland. Bradykinin had no effect on growth hormone secretion from cells of normal pituitary glands, or on prolactin secretion and phosphoinositide metabolism in GH3 rat pituitary tumour cells. Bradykinin receptor antagonists (both B1 and B2) had no effect on either bradykinin-stimulated inositol phosphate accumulation or prolactin secretion. Kallikreins, the enzymes responsible for the generation of kinins, are known to be present in the adenohypophysis. Therefore, the results presented here would suggest that kinins may have a role as paracrine agents in the pituitary gland.
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Search for other papers by B.L. Brown in
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ABSTRACT
The mechanism of action of the cytokine, interleukin-l (IL-1), has been investigated. Mouse thymoma (EL4 6.1) cells were preincubated with [3H]-glycerol and then incubated with recombinant IL-1β for varying periods. Interleukin-1 caused a rapid increase in diacylglycerol production (approx. 2 fold at 30 sees). This reproducible enhancement of diacylglycerol accumulation was abolished by pretreatment of the cells with pertussis toxin. Interestingly, a similar IL-1 induced increase in diacylglycerol was observed when the cells were preincubated with [3H]-myristic acid. These results appear to suggest a novel mode of action of interleukin-1 which involves a G-protein mediated breakdown of a membrane lipid resulting in the production of diacylglycerol. It is suggested that one possible candidate for this parent lipid may be a phosphatidylinositol glycan.
Search for other papers by J. R. McNeilly in
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Search for other papers by A. J. Clark in
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Search for other papers by A. S. McNeilly in
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ABSTRACT
While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free α subunit.
Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-β mRNA and FSH-β mRNA levels (to 5 and 30% of luteal control values respectively), but not α mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in α mRNA, there was a decrease in free α subunit in both the pituitary and plasma (to 30 and 80% of control levels).
These results suggest that, while GnRH positively regulates the expression of both gonadotrophin β-subunit genes, it can, under certain circumstances, negatively regulate α-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of β-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway. The dramatic reduction in LH pituitary content indicates that GnRH is required for the operation of a regulatory/storage pathway for the secretion of LH. There appears to be no similar mechanism for FSH. The LH-specific pathway is probably dependent upon the availability of LH-β subunits which subsequently plays a role in regulating α subunit by sequestering, assembling and storing the intact hormone in the presence of GnRH. Finally, in the absence of responsiveness to GnRH, the regulation of α-subunit production is not at the level of gene transcription. Inefficient translation of the mRNA or rapid degradation of the free α chain may account for the observed dramatic decrease in production of α subunit.
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Search for other papers by R J Brown in
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It has been 75 years since Evans and Long identified a somatic growth-promoting substance in pituitary extracts, yet it is only in the last 20 years that the molecular basis for this action has been established. Three key elements in this elucidation were the cloning of the GH receptor, the identification of Janus kinase (JAK) 2 as the receptor-associated tyrosine kinase, and the delineation of signal transduction and activators of transcription (STAT) 5a/b as the key transcription factor(s) activated by JAK2. The interaction between these three elements results in enhanced postnatal growth and is the subject of this review. We describe a new model for GH receptor activation based on subunit rotation within a constitutive dimer, together with the phenotype and hepatic transcript profile of mice with targeted knockins to the receptor cytoplasmic domain. These support a central role for STAT5a/b in postnatal growth.
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Search for other papers by C R Brown in
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Search for other papers by P J Barnes in
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ABSTRACT
Substance P has several inflammatory effects on the airways mediated via neurokinin 1 receptors (NK1Rs) and, if released from sensory nerves, may amplify the chronic inflammation seen in asthma. Northern blot analysis of NK1R mRNA in lung showed a 52 ± 10% (s.e.m.; P<0·01) increase in mRNA in the asthmatic lung compared with non-asthmatic control tissue. NK1R mRNA was reduced by 84·5 ± 1·9% after incubation with dexamethasone (1 μm) for 3 h (P<0·01). In contrast, NK2R mRNA was unaltered in asthmatic lungs and dexamethasone treatment had no effect on the level of NK2R mRNA. These results suggest that chronic inflammation in asthma may result in increased NK1R gene expression and that this effect is reversed by glucocorticosteroids.
