GSK3-controlled sympathetic activity in FGF23 production and FGF23 gene regulation by actin cytoskeleton reorganization

Fibroblast growth factor (FGF) 23 is a bone derived phosphaturic hormone. It is a potent regulator of vitamin D metabolism and phosphate homeostasis. FGF23 is secreted from the bone and exerts its function on the kidney. Further, it inhibits 25-dihydroxyvitamin D 1α-hydroxylase and reduces formation of active vitamin D and stimulates 25-dihydroxyvitamin D 24-hydroxylase, which in turn favors degradation of vitamin D. FGF23 inhibits renal phosphate reabsorption in the proximal tubules, thereby lowering serum phosphate and active vitamin D. To exert its renal function, FGF23 requires klotho as a co-receptor. Both FGF23 and klotho deficiency, lead to vascular calcification, hyperphosphatemia, hypercalcemia, muscular atrophy and profound aging like phenotypes. Glycogen synthase kinase (GSK) 3 is a serine-threonine kinase, which is ubiquitously expressed and involved in a variety of cellular process including; glycogen metabolism, transcription, translation, proliferation, survival, cell cycle regulation, cytoskeleton reorganization and apoptosis. GSK3 is a downstream signaling molecule of phosphoinositide-3 kinase (PI3 kinase)/PKB/Akt pathway. Mice expressing PKB insensitive GSK3α/β (gsk-3ki) show enhanced sympathetic nervous activity and phosphaturia with low bone density. The sympathetic nervous system was shown to stimulate FGF23 release. In this thesis, I investigated the role of GSK3-controlled sympathetic activity in the production of FGF23 and phosphate metabolism. Serum FGF23, urinary epinephrine, Vanillylmandelic acid (VMA), phosphate and calcium excretion were significantly higher in gsk-3ki mice compared to gsk-3WT mice. Serum FGF23 and 1,25(OH)2D3 concentrations were lower in gsk3KI mice than in gsk-3WT mice. Mice were treated with β-blocker (propranolol) for one week resulted in decreased serum FGF23 and renal phosphate and calcium loss and elevated serum phosphate concentration in gsk-3KI mice. Thus, these data suggest that PI3K insensitive GSK3 participates in the regulation of FGF23 formation, vitamin D metabolism and, thereby mineral metabolism by sympathetic nervous system. Next, I explored the role of actin cytoskeleton reorganization in controlling FGF23 production. Previous findings have suggested that 1,25(OH)2D3 and NF-κB stimulates FGF23 production. Therefore, the role of 1,25(OH)2D3-induced actin polymerization on Fgf23 expression in UMR 106 osteoblast-like cells was explored. Actin polymerization dynamics was determined by Western blotting and confocal imaging and Fgf23 transcript levels was measured by qRT-PCR. Western blotting and confocal imaging data showed 1,25(OH)2D3 induces actin polymerization in UMR 106 osteoblast-like cells. Thus induction of FGF23 production by 1,25(OH)2D3 resulted in actin polymerization, an effect blocked by the pharmacological inhibitor of NF-κB wogonin (100 μM). Cytochalasin B (100 nM) is a actin microfilament disrupting agent which abolished 1,25(OH)2D3-induced Fgf23 gene transcription, pointing a role of actin cytoskeleton in Fgf23 expression. Both Rac1 inhibitor NSC23766 (50 μM) and IPA3 (10 μM) blocked 1,25(OH)2D3-induced Fgf23 expression, suggesting the mechanism involved in actin cytoskeleton- controlled Fgf23 expression in UMR 106 cells involves Rac1 small GTPase signaling. These results provide strong evidence that actin reorganization regulated by Rac1 signaling contributes to 1,25(OH)2D3-induced Fgf23 gene transcription

Dose-response relationship between arsenic exposure and the serum enzymes for liver function tests in the individuals exposed to arsenic: a cross sectional study in Bangladesh

