Yutaka Shizuta

Altered expression of fatty acid–metabolizing enzymes in aromatase-deficient mice

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Congenitally defective aldosterone biosynthesis in humans: The involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients

The gene for steroid 18-hydroxylase (P-450C18) has been recently assigned to encode corticosterone methyl oxidases Type I and Type II which were previously postulated to catalyze the final two steps in the biosynthesis of aldosterone in humans. Molecular genetic analysis of the P-450C18 gene is three patients from three different families affected with CMO II deficiency has indicated that a point mutation of CGG----TGG (181Arg----Trp) in exon 3 and one of GTG----GCG (386Val----Ala) in exon 7 occur exclusively in the gene of the patients. Analysis of PCR products by restriction enzymes (HapII and HphI) has indicated that the patients are homozygous and the unaffected parent is heterozygous for both mutations, in accordance with the established concept that CMO II deficiency is inherited in an autosomal recessive manner. These data clearly provide the molecular genetic basis for the characteristic biochemical phenotype of CMO II clinical variants.

Alternative usage of different poly(A) addition signals for two major species of mRNA encoding human aromatase P-450.

Two cDNA clones for human placental aromatase P‐450 (P‐450AROM) have been isolated and sequenced. The insert of one clone (2894 bp) contains an open reading frame encoding a protein consisting of 503 amino acid residues together with a 49 bp 5′‐untranslated stretch and a 1336 bp 3′‐noncoding region to which a poly(A) tract is attached. Three potential poly(A) addition signals are detected in this 3′‐noncoding region. The other clone contains a shorter cDNA insert, the nucleotide sequence of which overlaps with most of the sequence of the longer cDNA insert (nucleotides 36–2355) except for one nucleotide substitution. The 3′‐noncoding region of this shorter cDNA is only 846 bp in length, but a poly(A) tract is also attached to its 3′‐terminus. Northern blot analysis of human placental RNA reveals the presence of two major mRNA species of 3.4 and 2.9 kb when probes excised from the overlapping region of these two cDNAs are employed. The 2.9 kb mRNA is not detected, however, when a fragment of the non‐overlapping region of the longer cDNA is used as a probe. It is therefore concluded that the two major species of P‐450AROM mRNA are formed as a consequence of alternative processing of precursor mRNA(s).

TISSUE FACTOR PATHWAY INHIBITOR-2 IS A NOVEL MITOGEN FOR VASCULAR SMOOTH MUSCLE CELLS

A mitogen for growth-arrested cultured bovine aortic smooth muscle cells was purified to homogeneity from the supernatant of cultured human umbilical vein endothelial cells by heparin affinity chromatography and reverse-phase high performance liquid chromatography. This mitogen was revealed to be tissue factor pathway inhibitor-2 (TFPI-2), which is a Kunitz-type serine protease inhibitor. TFPI-2 was expressed in baby hamster kidney cells using a mammalian expression vector. Recombinant TFPI-2 (rTFPI-2) stimulated DNA synthesis and cell proliferation in a dose-dependent manner (1–500 nm). rTFPI-2 activated mitogen-activated protein kinase (MAPK) activity and stimulated early proto-oncogene c-fos mRNA expression in smooth muscle cells. MAPK, c-fos expression and the mitogenic activity were inhibited by a specific inhibitor of MAPK kinase, PD098059. Thus, the mitogenic function of rTFPI-2 is considered to be mediated through MAPK pathway. TFPI has been reported to exhibit antiproliferative action after vascular smooth muscle injury in addition to the ability to inhibit activation of the extrinsic coagulation cascade. However, structurally similar TFPI-2 was found to have a mitogenic activity for the smooth muscle cell.

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17β-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.

Poly(ADP-ribosyl)ation of RNA polymerase II from wheat germ

Wheat germ RNA polymerase II is poly (ADP-ribosyl)ated in vitro by poly (ADP-ribose) synthetase purified from bovine thymus. RNA polymerase activity is decreased by 40% by the modification in conventional assays.

Estrogen deficiency results in enhanced expression of Smoothened of the Hedgehog signaling in the thymus and affects thymocyte development

