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Background/Aims: Fetuin-A (alpha-2-HS-glycoprotein, AHSG), a liver borne plasma protein, contributes to the prevention of soft tissue calcification, modulates inflammation, reduces insulin sensitivity and fosters weight gain following high fat diet or ageing. In polycystic ovary syndrome, fetuin-A levels correlate with free androgen levels, an observation pointing to androgen sensitivity of fetuin-A expression. The present study thus explored whether the expression of hepatic fetuin-A is modified by testosterone. Methods: HepG2 cells were treated with testosterone and androgen receptor antagonist flutamide, and were silenced with androgen receptor siRNA. To test the in vivo relevance, male mice were subjected to androgen deprivation therapy (ADT) for 7 weeks. AHSG mRNA levels were determined by quantitative RT-PCR and fetuin-A protein abundance by Western blotting. Results: In HepG2 cells, AHSG mRNA expression and fetuin-A protein abundance were both up-regulated following testosterone treatment. The human alpha-2-HS-glycoprotein gene harbors putative androgen receptor response elements in the proximal 5 kb promoter sequence relative to TSS. The effect of testosterone on AHSG mRNA levels was abrogated by silencing of the androgen receptor in HepG2 cells. Moreover, treatment of HepG2 cells with the androgen receptor antagonist flutamide in presence of endogenous ligands in the medium significantly down-regulated AHSG mRNA expression and fetuin-A protein abundance. In addition, ADT of male mice was followed by a significant decrease of hepatic Ahsg mRNA expression and fetuin-A protein levels. Conclusions: Testosterone participates in the regulation of hepatic fetuin-A expression, an effect mediated, at least partially, by androgen receptor activation. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Human ether-a-go-go related-gene K+ channels (hERG) participate in the regulation of tumor cell proliferation and apoptosis. HERG channel activity is up-regulated by growth factors. Kinases sensitive to growth factor signaling include the serine/threonine protein kinase B-RAF. The present study thus explored whether B-RAF influences hERG channel expression and activity. To this end, hERG channels were expressed in Xenopus oocytes with or without wild-type B-RAF, hERG channel activity was determined utilizing dual-electrode voltage clamp and hERG protein abundance in the cell membrane was analyzed utilizing confocal microscopy as well as chemiluminescence. Moreover, in rhabdomyosarcoma RD cells the effect of B-RAF inhibitor PLX-4720 on hERG-mediated current was quantified by whole-cell patch clamp and hERG cell surface protein abundance by utilizing biotinylation of cell surface proteins as well as flow cytometry. As a result, co-expression of wild-type B-RAF in hERG-expressing Xenopus oocytes significantly increased hERG channel activity and hERG channel protein abundance in the cell membrane. Treatment for 24 hours of B-RAF and hERG-expressing Xenopus oocytes with B-RAF inhibitor PLX-4720 (10 µM) significantly decreased hERG-mediated current and hERG cell surface expression. Similarly, in rhabdomyosarcoma RD cells, treatment for 24 hours with B-RAF inhibitor PLX-4720 significantly decreased hERG cell membrane protein abundance and hERG-mediated current. In conclusion, B-RAF is a powerful regulator of hERG channel activity and cell surface hERG protein abundance. [ABSTRACT FROM AUTHOR]

Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by renal 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)2D3 regulates Ca2+ and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)2D3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)2D3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 µM CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)2D3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)2D3 synthesis. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]