Effect of endogenous heme generation on delta-aminolevulinic acid synthase activity in rat liver mitochondria.

This study examined the possibility that generation of heme within mitochondria may provide a local concentration sufficient to inhibit the activity of delta-aminolevulinic acid (ALA) synthase, the enzyme that catalyzes the rate-limiting step in hepatic heme biosynthesis. This was accomplished by simultaneously running ALA synthase and heme synthase activities in intact mitochondria isolated from rat liver. Radiochemical assays were used to measure the enzyme activities. ALA synthase activity did not decrease as the rate of heme formation was increased by varying the concentration of substrates for heme synthase. Even at a rate of heme generation estimated to be at least 75 times the rate occuring in vivo, ALA synthase activity was unchanged. We conclude that end product inhibition of ALA synthase activity by heme is not an important physiological mechanism for regulation of hepatic heme biosynthesis.     

Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms

Regulation of delta-aminolevulinic acid (ALA) synthase and heme oxygenase was analyzed in primary rat hepatocytes and in two immortalized cell lines, CWSV16 and CWSV17 cells. ALA synthase was induced by 4,6-dioxohepatnoic acid (4,6-DHA), a specific inhibitor of ALA dehydratase, in all three systems; however, the induction in CWSV17 cells was greater than in either of the other two systems. Therefore, CWSV17 cells were used to explore the regulation of both enzymes by heme and 4,6-DHA. Data obtained from detailed concentration curves demonstrated that 4,6-DHA induced the activity of ALA synthase once ALA dehydratase activity became rate-limiting for heme biosynthesis. Heme induced heme oxygenase activity with increases occurring at concentrations of 10 microM or greater. Heme blocked the 4,6-DHA-dependent induction of ALA synthase with an EC50 of 1.25 microM. Heme-dependent decreases of ALA synthase mRNA levels occurred more quickly and at lower concentrations than heme-dependent increases of heme oxygenase mRNA levels. ALA synthase mRNA remained at reduced levels for extended periods of time, while the increases in heme oxygenase mRNA were much more transient. The drastic differences in concentrations and times at which heme-dependent effects were observed strongly suggest that two-different heme-dependent mechanisms control the ALA synthase and heme oxygenase mRNAs. In CWSV17 cells, heme decreased the stability of ALA synthase mRNA from 2.5 to 1.3 h, while 4,6-DHA increased the stability of the mRNA to 5.2 h. These studies demonstrate that regulation of ALA synthase mRNA levels by heme in a mammalian system is mediated by a change in ALA synthase mRNA stability. The results reported here demonstrate the function of the regulatory heme pool on both ALA synthase and heme oxygenase in a mammalian hepatocyte system.     

Nucleotide sequence of mouse 5-aminolevulinic acid synthase cDNA and expression of its gene in hepatic and erythroid tissues

The cDNA coding for 5-aminolevulinic acid (ALA) synthase (EC 2.3.1.37) in both liver and anemic spleen of the mouse has been cloned. The liver clone was selected by complementation of an Escherichia coli hemA mutant. Erythroid clones were obtained by screening a cDNA library made from mouse anemic spleen RNA, using the liver cDNA as a probe. The sequences of the spleen-derived and liver-derived cDNAs are identical. The nucleotide sequence and predicted amino acid (aa) sequence of a 1.85-kb spleen-derived cDNA is presented. The mouse ALA synthase as sequence displays extensive homology to ALA synthase of chick embryonic liver. The ALA synthase mRNA, detected by Northern blot analysis, was the same size, approx. 2.3 kb, in mouse liver, anemic spleen, and mouse erythroleukemia cells. It is therefore unlikely that different isozymic forms of ALA synthase are present in mouse erythroid and hepatic tissue and this is not the basis for the different effects of heme and porphyrinogenic compounds on the expression of liver and erythroid ALA synthase.     

