Shin Takasawa

Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetes-induced vascular injury.

The binding of advanced glycation end-products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. In the present study, we show that the cellular constituents of small vessels, endothelial cells (EC) and pericytes express novel splice variants of RAGE mRNA coding for the isoforms that lack the N-terminal V-type immunoglobulin-like domain (N-truncated) or the C-terminal transmembrane domain (C-truncated), as well as the known full-length mRNA. The ratio of the expression of the three variants was different between EC and pericytes; the content of the C-truncated form was highest in EC, whereas the full-length form was the most abundant in pericytes. Transfection experiments with COS-7 cells demonstrated that those variant mRNAs were translated into proteins as deduced; C-truncated RAGE was efficiently secreted into the culture media, and N-truncated RAGE was located mainly on the plasma membrane. The three isoforms were also detected in primary cultured human EC and pericytes. Further, full-length and C-truncated forms of RAGE bound to an AGE-conjugated column, whereas N-truncated RAGE did not. The AGE induction of extracellular-signal-related kinase phosphorylation and vascular endothelial growth factor in EC and of the growth and cord-like structure formation of EC was abolished completely by C-truncated RAGE, indicating that this endogenous secretory receptor (endogenous secretory RAGE) is cytoprotective against AGE. The results may contribute to our understanding of the molecular basis for the diversity of cellular responses to AGE and for individual variations in the susceptibility to diabetic vascular complications.

CD38 is critical for social behaviour by regulating oxytocin secretion

CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.

Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice

Vascular complications arising from multiple environmental and genetic factors are responsible for many of the disabilities and short life expectancy associated with diabetes mellitus. Here we provide the first direct in vivo evidence that interactions between advanced glycation end products (AGEs; nonenzymatically glycosylated protein derivatives formed during prolonged hyperglycemic exposure) and their receptor, RAGE, lead to diabetic vascular derangement. We created transgenic mice that overexpress human RAGE in vascular cells and crossbred them with another transgenic line that develops insulin-dependent diabetes shortly after birth. The resultant double transgenic mice exhibited increased hemoglobin A(1c) and serum AGE levels, as did the diabetic controls. The double transgenic mice demonstrated enlargement of the kidney, glomerular hypertrophy, increased albuminuria, mesangial expansion, advanced glomerulosclerosis, and increased serum creatinine compared with diabetic littermates lacking the RAGE transgene. To our knowledge, the development of this double transgenic mouse provides the first animal model that exhibits the renal changes seen in humans. Furthermore, the phenotypes of advanced diabetic nephropathy were prevented by administering an AGE inhibitor, (+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195), thus establishing the AGE-RAGE system as a promising target for overcoming this aspect of diabetic pathogenesis.

Cyclic ADP-ribose Binds to FK506-binding Protein 12.6 to Release Ca2+ from Islet Microsomes

Cyclic ADP-ribose (cADPR) is a second messenger for Ca2+ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca2+ from islet microsomes. After islet microsomes were treated with FK506, the Ca2+ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca2+ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca2+ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca2+.

RAGE Control of Diabetic Nephropathy in a Mouse Model Effects of RAGE Gene Disruption and Administration of Low-Molecular Weight Heparin

Diabetic nephropathy is a major microvascular complication in long-standing diabetic patients who eventually undergo renal dialysis or transplantation. To prevent development of this disease and to improve advanced kidney injury, effective therapies directed toward the key molecular target are required. In this study, we examined whether inhibition of the receptor for advanced glycation end products (RAGE) could attenuate changes in the diabetic kidney. Here, we show that inactivation of the RAGE gene in a mouse model of diabetic nephropathy results in significant suppression of kidney changes, including kidney enlargement, increased glomerular cell number, mesangial expansion, advanced glomerulosclerosis, increased albuminuria, and increased serum creatinine compared with wild-type diabetic mice. The degree of kidney injury was proportional to RAGE gene dosage. Furthermore, we show that low–molecular weight heparin (LMWH) can bind RAGE at a mean equilibrium dissociation constant (Kd) value of ∼17 nmol/l and act as an antagonist to RAGE. LMWH treatment of mice significantly prevented albuminuria and increased glomerular cell number, mesangial expansion, and glomerulosclerosis in a dose-dependent manner; it also significantly improved the indexes of advanced-stage diabetic nephropathy. This study provides insight into the pathological role of RAGE in both early- and advanced-phase diabetic nephropathy and suggests that RAGE antagonists will be a useful remedy in the treatment of diabetic nephropathy.

ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1

Human BST‐1, a bone marrow stromal cell surface molecule, is a GPI‐anchored protein that facilitates the growth of pre‐B cells. The deduced amino acid sequences of human and mouse BST‐1 show around 30% homology with those of CD38 and Aplysia ADP ribosyl cyclase. Therefore, like CD38, BST‐1 might possess ADP ribosyl cyclase activity. Here, we report the establishment of a stable transformant CHO cell line which secretes truncated human soluble BST‐1, and show that purified soluble BST‐1 displays both ADP ribosyl cyclase cADPR hydrolase activities.

Cyclic ADP-ribose modulates Ca2+ release channels for activation by physiological Ca2+ entry in bullfrog sympathetic neurons

Although Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors has been found to occur in intact neurons, little is known about the physiological processes that regulate it. We studied the effects of cyclic ADP-ribose (cADPR) on CICR in cultured bullfrog sympathetic neurons by fura-2 fluorescence recording and patch-clamp techniques. cADPR applied through a patch pipette augmented action potential- or depolarizing pulse-induced rises in intracellular Ca2+ without a change in Ca2+ entry initiating the responses, but not in the presence of ryanodine. Likewise, cADPR enhanced a single or oscillatory rise(s) in intracellular Ca2+ induced by caffeine. These results strongly suggest that cADPR can be an endogenous modulator of ryanodine receptors in neurons.

Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model.

Although microorganisms play a role in gut inflammation, it remains uncertain which epithelial genes are expressed in response to luminal flora and whether these molecules are also involved in pathologic mucosal inflammation. Germ-free mice were orally challenged with a bacterial suspension prepared from conventionally housed mice (bacterial reconstitution). Thereafter, the differential gene expression in gut epithelial cells was identified by differential display. The expression of the identified genes was also examined in dextran sulfate sodium (DSS)-induced colitis and human inflammatory bowel disease (IBD) epithelial cells. Regenerating gene III (Reg III) was strongly induced in gut epithelial cells following bacterial reconstitution, as well as in the colitis initiated by DSS. The mRNA expression of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP), a human counterpart of Reg III, was enhanced in colonic epithelial cells of patients with IBD. Reg III mRNA expression was localized in the epithelial cells including goblet cells and columnar cells in mice; on the other hand, HIP/PAP-expressing cells were correlated with Paneth cell metaplasia in human colon. Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon.

Regulatory role of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) in insulin secretion by glucose in pancreatic beta cells. Enhanced insulin secretion in CD38-expressing transgenic mice.

Cyclic ADP-ribose (cADPR) serves as a second messenger for Ca2+ mobilization in insulin secretion, and CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activities (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H.(1993) J. Biol. Chem. 268, 26052-26054). Here, we produced transgenic mice overexpressing human CD38 in pancreatic β cells. The enzymatic activity of CD38 in transgenic islets was greatly increased, and ATP efficiently inhibited the cADPR hydrolase activity. The Ca2+ mobilizing activity of cell extracts from transgenic islets incubated in high glucose was 3-fold higher than that of the control, suggesting that ATP produced by glucose metabolism increased cADPR accumulation in transgenic islets. Glucose- and ketoisocaproate-induced but not tolbutamide- nor KCl-induced insulin secretions from transgenic islets were 1.7-2.3-fold higher than that of control. In glucose-tolerance tests, the transgenic serum insulin level was higher than that of control. The present study provides the first evidence that CD38 has a regulatory role in insulin secretion by glucose in β cells, suggesting that the Ca2+ release from intracellular cADPR-sensitive Ca2+ stores as well as the Ca2+ influx from extracellular sources play important roles in insulin secretion.

Cloning and characterization of cDNA encoding rat ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase (homologue to human CD38) from islets of Langerhans

We report the cloning and cDNA sequence of rat CD38, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase. Rat CD38 is composed of 303 amino acids and shares a high degree of homology with human and mouse CD38. Rat CD38 mRNA is expressed in various tissues including pancreatic islets but not in RINm5F cells.