22 research outputs found
Role of AMP-Activated Protein Kinase on Steroid Hormone Biosynthesis in Adrenal NCI-H295R Cells
Regulation of human androgen biosynthesis is poorly understood. However, detailed knowledge is needed to eventually solve disorders with androgen dysbalance. We showed that starvation growth conditions shift steroidogenesis of human adrenal NCI-H295R cells towards androgen production attributable to decreased HSD3B2 expression and activity and increased CYP17A1 phosphorylation and 17,20-lyase activity. Generally, starvation induces stress and energy deprivation that need to be counteracted to maintain proper cell functions. AMP-activated protein kinase (AMPK) is a master energy sensor that regulates cellular energy balance. AMPK regulates steroidogenesis in the gonad. Therefore, we investigated whether AMPK is also a regulator of adrenal steroidogenesis. We hypothesized that starvation uses AMPK signaling to enhance androgen production in NCI-H295R cells. We found that AMPK subunits are expressed in NCI-H295 cells, normal adrenal tissue and human as well as pig ovary cells. Starvation growth conditions decreased phosphorylation, but not activity of AMPK in NCI-H295 cells. In contrast, the AMPK activator 5-aminoimidazole-4-carboxamide (AICAR) increased AMPKα phosphorylation and increased CYP17A1-17,20 lyase activity. Compound C (an AMPK inhibitor), directly inhibited CYP17A1 activities and can therefore not be used for AMPK signaling studies in steroidogenesis. HSD3B2 activity was neither altered by AICAR nor compound C. Starvation did not affect mitochondrial respiratory chain function in NCI-H295R cells suggesting that there is no indirect energy effect on AMPK through this avenue. In summary, starvation-mediated increase of androgen production in NCI-H295 cells does not seem to be mediated by AMPK signaling. But AMPK activation can enhance androgen production through a specific increase in CYP17A1-17,20 lyase activity
Isoelectric point mobility shift assay for rapid screening of charged and uncharged ligands bound to proteins.
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Cloning of novel human SEC14p-like proteins: ligand binding and functional properties.
We describe the cloning and expression of two novel genes highly similar to the tocopherol-associated protein (hTAP/SEC14L2/SPF). Immunoprecipitation of the three recombinant hTAPs and extraction of their associated lipid-soluble molecules indicates that they bind not just tocopherols, but also phosphatidylinositol, phosphatidylcholine, and phosphatidylglycerol. Ligand competition analysis by isoelectric point mobility shift assay indicates that phosphatidylcholine, tocopherols, and tocopheryl-succinate compete with phosphatidylinositol binding to hTAPs. To investigate a possible function of hTAPs on enzymes involved in phospholipids metabolism, the activity of recombinant phosphatidylinositol 3-kinase (PI3Kγ/p110γ) was tested. Recombinant hTAPs reduce in vitro the activity of the recombinant catalytic subunit of PI3Kγ and stimulate it in the presence of α-tocopherol up to 5-fold. Immunoprecipitation of hTAP1 from cells results in co-precipitation of PI3-kinase activity, indicating a physical contact between the two proteins at a cellular level. In summary, hTAPs may modulate, in a tocopherol-sensitive manner, phosphatidylinositol-3-kinase, a central enzyme in signal transduction, cell proliferation, and apoptosis. It is possible that other phosphatidylinositol- and phosphatidylcholine-dependent signaling pathways are modulated by hTAPs and tocopherols, possibly by transporting and presenting these ligands to the corresponding enzymes
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Characterization of three human sec14p-like proteins: a-Tocopherol
Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled α-tocopherol to mitochondria in the same order of magnitude as the human α-tocopherol transfer protein (α-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites