Yoshitaka Miura

Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation

In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.

Enhanced Expression of the Inorganic Phosphate Transporter Pit-1 Is Involved in BMP-2–Induced Matrix Mineralization in Osteoblast-Like Cells†

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP‐2–induced matrix calcification was studied. BMP‐2 enhances Pit‐1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification.

Protection of pancreatic -cells by exendin-4 may involve the reduction of endoplasmic reticulum stress; in vivo and in vitro studies

The aim of this study was to investigate the in vivo and in vitro effectsofexendin-4,apotentglucagon-likepeptide1agonist,on theprotectionofthepancreaticb-cellsagainsttheircelldeath.In in vivo experiments, we used b-cell-specific calmodulinoverexpressing mice where massive apoptosis takes place in their b-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1 a ,X -box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in b-cellspecific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess ofERstressoccursinthetransgenicb-cells,andthesuppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.

Idiopathic spinal cord herniation

Idiopathic spinal cord herniation is a rare disease, few cases having been reported. We encountered a case of idiopathic spinal cord herniation presenting with severe spasticity in the right leg and urinary dysfunction. The spinal cord was herniated into a cavity created by duplication of the dura mater and resection of the inner layer improved the neurological deficits. MRI, myelography, and CT myelography were useful for diagnosing this disease. Four radiological signs of spinal cord herniation are described.

The expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in rat brain: possible role of endogenous PACAP in vasopressin release.

We investigated the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in rat brain by in situ hybridization. PACAP mRNA was prominently expressed in arcuate nucleus (ARC). Three days of water deprivation significantly increased plasma arginine vasopressin and markedly potentiated the expression of PACAP mRNA in ARC. These results suggest that PACAP in ARC may play some physiological role, possibly one of which may be the control of vasopressin release.

Stimulatory effect of endothelin-1 on Na-dependent phosphate transport and its signaling mechanism in osteoblast-like cells.

Endothelin‐1 (ET‐1) has been reported to modulate bone metabolism both in vivo and in vitro. In the present study, we investigated the effect of ET‐1 on inorganic phosphate (Pi) transport in osteoblast‐like cells, which is now considered to be important for the initiation of bone matrix calcification. ET‐1 time‐ and dose‐dependently stimulated Na‐dependent Pi transport in mouse calvaria‐derived osteoblast‐like MC3T3‐E1 cells, and this effect was dependent on transcriptional and translational process. Kinetic analysis indicated that the change in Pi transport activity induced by ET‐1 was due to alteration in the number of the Pi transporter. BQ123, a selective antagonist for ETA receptor, suppressed the ET‐1‐induced Pi transport, but BQ788, a selective antagonist for ETB receptor, had no effect. The inhibition of phosphoinositide hydrolysis by phospholipase C (PLC) partially attenuated the Pi transport by ET‐1. Propranolol, which inhibits phosphatidic acid phosphohydrolase, also suppressed ET‐1‐induced Pi transport. On the contrary, indomethacin did not affect the stimulatory effect of Pi transport by ET‐1. Calphostin C, a protein kinase C (PKC) inhibitor, significantly blunted the stimulatory effect of ET‐1 on Pi transport. Combined effect of PMA and ET‐1 on Pi transport was not additive. Pi transport induced by ET‐1 was also suppressed in PKC down‐regulated cells. In conclusion, the results of the present study indicate that in MC3T3‐E1 osteoblast‐like cells, ET‐1 acting through ET receptor links to a stimulation of Pi transport via activation of PKC through both phosphoinositide and phosphatidylcholine hydrolyses.

Precise localization of the human thyroxine-binding globulin gene to chromosome Xq22.2 by fluorescence in situ hybridization

The human thyroxine-binding globulin (TBG) gene has been localized to X chromosome (Xq22.2) by in situ hybridization using a biotinylated gDNA probe. This is consistent with previous mapping of the TBG gene to chromosome Xq21-q22.

Calmodulin Overexpression Causes Ca2+-Dependent Apoptosis of Pancreatic β Cells, Which Can Be Prevented by Inhibition of Nitric Oxide Synthase

We investigated the mechanism of β-cell loss in transgenic mice with elevated levels of β cell calmodulin. The transgenic mice experienced a sudden rise in blood glucose levels between 21 and 28 days of age. This change was associated with development of severe hypoinsulinemia and loss of β cells from the islets. Ultrastructural analysis revealed that compromised granule formation and apoptotic changes in the transgenic β cells preceded the onset of hyperglycemia. Intraperitoneal injection of tolbutamide, an antidiabetic sulfonylurea, decreased blood glucose levels but increased the number of apoptotic β cells. Finally, injection of transgenic mice with Nω-nitro-l-arginine methyl ester, which inhibits nitric oxide synthase activity, prevented hyperglycemia and lessened the changes in number and size of β cells. Because immunofluorescent staining revealed preferential distribution of neural nitric oxide synthase in pancreatic β cells, we speculate that overexpression of calmodulin sensitizes the β cells to Ca2+-dependent activation of neural nitric oxide synthase, which mediates apoptosis.

A novel deletion mutation in the arginine vasopressin receptor 2 gene and skewed X chromosome inactivation in a female patient with congenital nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare inherited disorder caused by mutations in the arginine vasopressin receptor 2 (V2R) gene. The clinical phenotype is fully expressed in hemizygous male patients and is usually asymptomatic in heterozygous females. In the present study, a 51-yr-old Japanese female with congenital NDI and her family members were examined. The patient developed severe hypernatremia accompanied by hypoosmotic polyuria after gynecological surgery, and was unable to concentrate urinary osmolality in response to exogenous vasopressin. Direct sequencing analysis of the propositus and her two affected sons revealed a two-nucleotide deletion change at codon 30 (g.452–453delAC) in the V2R gene, resulting in a frameshift and premature termination in translation at codon 190. The X chromosome inactivation pattern was investigated in the propositus using methylation analysis of the polymorphic CAG repeat in the androgen receptor gene, and the value for relative X chromosome inactivation of one allele was 70.2%. In conclusion, we identified a novel V2R gene mutation in a female patient and her sons with congenital NDI, and her phenotype may be caused by skewed X chromosome inactivation.

A Case of Type 1 Diabetes Followed by Methimazole-Induced Hypersensitivity Syndrome

Viruses have generally been considered to be a major environmental factor in the etiology of type 1 diabetes. Drug-induced hypersensitivity syndrome (DIHS) is characterized by a severe drug eruption and multiorgan involvement, and reactivation of human herpesvirus-6 (HHV-6) may contribute to its pathology (1). This is the first reported case of type 1 diabetes followed by DIHS. Recently, we have reported a case of DIHS induced by methimazole for Graves’ disease (2). This patient developed type 1 diabetes during treatment of DIHS. Briefly, a 50-year-old Japanese male subject …