Toshiaki Monkawa

Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells

cDNA (CIC-3) encoding a protein kinase C-regulated chloride channel was cloned and characterized. The open reading frame encodes 760 amino acids, which possess significantly amino acid identity with previously cloned CIC chloride channels. The chloride currents expressed in Xenopus oocytes injected with CIC-3 cRNA were completely blocked by activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate. Abundant expression of CIC-3 mRNA was observed in rat brain, especially in the olfactory bulb, hippocampus, and cerebellum. These findings suggest that CIC-3 may play an important role in neuronal cell function through regulation of membrane excitability by protein kinase C.

Mutational Analysis of the Ligand Binding Site of the Inositol 1,4,5-Trisphosphate Receptor

To define the structural determinants for inositol 1,4,5-trisphosphate (IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding “core.” Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.

Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney.

To examine whether expression and distribution of aquaporin of collecting duct (AQP-CD) are regulated by vasopressin V2 receptor (V2R), we performed immunohistochemical studies with specific antibody against AQP-CD. Normal Wistar rats were divided into four groups and treated for 3 d; control, dehydration, vasopressin V1 receptor (V1R) antagonist (OPC-21268 120 mg/kg), V2R antagonist (OPC-31260 30 mg/kg). At time of death, urine osmolality (Uosm) in the dehydration group (1884 +/- 245 mOsm/kg) was significantly higher than that in the control (938 +/- 91). In the V2R antagonist group, Uosm was significantly decreased to 249 +/- 29, whereas V1R antagonist showed no effect on Uosm. In the control and V1R antagonist groups, immunofluorescence studies showed the AQP-CD staining of both apical membrane and subapical cytoplasm of CD cells of the cortex and the inner medulla. Dehydration increased the immunostaining of both apical membrane and subapical cytoplasm of CD cells of the inner medulla, and the degree of increase was dominant in apical membrane. In the V2R antagonist group, only faint staining of apical membrane and weak labeling of cytoplasm of CD cells of the inner medulla were observed. These changes in the localization and protein amount of AQP-CD by dehydration and V2R antagonist were quantitatively confirmed by immunogold studies and immunoblot analysis of the inner medulla. The present results indicate that the distribution and amount of AQP-CD in the CD cells are regulated by vasopressin V2 receptor.

A new member of the HCO3- transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney

The kidneys play pivotal roles in acid-base homeostasis, and the acid-secreting (α-type) and bicarbonate-secreting (β-type) intercalated cells in the collecting ducts are major sites for the final modulation of urinary acid secretion. Since the H+-ATPase and anion exchanger activities in these two types of intercalated cells exhibit opposite polarities, it has been suggested that the α- and β-intercalated cells are interchangeable via a cell polarity change. Immunohistological studies, however, have failed to confirm that the apical anion exchanger of β-intercalated cells is the band 3 protein localized to the basolateral membrane of α-intercalated cells. In the present study, we show the evidence that a novel member of the anion exchanger and sodium bicarbonate cotransporter superfamily is an apical anion exchanger of β-intercalated cells. Cloned cDNA from the β-intercalated cells shows about 30% homology with anion exchanger types 1–3, and functional expression of this protein in COS-7 cells and Xenopus oocytes showed sodium-independent and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-insensitive anion exchanger activity. Furthermore, immunohistological studies revealed that this novel anion exchanger is present on the apical membrane of β-intercalated cells, although some β-intercalated cells were negative for AE4 staining. We conclude that our newly cloned transporter is an apical anion exchanger of the β-intercalated cells, whereas our data do not exclude the possibility that there may be another form of anion exchanger in these cells.

Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5-trisphosphate receptor: Application to the studies on inflammatory cells

Monoclonal antibodies were raised that specifically recognize the COOH‐terminal sequences and the loop sequences between the fifth and the sixth transmembrane spanning regions of human inositol 1,4,5‐trisphosphate receptor (IP3R) type 1, 2 and 3. Western blot analysis using Jurkat cells, mouse cerebellum, COS‐7 expressing IP3R type 3 cDNA showed that those monoclonal antibodies reacted specifically with each of these three IP3R subtypes and that they do not cross‐react. These antibodies could be used for the specific immunoprecipitation of IP3Rs. Using these monoclonal antibodies, the expression profiles of IP3R‐subtype proteins were found to be different among inflammatory cells such as macrophages, polymorphonuclear cells, mast cells, eosinophils, splenocytes, thymocytes and megakaryocytic cells. Usually, more than one type of IP3R were expressed in a cell simultaneously. The observation of CMK cells under immunofluorescence confocal microscopy revealed that IP3R type 1 and type 2 are located at different subcellular fractions.

