Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme catabolism and presumably is involved in cellular iron homeostasis. It is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. We examined induction of HO-1 mRNA in skin fibroblasts and investigated the mechanism by which it occurs. Hypoxia did not appear to act via induction of oxygen free radicals: induction of HO-1 was not sensitive to the free radical scavenger GSH or other antioxidants. Moreover, hypoxia did not increase steady-state levels of free radicals generated by fibroblasts. In contrast, HO-1 induction by the oxidants, H(2)O(2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) was significantly attenuated in the presence of free radical scavengers. This correlated with increased levels of free radical production in fibroblasts treated with these oxidants. Iron depletion by desferrioxamine mesylate, a specific iron complexon, completely inhibited hypoxic stimulation of HO-1 but did not attenuate the effect of H(2)O(2) and CCCP on HO-1 mRNA. Addition of Fe(2+), Fe(3+), or holo-transferrin to fibroblasts increased levels of HO-1 mRNA. Treatment of cells with hypoxia, but not H(2)O(2) or an exogenous source of iron, significantly increased the half-life of HO-1 mRNA. The data suggest hypoxia regulates HO-1 gene expression by a specific posttranscriptional mechanism: stabilization of mRNA. Hypoxia has previously been shown to increase fibroblast collagen synthesis and is thought to play a role in pathogenesis of systemic sclerosis (SSc). Skin fibroblasts isolated from patients with SSc demonstrated significantly stronger induction of HO-1 by hypoxia than did fibroblasts from normal controls. We hypothesize that exposure of SSc fibroblasts to hypoxic conditions leads to in vivo selective proliferation of cells that adapt to hypoxia.

Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase.

Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

Expression of lysyl oxidase from cDNA constructs in mammalian cells: the propeptide region is not essential to the folding and secretion of the functional enzyme.

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32-2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including beta-aminopropionitrile, phenylhydrazine, ethylenediamine, alpha, alpha'-dipyridyl, and diethyldithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase.

Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia.

Exposure of endothelial cells (EC) to hypoxia results in the increased expression of a distinct set of proteins with molecular masses of 56, 47, 39, 36, and 34 kDa. Their induction appears to be unique to EC and the stress of decreased oxygen tension. To understand the mechanism(s) and significance of the up-regulation of these proteins we have identified the 36-kDa protein by limited amino-terminal amino acid sequencing. The 21-amino acid sequence from the bovine protein exhibited 90.5% identity with the human sequence of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blot analysis showed that the time course and extent of EC GAPDH mRNA up-regulation correlated with the increase in 36-kDa protein synthesis. Nuclear runoff analysis demonstrated that this increase in GAPDH expression is regulated, in part, at the transcriptional level; however, the increase in the rate of transcription did not account for the entire mRNA accumulation, suggesting that GAPDH, like other hypoxia-regulated proteins, is posttranscriptionally regulated. Subcellular fractionation of hypoxic EC showed up-regulation of the 36-kDa protein in the cytoplasmic fraction and, to a lesser extent, in the nuclear fraction. The up-regulation of GAPDH in EC may be related to their relative hypoxia tolerance. Alternatively, the up-regulation of GAPDH in EC during hypoxia may be related to the potential nonglycolytic functions of this enzyme.

Direct demonstration of enol-oxaloacetate as an immediate product of malate oxidation by the mammalian succinate dehydrogenase.

Rapid malonate-sensitive transitory formation of enol-oxaloacetate followed by slow ketonization of the product was observed after addition of malate to the mammalian succinate-ubiquinone reductase in the presence of electron acceptor. The initial rate of enol-oxaloacetate production was equal to that of malate oxidation. Oxaloacetate keto-enol tautomerase had no effect on the initial rate of enol-oxaloacetate production nor on the kinetics of malate oxidation; the enzyme drastically accelerated the ketonization of the product. The solubilized and partially purified membrane-bound flavine adenine dinucleotide-dependent malate dehydrogenase from Acetobacter xylinum catalyzed oxidation of L- and D-malate without formation of enol-oxaloacetate as an intermediate of the reaction.