<p>Abstract</p> <p>Background</p> <p>Chronic arsenic exposure has been shown to cause liver damage. However, serum hepatic enzyme activity as recognized on liver function tests (LFTs) showing a dose-response relationship with arsenic exposure has not yet been clearly documented. The aim of our study was to investigate the dose-response relationship between arsenic exposure and major serum enzyme marker activity associated with LFTs in the population living in arsenic-endemic areas in Bangladesh.</p> <p>Methods</p> <p>A total of 200 residents living in arsenic-endemic areas in Bangladesh were selected as study subjects. Arsenic concentrations in the drinking water, hair and nails were measured by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). The study subjects were stratified into quartile groups as follows, based on concentrations of arsenic in the drinking water, as well as in subjects' hair and nails: lowest, low, medium and high. The serum hepatic enzyme activities of alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT) were then assayed.</p> <p>Results</p> <p>Arsenic concentrations in the subjects' hair and nails were positively correlated with arsenic levels in the drinking water. As regards the exposure-response relationship with arsenic in the drinking water, the respective activities of ALP, AST and ALT were found to be significantly increased in the high-exposure groups compared to the lowest-exposure groups before and after adjustments were made for different covariates. With internal exposure markers (arsenic in hair and nails), the ALP, AST and ALT activity profiles assumed a similar shape of dose-response relationship, with very few differences seen in the higher groups compared to the lowest group, most likely due to the temporalities of exposure metrics.</p> <p>Conclusions</p> <p>The present study demonstrated that arsenic concentrations in the drinking water were strongly correlated with arsenic concentrations in the subjects' hair and nails. Further, this study revealed a novel exposure- and dose- response relationship between arsenic exposure metrics and serum hepatic enzyme activity. Elevated serum hepatic enzyme activities in the higher exposure gradients provided new insights into arsenic-induced liver toxicity that might be helpful for the early prognosis of arsenic-induced liver diseases.</p

miR-204 suppresses the development and progression of human glioblastoma by targeting ATF2

In human cancers, miRNAs are important regulators of multiple cellular processes, and aberrant miRNA expression has been observed, and their alterations contribute to multiple cancer development and progression. Till now, little has been known about the role of miR-204 in human glioblastoma (GBM). In the present study, we used in-vitro assays to investigate the mechanisms of miR-204 in GBM cell lines and 60 cases of GBM tissues. Here, we found that miR-204 expression is downregulated in both GBM cell lines A172, U87 and U251 cells and GBM tissues as compared with NHA cells and normal tissues (all p < 0.001). In addition, the ectopic expression of miR-204 suppressed A172 and U87 cell proliferation, migration and invasion. Meanwhile, miR-204 over-expression extremely inhibited the protein expression of ATF2. Notably, the enforced expression of ATF2 in A172 and U87 cells with the over-expression of miR-204 attenuated the inhibitory effects of miR-204 on proliferation, migration and invasion. In conclusion, our findings suggest that miR-204 suppressed cell proliferation, migration and invasion through inhibition of ATF2, thus, miR-204 may function as a useful drug target in the treatment and diagnosis of GBM

Influence of annexin A7 on insulin sensitivity of cellular glucose uptake

Insulin sensitivity is decreased by prostaglandin E-2 (PGE(2)), a major product of cyclooxygenase (COX). As shown in erythrocytes, PGE(2) formation is inhibited by annexin A7. The present study defined the role of annexin A7 in glucose metabolism. Gene-targeted mice lacking annexin A7 (annexin7 (-/-)) were compared to wild-type mice (annexin7 (+/+)). The serum 6-Keto-prostaglandin-F-1 alpha (6-Keto-PGF(1)alpha) concentration was measured by ELISA and hepatic COX activity determined by an enzyme assay. Expression of COX-1, COX-2, prostaglandin E synthase, GLUT-4, and insulin receptor was determined by Western blotting. Glucose and insulin serum concentrations were analyzed following an intraperitoneal glucose load and glucose serum levels after intraperitoneal injection of insulin. Experiments were done without and with pretreatment of the mice with COX-inhibitor aspirin. The serum 6-Keto-PGF(1)alpha level and hepatic COX activity were significantly higher in annexin7 (-/-) than in annexin7 (+/+) mice. Hepatic COX-1 expression was higher in annexin7 (-/-) mice. Glucose tolerance was decreased in annexin7 (-/-) mice. Intraperitoneal insulin injection decreased the serum glucose level in both genotypes, an effect significantly less pronounced in annexin7 (-/-) mice. Glucose-induced insulin secretion was higher in annexin7 (-/-) mice. GLUT-4 expression in skeletal muscle from annexin7 (-/-) mice was reduced. Aspirin pretreatment lowered the increase in insulin concentration following glucose injection in both genotypes and virtually abrogated the differences in serum insulin between the genotypes. Aspirin pretreatment improved glucose tolerance in annexin7 (-/-) mice. In conclusion, annexin A7 influences insulin sensitivity of cellular glucose uptake and thus glucose tolerance. These effects depend on COX activity

