Kazumasa Ikeda

Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia

1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3], a vitamin D receptor (VDR) ligand, regulates calcium homeostasis and also exhibits noncalcemic actions on immunity and cell differentiation. In addition to disorders of bone and calcium metabolism, VDR ligands are potential therapeutic agents in the treatment of immune disorders, microbial infections, and malignancies. Hypercalcemia, the major adverse effect of vitamin D3 derivatives, limits their clinical application. The secondary bile acid lithocholic acid (LCA) is an additional physiological ligand for VDR, and its synthetic derivative, LCA acetate, is a potent VDR agonist. In this study, we found that an additional derivative, LCA propionate, is a more selective VDR activator than LCA acetate. LCA acetate and LCA propionate induced the expression of the calcium channel transient receptor potential vanilloid type 6 (TRPV6) as effectively as that of 1α,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1), whereas 1,25(OH)2D3 was more effective on TRPV6 than on CYP24A1 in intestinal cells. In vivo experiments showed that LCA acetate and LCA propionate effectively induced tissue VDR activation without causing hypercalcemia. These bile acid derivatives have the ability to function as selective VDR modulators.

Vitamin D Receptor Activation Enhances Benzo[a]pyrene Metabolism via CYP1A1 Expression in Macrophages

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of genes involved in xenobiotic metabolism, including CYP1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I subfamily of the nuclear receptor superfamily, which also regulates expression of xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR signaling pathways and found that 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], a potent physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human monocytic U937 cells and THP-1 cells, breast cancer cells, and kidney epithelium-derived cells. 1,25(OH)2D3 alone did not induce CYP1A1, and 1,25(OH)2D3 plus BaP did not increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. The combination of 1,25(OH)2D3 and BaP increased CYP1A1 protein levels, BaP hydroxylation activity, and BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP alone. The combined effect of 1,25(OH)2D3 and BaP on CYP1A1 mRNA expression in U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat (ER) 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP-mediated toxicity and the physiological function of this enzyme.

The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells

The nervous system is highly sensitive to various environmental stresses, such as ischemia. Stress response mechanisms that result in neuroprotection, including the induction of heat shock proteins (HSP), are not well understood. We examined the effect of KNK437, a compound that inhibits the synthesis of inducible heat shock proteins, on neuronal differentiation in rat pheochromocytoma PC12 cells. KNK437 decreased the expression of HSP70, and induced the neurite outgrowth of PC12 cells in the absence of stress stimulation, although with lower efficacy than nerve growth factor (NGF). Neurite outgrowth stimulated by KNK437 and NGF was blocked by inhibitors of ERK mitogen-activated protein (MAP) kinase, p38 MAP kinase, and glycogen synthase kinase 3beta signaling pathways. NGF, and not KNK437, induced acetylcholine esterase (AChE) activity, a functional differentiation marker, indicating that KNK437 utilizes a mechanism distinct from that of NGF. KNK437 enhanced the activity of low dose NGF treatment on neurite outgrowth induction and ERK phosphorylation in PC12 cells, a finding that identifies KNK437 as a possible nerve regeneration agent. This compound may be a useful tool for the investigation of neuronal differentiation and neuroprotection against environmental stress.

Postnatal expression of hypoxanthine guanine phosphoribosyltransferase in the mouse brain

The distributional and activity changes of hypoxanthine guanine phosphoribosyltransferase (HGPRT) were investigated in the developing mouse brain. The HGPRT activity level was low at birth, increased rapidly during the first 7 days of life, and underwent a gradual increase thereafter to the mature level. Polyclonal antibody against HGPRT purified from mouse brain was prepared for immunohistochemical demonstration of the enzyme during brain development. In the cerebellum, part of the Purkinje cells was consistently immunostained throughout growth, and the presence of HGPRT was observed in the dendrites of mature Purkinje cells. The most dominant change in HGPRT localization was observed in the hippocampus. Little HGPRT was detectable in the newborn mouse hippocampus. At postnatal day 7, cytoplasmic HGPRT appeared sporadically in the granular cells independently of the region of the hippocampus. The number of positive immunoreactive cells increased with growth, and the dendrites of granular cells were also immunostained on postnatal day 28. Further immunostaining was noted in the granule cells of the dentate gyrus on postnatal day 35. The above results suggest that HGPRT may play an important role in the developing hippocampus. Further investigations of the HGPRT in the human hippocampus may help to clarify the mechanism underlying the neurological disorders encountered in the Lesch-Nyhan syndrome.

Purification and characterization of canine manganese superoxide dismutase and its immunohistochemical localization in canine heart compared with that of copper-zinc superoxide dismutase

Several forms of superoxide dismutase (SOD; EC 1.15.1.1) are known to be major scavengers of superoxide radicals which are involved in various kinds of injuries including heart failure. In an attempt to elucidate the protective system of the canine heart against superoxide radical, the present study immunohistochemically examined the localizations of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in the canine heart. Mn-SOD was purified homogeneously from canine liver by chromatography on a DE-52 column and a CM-52 column, followed by gel filtration on a Sephadex G-200 column: the purified enzyme consisted of two identical subunits of molecular weight 22,000. Polyclonal antibody was prepared against the purified Mn-SOD, and its specificity was confirmed by Western blotting. The Mn-SOD and Cu,Zn-SOD in canine hearts were examined by immunocytochemical staining using specific anti Mn-SOD serum and anti-canine Cu,Zn-SOD serum prepared previously. Cu,Zn-SOD was localized in vessel cells including the endothelium, whereas little Mn-SOD occurred in the vessel cells but some was located in the endothelium. The corrected Cu,Zn-SOD activity (714 ± 27 U/ml; mean ± SEM, n = 4) was similar to that of the corrected Mn-SOD activity (633 ± 42 U/ml; n = 4) in the canine heart. The present study shows that both forms of SOD are present in the endothelium. It is concluded that they may cooperatively scavenge superoxide radicals generated around the canine heart vessels, and Cu,Zn-SOD may be functional in secondary protection of the myocytes against more severe injury derived from superoxide radicals in the vessel cells.