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Hypoxia-inducible factor (HIF) plays an important role in regulating gene expression in response to ischemia. Although activation of HIF-1 in muscle tissue was found during ischemia in vivo, the meaning and mechanisms in isolated cells are still incompletely understood. We studied activation of HIF-1 in skeletal muscle cells cultured in either their undifferentiated myoblast state or differentiated into myotubes. HIF-1 was activated in myoblasts and myotubes by hypoxia and simulated ischemia. Induction of adrenomedullin mRNA and, to a lesser extent, VEGF mRNA correlated well with the induction of HIF-1[alpha] protein in both cell types. Enzymes of glycolysis-like lactate dehydrogenase and pyruvate kinase showed upregulation of their mRNA only under hypoxic conditions but not during simulated ischemia. Phosphofructokinase mRNA showed no significant upregulation at all. Although HIF-1 was activated in myotubes during simulated ischemia, myotubes died preceded by a loss of ATP. Myoblasts survived simulated ischemia with no decrease in ATP or ATP turnover. Furthermore, pharmacological inhibition of HIF-1 hydroxylases by dimethyloxalylglycine (DMOG) increased HIF-1[alpha] accumulation and significantly upregulated the expression of adrenomedullin, VEGF, lactate dehydrogenase, and pyruvate kinase in myoblasts and myotubes. However, DMOG provided no protection from cell death. Our data indicate that HIF-1, although activated in myotubes during simulated ischemia, cannot protect against the loss of ATP and cell viability. In contrast, myoblasts survive ischemia and thus may play an important role during regeneration and HIF-1-induced revascularization. hypoxia; myoblasts; adrenomedullin; vascular endothelial growth factor; lactate dehydrogenase

Hypoxia-inducible factor-1 (HIF-1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia-inducible genes that ensure an adequate energy supply. Assembly of the HIF-1 complex by its oxygen-regulated subunit HIF-1α and its constitutive β subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. By two-photon microscopy, we studied HIF-1 assembly in living cells and the mobility of fluorophorelabeled HIF-1 subtmits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF-1α than of HIF-1β, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between α and β subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits were localized as close as 6.2 nm, but even the N and C terminus of the HIF-1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3-dimensional organization of the HIF complex than described so far by 2-dimensional models. [ABSTRACT FROM AUTHOR]

A unique feature of the germinal center B cell-derived Hodgkin and Reed/Sternberg cells of classical Hodgkin lymphoma is their lost B cell phenotype and the aberrant expression of factors of other hematopoietic cell types, including ID2 and NOTCH1. As cellular dedifferentiation and upregulation of ID2 and NOTCH1 are typical consequences of a hypoxic response, we wondered whether hypoxia may impose an HRS cell-like phenotype in B cells. Culturing normal B cells or cell lines of germinal center-type diffuse large B-cell lymphoma under hypoxic conditions caused partial downregulation of several B cell markers, ID2 upregulation, and increased NOTCH1 activity. The hypoxic cells acquired further features of Hodgkin and Reed/Sternberg cells, including increased JUN expression, and enhanced NFκB activity. The Hodgkin and Reed/Sternberg cell-expressed epigenetic regulators KDM4C and PCGF2, as well as the phosphatase DUSP1 were partially induced in hypoxic B cells. Inhibition of DUSP1 was toxic for classical Hodgkin lymphoma cell lines. Thus, hypoxia induces key Hodgkin and Reed/Sternberg cell characteristics in mature B cells. We speculate that hypoxic conditions in the germinal center may impose phenotypic changes in germinal center B cells, promoting their survival and initiating their differentiation towards a Hodgkin and Reed/Sternberg cell-like phenotype. These may then be stabilized by transforming events in the Hodgkin and Reed/Sternberg precursor cells., (Copyright© Ferrata Storti Foundation.)

Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression.

Thyroid hormones are important regulators of differentiation, growth, metabolism, and physiological function of virtually all tissues. Active thyroid hormone T(3) affects expression of genes that encode for angiogenic proteins like adrenomedullin or vascular endothelial growth factor and erythropoietin, as well as for glucose transporters and phospho fructokinase that determine glucose use. Interestingly, those target genes are also hypoxia inducible and under the control of the oxygen-dependent transcription factor hypoxia-inducible factor (HIF)-1). We and others have reported that T(3) stimulates HIF-1 activation, which intimately links T(3) and HIF-1 induced gene expression. Here, we studied intracellular pathways that mediate HIF-1alpha regulation by T(3). We found that T(3)-dependent HIF-1 activation is not limited to hepatoma cells but is also observed in primary human hepatocytes, kidney and lung carcinoma cells. T(3) increased the HIF-1alpha subunit mRNA and protein within a few hours through activation of the thyroid hormone receptor beta retinoid X receptor alpha heterodimer because knockdown of each of the partners abrogated the stimulation by T(3). However, T(3) had no direct effect on transcription of HIF-1alpha, but activation of the thyroid hormone receptor beta/retinoid X receptor alpha heterodimer by T(3) stimulated expression of the hepatic leukemia factor, which increases HIF-1alpha gene expression.