Search for other papers by James E P Brown in
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The role of the adipocyte-derived factor visfatin in metabolism remains controversial, although some pancreatic β-cell-specific effects have been reported. This study investigated the effects of visfatin upon insulin secretion, insulin receptor activation and mRNA expression of key diabetes-related genes in clonal mouse pancreatic β-cells. β-TC6 cells were cultured in RPMI 1640 and were subsequently treated with recombinant visfatin. One-hour static insulin secretion was measured by ELISA. Phospho-specific ELISA and western blotting were used to detect insulin receptor activation. Real-time SYBR Green PCR array technology was used to measure the expression of 84 diabetes-related genes in both treatment and control cells. Incubation with visfatin caused significant changes in the mRNA expression of several key diabetes-related genes, including marked up-regulation of insulin (9-fold increase), hepatocyte nuclear factor (HNF)1β (32-fold increase), HNF4α (16-fold increase) and nuclear factor κB (40-fold increase). Significant down-regulation was seen in angiotensin-converting enzyme (−3.73-fold) and UCP2 (−1.3-fold). Visfatin also caused a significant 46% increase in insulin secretion compared to control (P<0.003) at low glucose, and this increase was blocked by co-incubation with the specific nicotinamide phosphoribosyltransferase inhibitor FK866. Both visfatin and nicotinamide mononucleotide induced activation of both insulin receptor and extracellular signal-regulated kinase (ERK)1/2, with visfatin-induced insulin receptor/ERK1/2 activation being inhibited by FK866. We conclude that visfatin can significantly regulate insulin secretion, insulin receptor phosphorylation and intracellular signalling and the expression of a number of β-cell function-associated genes in mouse β-cells.
Search for other papers by Qi Cheng in
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Search for other papers by Stanley M H Chan in
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Search for other papers by Jeremy R Brown in
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Search for other papers by Alan Bevington in
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Search for other papers by Terence P Herbert in
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Abstract
The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into the mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influence β-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets, and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L amino acid transporter 1 (LAT1) is abundantly expressed in the islets, and that knockdown of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the LAT1 is required for regulating β-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes.
Search for other papers by Kathryn L Auld in
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Search for other papers by Ying Zhang in
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Search for other papers by Bheem M Bhat in
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Search for other papers by Eugene L Brown in
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Search for other papers by Robert V Martinez in
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Based on its homology to the estrogen receptor and its roles in osteoblast and chondrocyte differentiation, the orphan nuclear receptor estrogen-related receptor α (ERRα (ESRRA)) is an intriguing therapeutic target for osteoporosis and other bone diseases. The objective of this study was to better characterize the molecular mechanisms by which ERRα modulates osteoblastogenesis. Experiments from multiple systems demonstrated that ERRα modulates Wnt signaling, a crucial pathway for proper regulation of bone development. This was validated using a Wnt-luciferase reporter, where ERRα showed co-activator-dependent (peroxisome proliferator-activated receptor gamma co-activator 1α, PGC-1α) stimulatory effects. Interestingly, knockdown of ERR α expression also enhanced WNT signaling. In combination, these data indicated that ERRα could serve to either activate or repress Wnt signaling depending on the presence or absence of its co-activator PGC-1α. The observed Wnt pathway modulation was cell intrinsic and did not alter β-catenin nuclear translocation but was dependent on DNA binding of ERRα. We also found that expression of active ERRα correlated with Wnt pathway effects on osteoblastic differentiation in two cell types, consistent with a role for ERRα in modulating the Wnt pathway. In conclusion, this work identifies ERRα, in conjunction with co-activators such as PGC-1α, as a new regulator of the Wnt-signaling pathway during osteoblast differentiation, through a cell-intrinsic mechanism not affecting β-catenin nuclear translocation.