Aromatase is an essential enzyme for estrogen synthesis. We investigated the role of estrogen in thymocyte development using aromatase-deficient (ArKO) mice. Like its role as a regulator of bone metabolism through regulating osteoprotegerin (OPG) production, estrogen is involved in the processes of thymocyte development although aromatase mRNA was not detectable in the thymus. Thymic regression and reduced cellularity were evident in ArKO mice. The major difficulties in thymocyte development of ArKO mice were observed during the CD44+ CD25- stage at the cortico-medullary junction and during the CD44- CD25- stage at the subcapsular region where the estrogen receptor was expressed in the stromal cells. The proportion of thymocytes during the CD44+ CD25- stage was reduced. The progression of CD44- CD25+ cells to the CD44- CD25- stage was accelerated in ArKO mice possibly due to insufficient osteoprotegerin production in estrogen-deficiency. However, the expression of Smoothened of the Hedgehog signaling was enhanced in CD4- CD8- double negative cells. This enhancement may result in impaired progression of CD44- CD25- cells to the CD4+ CD8+ double positive stage and impaired proliferation of CD4+ CD8+ double positive cells since Smoothened (Smo) is known to arrest cells as non-proliferating cells. This could be the reason why the proportion of CD3+ TCRbeta(high) cells during the late phase of thymocyte maturation was reduced in ArKO mice. From these observations, we propose that estrogen supports thymocyte development and maturation at many stages through many regulatory pathways including the sonic hedgehog- and the osteoprotegerin ligand (OPGL)-mediated signaling.

Aromatase-deficient (ArKO) mice are retrieved from severe hepatic steatosis by peroxisome proliferator administration

Tamoxifen is a potent antagonist of estrogen, and hepatic steatosis is a frequent complication in adjuvant tamoxifen for breast cancer. Recently, aromatase-deficient (ArKO, Ar-/-) mice lacking intrinsic estrogen was developed and the molecular mechanism involved in progression of massive hepatic steatosis in estrogen-deficiency was elucidated; impairment in hepatic fatty acid beta-oxidation of peroxisomes, microsomes and mitochondria. This impairment is latent, but is potentially serious, because hepatic energy supply depends greatly on fatty acid beta-oxidation. Therefore in the present study, we tried to conquer impaired hepatic fatty acid beta-oxidation by administrating bezafibrate, a potent peroxisome proliferator, to Ar-/- mice through activating fatty acid beta-oxidation via the peroxisome proliferator activated receptor-alpha mediated signaling pathway. Northern blot analysis of Ar-/- mice liver revealed a significant restoration of mRNA expression of very long fatty acyl-CoA synthetase in peroxisome, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase in mitochondria, essential enzymes in fatty acid beta-oxidation by administration of bezafibrate. Severe hepatic steatosis observed in Ar-/- mice regressed dramatically. Consistent findings were obtained in the in vitro assays of fatty acid beta-oxidation activity. These findings demonstrate that bezafibrate is capable of restoring impaired fatty acid beta-oxidation in vivo via the peroxisome proliferator-activated receptor-alpha mediated signaling pathway and is potent enough to regress severe hepatic steatosis in mice deficient in intrinsic estrogen.

Regulation of biodegradative threonine deaminase.

Publisher Summary The biodegradative threonine deaminases have been purified to homogeneity and crystallized from the extracts of Escherichia coli ( E. coli ) and Clostridium tetranomorphum , respectively. It has been demonstrated that deaminases are composed of four identical subunits, each monomer containing one mole of pyridoxal phosphate. The deamination reaction mechanism studied using the E . coli enzyme is compatible with the mechanism derived from the pyridoxal-catalyzed α,β-elimination reaction in the non-enzymic model system, although direct evidence for the participation of pyridoxal phosphate in the enzyme reaction is lacking. Spectral and circular dichroic changes of the enzyme-bound pyridoxal phosphate after addition of the substrate, L-threonine, were postulated to represent the formation of a complex between pyridoxal phosphate and the dehydrated reaction intermediate. Optical studies indicate that optical changes occur prior to the β -elimination reaction. Kinetic analyses have indicated that the enzyme exists as an inactive form under the conditions of these optical experiments. These phenomena could be interpreted as indicating that the conversion of this inactive enzyme into an active form occurs after addition of L-threonine. These optical changes are observed during the reaction and this conversion step appears to be the rate-limiting step of the overall reaction under the conditions employed.

Antigenic determinant and interspecies cross-reactivity of a monoclonal antibody to poly(ADP-ribose) synthetase

A monoclonal antibody (1F4) was prepared against calf thymus poly(ADP‐ribose) synthetase. It was classified as IgG1/κ and its antigenic determinant was localized on the 46 kDa portion of the enzyme molecule which contains the site for the binding of DNA. When calf thymus DNA‐binding proteins were subjected to immunostaining after electrophoresis and transblotting to a nitrocellulose filter, the native enzyme (120 kDa) and its endogenous degradation products (80, 64 and 32 kDa) were detected. When the interspecies cross‐reactivity was examined using DNA‐binding proteins from 6 different sources, 1F4 reacted with the 120‐ and 32‐kDa protein bands in HeLa cells, mouse testis and chicken liver as in the case of calf thymus. These results indicate that the antigenic structures ofpoly(ADP‐ribose) synthetase and its degradation products are highly conserved in various animal cells.