[Interaction of ATPase from submitochondrial fragments and a natural inhibitor protein during delta-mu-H+ generation on a membrane]. / Vzaimodeistvie ATPazy submitokhondrial'nykh fragmentov i prirodnogo belka-ingibitora v usloviiakh generatsii delta-mu-H+ na membrane.

An addition of the inhibitor protein (IF1) to submitochondrial particles (SMP) essentially free of endogenous IF1 (AS-SMP) results in a synchroneous inhibition of ATP hydrolysis and ATP-dependent reduction of NAD+ by succinate without any effect on the oxidative phosphorylation rate. The binding of IF1 to the membrane-bound ATPase leads to the loss of the inhibitor protein sensitivity to trypsin despite the delta mu H+ generation. The data obtained are consistent with a model according to which there exist the hydrolase and synthetase forms of F1 and contradict the generally accepted concepts on the delta mu H+-dependent dissociation of the F1-IF1 complex.

[Kinetics of the interaction of ATPase of submitochondrial fragments and a natural protein-inhibitor]. / Kinetika vzaimodeistviia ATPazy submitokhondrial'nykh fragmentov i prirodnogo belka-ingiboitora.

Conditions were selected which enable a quantitative assay of the ATPase inhibitor protein in submitochondrial particles. It was found that the isolated soluble inhibitor exhibits a marked pH-dependent hysteretic behaviour, i. e., an instant jump of pH for the inhibitor solution from 4.8 to 8.2 induced a slow alteration of its activity as measured by the inhibition of ATP hydrolysis by submitochondrial particles. In acid media (pH less than 6.8), the inhibitor is in the active, whereas in alkaline media (pH greater than 6.8) in the inactive state; the apparent pKa value for the cooperative active/inactive transition is 6.8. Treatment of the inhibitor protein with diethylpyrocarbonate, a specific reagent for histidine, completely abolishes its inhibitory activity. Two types of the inhibitor protein--ATPase interaction were revealed, i.e., reversible (ATP-independent) and irreversible (ATP-dependent) ones. Both reactions, i.e., ATP hydrolysis and ATP inhibition by the inhibitor in the presence of Mg2+ are characterized by a hyperbolic dependence of the reaction rate on ATP concentration; however, for both reactions the apparent KmATP values (50 and 5 microM, respectively) differ significantly (pH 8.0). Thus, the inhibitor--ATPase interaction shows that there exists a specific site for ATP in the ATPase which is different from the catalytic one. A model for the inhibitor protein interaction with ATPase which takes account of a slow pH-dependent conformational transformation of the inhibitor protein is proposed.

Interaction between the mitochondrial ATP synthetase and ATPase inhibitor protein. Active/inactive slow pH-dependent transitions of the inhibitor protein.

The rate of mitochondrial ATPase inactivation by the naturally occurring inhibitor protein in the presence of saturating ATP and Mg2+ at pH 8.0 depends hyperbolically on the amount of inhibitor added; the upper limit of an apparent first-order constant for the inactivation process is 1.0(-1) at 25 degrees C. A dramatic difference in the inactivation rate is observed when the protein inhibitor is added to the same assay system from either acidic (pH 4.8) or alkaline (pH 8.2) solutions. The slow reversible transition of the inhibitor from its rapidly reacting 'acidic' form to the slow reacting 'alkaline' form occurs when the solution of the protein inhibitor is subjected to a pH-jump from 4.8 to 8.2 (t1/2 approximately 30s at 25 degrees C). The pH-profile of the inhibitor active/inactive equilibrium suggests that a group with pKa approximately 6.5 is involved in the transition. Treatment of the inhibitor protein with a histidine-specific reagent (e.g. diethyl pyrocarbonate) abolishes its inactivating effect on the ATPase activity. It is concluded that the protonation/deprotonation of the inhibitor protein followed by its slow conformational changes is the rate-limiting step in the inhibitor-ATP synthetase interaction.