Miho Ohta

Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily.

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by recombinant yeast cells expressing either rat CYP1A1 or CYP1A2 was examined. When each of the dibenzo-p-dioxins (DDs), mono-, di-, and tri-chloroDDs, was added to the cell culture of the recombinant yeast, a remarkable metabolism was observed. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and opening of dioxin ring. The distinct difference was observed in substrate specificity and reaction specificity between CYP1A1 and CYP1A2. Kinetic analysis using microsomal fractions prepared from the recombinant yeast cells revealed that 2,7-dichloroDD and 2,3,7-trichloroDD were good substrates for both CYP1A1 and CYP1A2. When 2,3,7-trichloroDD was added to the yeast cells expressing each of rat CYP1A1 and CYP1A2, most of 2,3,7-trichloroDD was first converted to 8-hydroxy-2,3,7-trichloroDD, and further metabolized to more hydrophilic compounds whose ethereal bridges were cleaved. These findings give essential information on the metabolism of PCDDs in mammalian liver. In addition, this study indicates the possibility of application of microorganisms expressing mammalian cytochrome P450 to bioremediation of contaminated soils with dioxins.

Structure-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid residues responsible for species-based difference of CYP24A1 between humans and rats.

Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500F showed quite a different metabolism of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] from both human and rat CYP24A1. These results strongly suggest that the amino acid residues at positions 416 and 500 play a crucial role in substrate binding and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1α,25(OH)2D3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use.

Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.

Metabolism of sesamin by cytochrome P450 in human liver microsomes.

Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest kcat/Km value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

Cloning, expression and characterization of horse L-ferritin in Escherichia coli

Horse L-ferritin cDNA was cloned from horse liver, and the base sequence was determined. The L-ferritin was expressed using pTZ18U encoding lac promoter, and found to possess an additional 8-amino acid sequence at the N-terminus as compared with commercially obtained horse spleen (natural) ferritin. It was determined that there was Pro at position 94 in both the recombinant and natural L-ferritin, although it was previously reported that Leu was in this position in the natural species. Transmission electron microscopy showed that this recombinant ferritin formed a 24-mer shell.

Anti-proliferative activity of 25-hydroxyvitamin D3 in human prostate cells.

1α-Hydroxylation of 25-hydroxyvitamin D3 is believed to be essential for its biological effects. In this study, we evaluated the biological activity of 25(OH)D3 itself comparing with the effect of cell-derived 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). First, we measured the cell-derived 1α,25(OH)2D3 level in immortalized human prostate cell (PZ-HPV-7) using [(3)H]-25(OH)D3. The effects of the cell-derived 1α,25(OH)2D3 on vitamin D3 24-hydroxylase (CYP24A1) mRNA level and the cell growth inhibition were significantly lower than the effects of 25(OH)D3 itself added to cell culture. 25-Hydroxyvitamin D3 1α-hydroxylase (CYP27B1) gene knockdown had no significant effects on the 25(OH)D3-dependent effects, whereas vitamin D receptor (VDR) gene knockdown resulted in a significant decrease in the 25(OH)D3-dependent effects. These results strongly suggest that 25(OH)D3 can directly bind to VDR and exerts its biological functions. DNA microarray and real-time RT-PCR analyses suggest that semaphorin 3B, cystatin E/M, and cystatin D may be involved in the antiproliferative effect of 25(OH)D3.

Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D3 to its biologically active form, 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3], suggesting the possibility for R73V/R84A to produce 1α,25(OH)2D3. Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25‐hydroxyvitamin D3 [25(OH)D3] and 1α,25(OH)2D3 were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)2D3, the second‐step metabolite of vitamin D3, was observed, although a considerable amount of vitamin D3 still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)3D3 and 1α,25(S),26(OH)3D3, both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5 : 1. The crystal structure of the double variant with 1α,25(OH)2D3 and a docking model of 1α,25(OH)2D3 in its active site strongly suggest a hydrogen‐bond network including the 1α‐hydroxyl group, and several water molecules play an important role in the substrate‐binding for 26‐hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D3 to produce biologically useful compounds.

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1alpha,25-dihydroxyvitamin D2 (1alpha,25(OH)2D2) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1alpha,25(OH)2D2 between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1alpha,25(OH)2D2 to 1alpha,24,25,26(OH)4D2, 1alpha,24,25,28(OH)4D2, and 24-oxo-25,26,27-trinor-1alpha(OH)D2 via 1alpha,24,25(OH)3D2. These results indicate that human CYP24A1 catalyzes the C24-C25 bond cleavage of 1alpha,24,25(OH)2D2, which is quite effective in the inactivation of the active form of vitamin D2. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D2 and its analogs in the human body.

Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes

Background: Salt-inducible kinases (SIKs) are capable of suppressing gluconeogenic gene expression in hepatocytes when they are overexpressed. Results: However, enhanced gluconeogenic programs are observed only in SIK3-defective hepatocytes. Conclusion: SIK3 is the major kinase that down-regulates gluconeogenesis. Significance: The present study proposes that SIK3 could be a new target of diabetic care. Salt-inducible kinases (SIKs), members of the 5′-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.

Mechanism of the anti-proliferative action of 25-hydroxy-19-nor-vitamin D3 in human prostate cells

According to the prevailing paradigm, 1α-hydroxylation of 25-hydroxyvitamin D(3) (25(OH)D(3)) and its analogs is a pre-requisite step for their biological effects. We previously reported that 25-hydroxy-19-nor-vitamin D(3) (25(OH)-19-nor-D(3)) had anti-proliferative activity in a cell line, PZ-HPV-7, which was derived from human non-cancerous prostate tissue, and suggested that 25(OH)-19-nor-D(3) acted after 1α-hydroxylation by vitamin D 1α-hydroxylase (CYP27B1). However, metabolic studies of 25(OH)-19-nor-D(3) using recombinant CYP27B1 revealed that 25(OH)-19-nor-D(3) was rarely subjected to 1α-hydroxylation. Therefore, in this report, we attempted to clarify the mechanism of 25(OH)-19-nor-D(3) action in intact cells using PZ-HPV-7 prostate cells. After incubating the cells with 25(OH)-19-nor-D(3), eight metabolites of 24-hydroxylase (CYP24A1) were detected, whereas no products of CYP27B1 including 1α,25-dihydroxy-19-nor-vitamin D(3) (1α,25(OH)(2)-19-nor-D(3)) were found. Furthermore, the time-dependent nuclear translocation of vitamin D receptor (VDR) and the subsequent transactivation of cyp24A1 gene in the presence of 25(OH)-19-nor-D(3) were almost identical as those induced by 1α,25(OH)(2)-19-nor-D(3). These results strongly suggest that 25(OH)-19-nor-D(3) directly binds to VDR as a ligand and transports VDR into the nucleus to induce transcription of cyp24A1 gene. In addition, knock down of cyp27B1 gene did not affect the anti-proliferative activity of 25(OH)-19-nor-D(3), whereas knock down of VDR attenuated the inhibitory effect. Thus, our results clearly demonstrate that the anti-proliferative activity of 25(OH)-19-nor-D(3) is VDR dependent but 1α-hydroxylation independent, suggesting that 25(OH)D(3) analogs such as 25(OH)-19-nor-D(3) could be attractive candidates for anticancer therapy.