Up-regulation of FGF23 release by aldosterone

The fibroblast growth factor (FGF23) plasma level is high in cardiac and renal failure and is associated with poor clinical prognosis of these disorders. Both diseases are paralleled by hyperaldosteronism. Excessive FGF23 levels and hyperaldosteronism are further observed in Klotho-deficient mice. The present study explored a putative aldosterone sensitivity of Fgf23 transcription and secretion the putative involvement of the aldosterone sensitive serum & glucocorticoid inducible kinase SGK1, SGK1 sensitive transcription factor NFκB and store operated Ca(2+) entry (SOCE). Serum FGF23 levels were determined by ELISA in mice following sham treatment or exposure to deoxycorticosterone acetate (DOCA) or salt depletion. In osteoblastic UMR106 cells transcript levels were quantified by qRT-PCR, cytosolic Ca(2+) concentration utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, DOCA treatment and salt depletion of mice elevated the serum C-terminal FGF23 concentration. In UMR106 cells aldosterone enhanced and spironolactone decreased SOCE. Aldosterone further increased Fgf23 transcript levels in UMR106 cells, an effect reversed by mineralocorticoid receptor blockers spironolactone and eplerenone, SGK1 inhibitor EMD638683, NFκB-inhibitor withaferin A, and Ca(2+) channel blocker YM58483. In conclusion, Fgf23 expression is up-regulated by aldosterone, an effect sensitive to SGK1, NFκB and store-operated Ca(2+) entry

NFκB-sensitive Orai1 expression in the regulation of FGF23 release

Fibroblast growth factor (FGF23) plasma levels are elevated in cardiac and renal failure and correlate with poor clinical prognosis of those disorders. Both disorders are associated with inflammation and activation of the inflammatory transcription factor NFκB. An excessive FGF23 level is further observed in Klotho-deficient mice. The present study explored a putative sensitivity of FGF23 expression to transcription factor NFκB, which is known to upregulate Orai1, the Ca(2+) channel accomplishing store-operated Ca(2+) entry (SOCE). In osteoblastic cells (UMR106) and immortalized primary periosteal (IPO) cells, protein abundance was determined by Western blotting, and in UMR106 cells, transcript levels were quantified by RT-PCR, cytosolic Ca(2+) activity utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, UMR106 and IPO cells expressed Ca(2+) channel Orai1. SOCE was lowered by NFκB inhibitor wogonin as well as by Orai1 inhibitors 2-APB and YM58483. UMR106 cell Fgf23 transcripts were increased by stimulation of SOCE and Ca(2+) ionophore ionomycin and decreased by Orai inhibitors 2-APB, YM58483 and SK&F96365, by Orai1 silencing, as well as by NFκB inhibitors wogonin, withaferin A, and CAS 545380-34-5. In conclusion, Fgf23 expression is upregulated by stimulation of NFκB-sensitive, store-operated Ca(2+) entry. KEY MESSAGES Osteoblast UMR106 and IPO cells express Ca(2+) channel Orai1. Osteoblast store-operated Ca(2+) entry is accomplished by NFκB-sensitive Orai1. Osteoblast Fgf23 transcription is upregulated by increase in the cytosolic Ca(2+) activity. Fgf23 transcription is decreased by Orai inhibitors and Orai1 silencing. Fgf23 transcription is lowered by NFκB inhibitors

AMPKα1-Sensitivity of Orai1 and Ca2+ Entry in T - Lymphocytes

Background/Aims: T-lymphocyte activation and function critically depends on Ca2+ signaling, which is regulated by store operated Ca2+ entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca2+ concentration ([Ca2+]i) by treatment of the cells with Ca2+ ionophore or following inhibition of endosomal Ca2+ ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca2+ entry and Ca2+-sensitive regulation of T-lymphocyte function. Methods: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk-/-) mice and from their wildtype (ampk+/+) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca2+]i estimated from Fura-2 fluorescence, SOCE from increase of [Ca2+]i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting. Results: Expression of surface markers in CD4+ and CD8+ T-cells were similar in ampk-/- and ampk+/+ T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk-/- and ampk+/+ T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk-/- than in ampk+/+ T-lymphocyte blasts. SOCE and increase of [Ca2+]i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk-/- than in ampk+/+ T-lymphocyte blasts. The difference of Ca2+ entry between ampk-/- and ampk+/+ T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk-/- lymphocytes was higher than proliferation of ampk+/+ T-lymphocytes, a difference reversed by Orai1 silencing. Conclusions: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca2+ activity