Effects of modulators of protein phosphorylation on heme metabolism in human hepatic cells: induction of delta-aminolevulinic synthase mRNA and protein by okadaic acid

Effects of modulators of protein phosphorylation on delta-aminolevulinic acid (ALA) synthase and heme oxygenase-1 mRNA were analyzed in the human hepatic cell lines Huh-7 and HepG2 using a quantitative RNase protection assay. Okadaic acid was found to induce ALA synthase mRNA in a concentration-dependent fashion in both Huh-7 and HepG2 cells. The EC(50) for induction of ALA synthase mRNA in Huh-7 cells was 13.5 nM, with maximum increases occurring at okadaic acid concentrations of 25-50 nM. The EC(50) for induction of ALA synthase mRNA in HepG2 cells was 35.5 nM, with maximum increases occurring at okadaic acid concentrations of 50 nM. Concentration-dependent induction of ALA synthase mRNA paralleled the increase in ALA synthase protein. Maximum induction of ALA synthase was observed between 5 and 10 h post-treatment in both cell lines. Induction of ALA synthase mRNA in Huh-7 cells, but not HepG2 cells, was associated with an increase in ALA synthase mRNA stability. Okadaic acid also induced heme oxygenase-1 mRNA in both cell lines, but the magnitude of induction was only twofold, and was rapid and transient. Okadaic acid and phorbol 12-myristate 13-acetate significantly decreased heme-mediated induction of heme oxygenase-1 mRNA in both Huh-7 and HepG2 cells. Wortmannin diminished the heme-mediated induction of heme oxygenase-1 mRNA in HepG2 cells, but not Huh-7 cells. These results report a novel property of okadaic acid to affect heme metabolism in human cell lines.     

Regulation of production of embryonic chick liver delta-aminolevulinate synthase: effects of testosterone and of hemin on the mRNA of the enzyme

The effects of testosterone and of hemin on the concentration of the mRNA of embryonic chick liver ALA synthase were investigated. Using cDNA-RNA liquid hybridization analyses, we determined that testosterone, when injected into the fluid surrounding chick embryos, caused a dose-dependent increase in the concentration of ALA synthase mRNA in liver. Similarly, addition of testosterone (5 micrograms/ml) or of 75 micrograms/ml of allylisopropylacetamide (AIA) into the medium of chick embryo hepatocytes maintained in culture caused an increase in the concentration of ALA synthase mRNA. Hemin (2 or 5 microM), when added to the culture medium, inhibited the elevations of ALA synthase mRNA concentration brought on by testosterone and by AIA.     

Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.     

Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture.

Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.     

Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line

A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biogenesis of embryonic chick liver delta-aminolevulinate synthase: regulation of the level of mRNA by hemin

The effects of hemin on the concentration of the mRNA for delta-aminolevulinate synthase (ALA synthase) and on the association of the messenger with polysomes were investigated in primary cultures of embryonic chick hepatocytes incubated with allylisopropylacetamide (AIA). A synthetic 24-mer DNA complementary to ALA synthase mRNA was used to determine by solution hybridization the effects of AIA and of AIA plus hemin on the ALA synthase-specific RNA sequences in the cells. The results indicated that ALA synthase mRNA concentrations increased significantly in hepatocytes incubated for 5 h with AIA (0.075 mg/ml), and that hemin in the medium (2 or 10 microM) blocked the increase in the messenger. When delta-aminolevulinic acid (ALA) and FeCl3 were added into the culture medium (1 mM and 5 microM, respectively), the increase in ALA synthase mRNA brought on by AIA was also inhibited. Neither ALA nor FeCl3, when individually added to the cultures, was as effective as the combination of the two. The results with ALA + FeCl3 suggested that stimulation of intracellular production of heme was also effective in blocking the increase in ALA synthase mRNA caused by AIA. Finally, the distributions of ALA synthase mRNA were compared in polysomes isolated from hepatocytes which had been incubated with AIA for 5 h in the presence and absence of 10 microM hemin in the medium. Although a drop was detected in the concentration of ALA synthase mRNA in polysomes from hepatocytes incubated with hemin for 30 min, the decrease was explained by the effect of hemin on the mRNA concentration in the cells.     

Expression of coproporphyrinogen oxidase and synthesis of hemoglobin in human erythroleukemia K562 cells.

Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with delta-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific delta-aminolevulinic acid synthase, as well as of alpha-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)beta 1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGF beta 1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGF beta 1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.     

