Accumulation and nuclear import of HIF1 alpha during high and low oxygen concentration in skeletal muscle cells in primary culture

AbstractThe hypoxia-inducible-factor-1 (HIF1) mediates the transcriptional upregulation of several target genes during hypoxia. HIF1 itself is known to be regulated essentially by ubiquitinylation and proteolytic degradation of the subunit HIF1α of the dimeric transcription factor HIF1. In contrast to other tissues, skeletal muscle expresses high amounts of HIF1α in normoxia as well as in hypoxia. In view of this, we aimed to investigate HIF1α accumulation and subcellular localization as well as the transcriptional activity of the HIF1α-regulated gene of glyceraldehyde dehydrogenase (GAPDH) in skeletal muscle cells exposed to low oxygen concentration (3% O2), normoxia (20% O2) or high oxygen concentration (42% O2). Immunofluorescence analysis reveals that under normoxic and high oxygen conditions, significant amounts of HIF1α can be found exclusively in the cytoplasm of the myotubes. Muscle cells treated with CoCl2, a known inhibitor of HIF1α degradation, show even higher levels of HIF1α, again exclusively in the cytoplasm. Under conditions of low oxygen, HIF1α in controls as well as in CoCl2-treated cells is found in the nuclei. CdCl2 inhibits nuclear import of HIF1α at low oxygen concentration and leads to a transcriptional downregulation of the marker enzyme of anaerobic glycolysis GAPDH. Immunoprecipitation with anti-HIF1α antibody co-precipitates HSP90 in an oxygen-dependent manner, more at high pO2 than at low pO2. Cadmium-treated samples also show high amounts of co-immunoprecipitated HSP90, independent of oxygen concentration. We conclude that in skeletal muscle cells, HIF1α, in contrast to other tissues, may, in addition to its regulation by degradation, also be regulated by binding to HSP90 and subsequent inhibition of its import into the nuclei

T Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle

We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV–CAIX–CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.Public Library of Scienc

Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme of the oxidative part of the pentose phosphate pathway and serves as the major source of NADPH for metabolic reactions and oxidative stress response in pro- and eukaryotic cells. We here report on a strain of the model yeast Saccharomyces cerevisiae which lacks the G6PD-encoding ZWF1 gene and displays distinct growth retardation on rich and synthetic media, as well as a strongly reduced chronological lifespan. This strain was used as a recipient to introduce plasmid-encoded heterologous G6PD genes, synthesized in the yeast codon usage and expressed under the control of the native PFK2 promotor. Complementation of the hypersensitivity of the zwf1 mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (HsG6PD1), the milk yeast Kluyveromyces lactis (KlZWF1), the bacteria Escherichia coli (EcZWF1) and Leuconostoc mesenteroides (LmZWF1), as well as the genes encoding three different plant G6PD isoforms from Arabidopsis thaliana (AtG6PD1, AtG6PD5, AtG6PD6). The plastidic AtG6PD1 isoform retained its redox-sensitive activity when produced in the yeast as a cytosolic enzyme, demonstrating the suitability of this host for determination of its physiological properties. Mutations precluding the formation of a disulfide bridge in AtG6PD1 abolished its redox-sensitivity but improved its capacity to complement the yeast zwf1 deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications

Cytosolic GAPDH as a redox-dependent regulator of energy metabolism

Abstract Background Plant cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GapC) displays redox-dependent changes in its subcellular localizations and activity. Apart from its fundamental role in glycolysis, it also exhibits moonlighting properties. Since the exceptional redox-sensitivity of GapC has been suggested to play a crucial role in its various functions, we here studied its redox-dependent subcellular localization and the influence of the redox-state on GapC protein interactions. Results In mesophyll protoplasts from Arabidopsis thaliana, colocalization of GapC with mitochondria was more pronounced under reducing conditions than upon oxidative stress. In accordance, reduced GapC showed an increased affinity to the mitochondrial voltage-dependent anion-selective channel (VDAC) compared to the oxidized one. On the other hand, nuclear localization of GapC was increased under oxidizing conditions. The essential role of the catalytic cysteine for nuclear translocation was shown by using the corresponding cysteine mutants. Furthermore, interaction of GapC with the thioredoxin Trx-h3 as a candidate to revert the redox-modifications, occurred in the nucleus of oxidized protoplasts. In a yeast complementation assay, we could demonstrate that the plant-specific non-phosphorylating glyceraldehyde 3-P dehydrogenase (GapN) can substitute for glucose 6-P dehydrogenase to generate NADPH for re-reduction of the Trx system and ROS defense. Conclusions The preferred association of reduced, glycolytically active GapC with VDAC suggests a substrate-channeling metabolon at the mitochondrial surface for efficient energy generation. Increased occurrence of oxidized GapC in the nucleus points to a function in signal transduction and gene expression. Furthermore, the interaction of GapC with Trx-h3 in the nucleus indicates reversal of the oxidative cysteine modification after re-establishment of cellular homeostasis. Both, energy metabolism and signal transfer for long-term adjustment and protection from redox-imbalances are mediated by the various functions of GapC. The molecular properties of GapC as a redox-switch are key to its multiple roles in orchestrating energy metabolism