Heart failure causes cholinergic transdifferentiation of cardiac sympathetic nerves via gp130-signaling cytokines in rodents

Although several cytokines and neurotrophic factors induce sympathetic neurons to transdifferentiate into cholinergic neurons in vitro, the physiological and pathophysiological roles of this remain unknown. During congestive heart failure (CHF), sympathetic neural tone is upregulated, but there is a paradoxical reduction in norepinephrine synthesis and reuptake in the cardiac sympathetic nervous system (SNS). Here we examined whether cholinergic transdifferentiation can occur in the cardiac SNS in rodent models of CHF and investigated the underlying molecular mechanism(s) using genetically modified mice. We used Dahl salt-sensitive rats to model CHF and found that, upon CHF induction, the cardiac SNS clearly acquired cholinergic characteristics. Of the various cholinergic differentiation factors, leukemia inhibitory factor (LIF) and cardiotrophin-1 were strongly upregulated in the ventricles of rats with CHF. Further, LIF and cardiotrophin-1 secreted from cultured failing rat cardiomyocytes induced cholinergic transdifferentiation in cultured sympathetic neurons, and this process was reversed by siRNAs targeting Lif and cardiotrophin-1. Consistent with the data in rats, heart-specific overexpression of LIF in mice caused cholinergic transdifferentiation in the cardiac SNS. Further, SNS-specific targeting of the gene encoding the gp130 subunit of the receptor for LIF and cardiotrophin-1 in mice prevented CHF-induced cholinergic transdifferentiation. Cholinergic transdifferentiation was also observed in the cardiac SNS of autopsied patients with CHF. Thus, CHF causes target-dependent cholinergic transdifferentiation of the cardiac SNS via gp130-signaling cytokines secreted from the failing myocardium.

The Hypertrophic Effect of Transforming Growth Factor-β is Reduced in the Absence of Cyclin-Dependent Kinase-Inhibitors p21 and p27

Transforming growth factor-beta (TGF-beta) has both antiproliferative and hypertrophic effects on mesangial cells (MC). However, it is not known if these processes are independent or if they share common signaling pathways. Proliferation and hypertrophy are regulated by specific cell-cycle regulatory proteins, where the cyclin-dependent kinase (CDK) inhibitors inhibit target cyclin-CDK complexes. This study examined whether the growth regulatory effects of TGF-beta were determined by the CDK inhibitors p21 and p27. Accordingly, cultured MC from wild type (+/+) and single and double null (-/-) p21 and p27 mice were grown in 5% serum in the presence or absence of TGF-beta1 (2 ng/ml). Proliferation ([(3)H]-thymidine incorporation, cell number, cell cycle) and hypertrophy ([(3)H]-leucine incorporation, total protein content, forward light scatter) were measured after 24 h, 48 h, and 96 h. TGF-beta inhibited proliferation in +/+ and p21/p27 double -/- MC to a similar extent. TGF-beta induced hypertrophy in +/+ MC (18.0% increase at 48 h), and to lesser extent in p21 -/- (12.8%) and p27 -/- MC (11.5%) measured by forward light scatter analysis. In p21/p27 double -/-, the hypertrophic effects of TGF-beta were significantly reduced (3.9% at 48 h). Similar results were obtained by measuring hypertrophy by total protein and [(3)H]-leucine incorporation. In conclusion, the CDK inhibitors p21 and p27 are not required for the antiproliferative effects of TGF-beta. However, the hypertrophic growth effects of TGF-beta are reduced in the absence of both p21 and p27. These data suggest that the regulation of the antiproliferative and hypertrophic effects of TGF-beta may be distinct processes.

Serum leptin concentrations in patients with thyroid disorders

Leptin, the obese gene product, is secreted exclusively by adipocytes and is thought to act as a lipostatic signal that regulates body weight homeostasis. We previously reported that thyroid hormone is one of the up‐regulating factors of leptin in vitro. T3, at physiological concentrations, stimulates leptin mRNA expression and leptin secretion by 3T3‐L1 adipocytes. The aim of this study was to explore the role of thyroid hormone in the regulation of leptin in humans.

Snail1 is involved in the renal epithelial-mesenchymal transition.

The pathological significance of the tubular epithelial-mesenchymal transition (EMT) in kidney diseases is becoming increasingly recognized, and the transcription factor Snail1 plays a critical role in EMT. The results of this study show that Snail1 mRNA and protein were upregulated in the tubular epithelial cells of the obstructed kidneys in a rat model of unilateral ureteral obstruction and in human proximal tubule HKC-8 cells treated with TGF-beta1. Glycogen synthase kinase-3beta (GSK-3beta) regulates the Snail1 level by degrading Snail1 protein. The level of the phosphorylated inactive form of GSK-3beta was increased in the tubular epithelial cells of the obstructed kidney. TGF-beta1 increased the phosphorylated form of GSK-3beta in HKC-8 cells, and inhibition of GSK-3beta by the selective inhibitors lithium and TDZD-8 caused Snail1 protein to accumulate. This study demonstrated that Snail1 is involved in renal tubular EMT and that TGF-beta1 regulates Snail1 at the transcription and protein degradation levels.