Synergistic induction of delta-aminolevulinate synthase by glutethimide and iron: relationship to the synergistic induction of heme oxygenase

Relationships between activities of delta-aminolevulinate synthase and heme oxygenase, respectively the rate-limiting enzymes of heme biosynthesis and degradation, have been studied in chick embryo liver cell cultures following exposure of the cultures to glutethimide and iron, a combination known to produce a synergistic induction of both enzymes. In time-course experiments, synergistic induction of heme oxygenase activity by glutethimide and iron preceded that of delta-aminolevulinate synthase by 4 h. Effects of selective inhibitors of both heme synthesis and degradation have also been studied with respect to effects on delta-aminolevulinate synthase and heme oxygenase activities. The synergistic induction of heme oxygenase by glutethimide and iron appears to be dependent upon cellular heme synthesis because addition of inhibitors of heme biosynthesis, 4,6-dioxoheptanoic acid or N-methyl-mesoporphyrin abolishes this synergistic induction. Exposure of cultures to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, prevented the synergistic induction of delta-aminolevulinate synthase produced by glutethimide and iron, or, when added after induction was already established, promptly halted any further induction. These results suggest that the level of activity of heme oxygenase can reciprocally modulate intracellular heme levels and thus activity of delta-aminolevulinate synthase.     

Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.

Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli (S. Hino and A. Ishida, Enzyme 16:42-49, 1973).     

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from experimental-diabetic rats

Isolated hepatocytes from rats with experimental diabetes exhibit increased content of cytochrome P-450 and cyclic AMP and normal activities of the regulatory enzymes delta-aminolevulinic acid synthase and ferrochelatase. The inducing effect exerted by phenobarbital on cytochrome P-450, delta-aminolevulinic acid synthase and ferrochelatase biosynthesis and cyclic AMP content in diabetic hepatic cells is markedly greater than that observed in normal hepatocytes. This stimulatory response is neither enhanced by added dibutyryl cyclic AMP nor repressed by glucose. The present results suggest that the heme pathway of diabetic hepatocytes is more susceptible to porphyrinogenic factors.     

Steroid induction of delta-aminolevulinic acid synthase and porphyrins in liver. Structure-activity studies and the permissive effects of hormones on the induction process.

Quantitative aspects and structure-activity relationships of the inducing effects of natural steroids on delta-aminolevulinic acid (ALA) synthase and porphyrins have been investigated in monolayer cultures of chick embryo liver cells maintained in a serum-free medium as well as in the chick embryo liver in ovo. Many 5 alpha and 5 beta metabolites of neutral C-19 and C-21 hormones and hormone precursors stimulated porphyrin formation and ALA-synthase induction in the cultured liver cells as we have previously described. In these inducing actions a number of 5 beta epimers (A:B cis) were found to be more potent than their corresponding 5 alpha epimers (A:B trans). The structure-activity relationship between 5 beta and 5 alpha steroid epimers with respect to ALA-synthase induction in culture was also found to prevail with respect to induction of this enzyme in chick embryo liver in ovo. Hemin in concentrations of 2 x 10(-7) M inhibited steroid induction of porphyrin formation, and CaMgEDTA enhanced the responsiveness of the cultured liver cells to steroids by approximately 10 times. The addition of insulin, or insulin plus hydrocortisone or insulin plus hydrocortisone plus triiodothyronine, was important for the maintenance of protein synthesis and essential for maximal expression of the ability of steroids to induce porphyrins and ALA-synthase in the "permissive" effect which insulin, hydrocortisone, and triiodothyronine exert on allylisopropylacetamide induction of porphyrins and ALA-synthase also extends to the induction process which is elicited by natural steroids. These findings also strongly suggest that the regulation of hepatic porphyrin-heme biosynthesis by endogenous as well as exogenous chemicals is significantly influenced by the internal hormonal milieu.     

Maturation of embryonic chick liver delta-aminolevulinate synthase: precursor pools and regulation by intra-cellularly produced heme

1. Immunoblot analyses were carried out to determine the relative distributions of delta-aminolevulinate synthase (ALA synthase) in mitochondrial and cytosol fractions prepared from embryos at different times after injections with allylisopropylacetamide (AIA). 2. The results indicated that the molecular mass of mature ALA synthase (Mr 65,000) increased with time in mitochondria. 3. At no time was the precursor form (Mr 75,000) of the enzyme detected either in mitochondria or in the cytosol. 4. In primary cultures of hepatocytes, where the increased production of ALA synthase had been induced with AIA, addition of delta-aminolevulinic acid (ALA) and Fe2(SO4)3 into the culture medium completely blocked the processing of the precursor form of the enzyme. 5. On the other hand, the addition of ALA together with deferoxamine mesylate into the medium had no detectable effect on the maturation of ALA synthase in the hepatocytes. 6. The results indicated: first, that upon induction of porphyria the pools of pre-ALA synthase in liver are relatively low in chick embryos when compared with those in other organisms; and second, that increased heme production by the hepatocytes caused the inhibition of processing of the precursor form of ALA synthase.