Mariko Nishiyama

Novel Mutations of the Endothelin B Receptor Gene in Patients with Hirschsprung’s Disease and Their Characterization

Hirschsprung’s disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely theRET protooncogene, the endothelin B (ETB) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations inEDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous.

Characterization of RGS5 in regulation of G protein-coupled receptor signaling.

RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.

Expression of regulator of G protein signalling protein 5 (RGS5) in the tumour vasculature of human renal cell carcinoma

The gene expression profiles of tumour and normal vasculature are distinctively different. The altered expression of various angiogenesis‐related genes in tumour‐derived endothelial cells has been investigated intensively, but there may be as yet unidentified molecules that regulate tumour angiogenesis. In the present study, the distinctive expression of regulator of G protein signalling protein 5 (RGS5) in tumour vessels in human renal cell carcinoma (RCC) has been clarified. RGS5 is a member of the RGS superfamily and acts as a negative regulator of heterotrimeric G protein‐mediated signalling through G protein‐coupled receptors (GPCRs). RT‐PCR showed strong expression of RGS5 in all RCCs examined, but expression was very weak or undetectable in normal kidneys. By real‐time RT‐PCR, the ratio of RGS5 mRNA in RCC to that in normal kidney was 6.6 : 1 (p = 0.0012). In situ hybridization showed strong expression of RGS5 in vessels within tumour cell nests. It was expressed neither in tumour cells nor in normal renal capillaries. Immunohistochemical staining using serial sections for endothelial cell markers (CD31 and CD34) and smooth muscle cell markers (α‐SMA and desmin), as well as fluorescence double staining, strongly suggested that tumour endothelial cells were the main location of RGS5 in RCC. These findings suggest that RGS5 may be involved in G protein‐mediated signalling in tumour vessels in human RCC. Copyright

Analysis of two pharmacologically predicted endothelin B receptor subtypes by using the endothelin B receptor gene knockout mouse

This study was performed to clarify whether the endothelin (ET) receptor subtypes mediating two pharmacologically heterogeneous response to ETB receptor agonists in normal mice are the product(s) of a single ETB receptor gene. Vasodilator responses to sarafotoxin S6c (S6c) in the thoracic aorta and contractile responses to ET‐1 and IRL1620 in the stomach were examined in tissues from normal and ETB receptor gene knockout mice, in the absence and presence of an ETA receptor antagonist, BQ‐123, or an ETA/ETB receptor antagonist, PD142893. In the normal mouse aorta precontracted with phenylephrine, S6c (0.1100 nm) caused concentration‐dependent relaxations (pD2=8.4). BQ‐123 had no effect on these responses. However, PD142893 almost abolished the relaxations induced by 0.1300 nm S6c. In aortae taken from ETB receptor gene knockout mice, S6c up to 1 μm failed to cause relaxations, confirming that ETB receptors are involved in mediating this response. In normal mouse gastric fundus, 0.1 nm1 μm ET‐1, S6c or IRL1620 caused dose‐dependent, BQ‐123‐insensitive contractions, which were much more resistant to PD142893 than S6c‐induced relaxations of the aorta. The pD2 values for S6c in the absence and presence of PD142893 (10 μm) were 8.12±0.11 and 7.70±0.11, respectively. In the gastric fundus of the ETB receptor gene knockout mouse, S6c and IRL1620 caused no contractions. ET‐1 (0.1 nm1 μm) caused contractions sensitive to both BQ‐123 and PD142893, indicating that only ETA receptors mediate ET‐1‐induced contractions of the knockout mouse gastric fundus. Since both the PD142893‐sensitive vasodilator response of the aorta and the PD142893‐resistant contractile response of the gastric fundus to S6c were completely absent in the ETB receptor gene knockout mouse, we conclude that the two pharmacologically heterogeneous responses to S6c are mediated by receptors derived from the same ETB receptor gene.

Up‐regulation of the interferon γ (IFN‐γ)‐inducible chemokines IFN‐inducible T‐cell α chemoattractant and monokine induced by IFN‐γ and of their receptor CXC receptor 3 in human renal cell carcinoma

The antiangiogenic CXC chemokines interferon γ (IFN‐γ)‐inducible T‐cell α chemoattractant (I‐TAC) and monokine induced by IFN‐γ (Mig) are known as members of IFN‐γ‐inducible antiangiogenic CXC chemokines. However, the expression of these chemokines in highly angiogenic tumors remains poorly understood. The authors examined expression of I‐TAC, Mig, and their receptor, CXCR3, in tissue samples from patients with renal cell carcinoma (RCC).

CD151 dynamics in carcinoma-stroma interaction: integrin expression, adhesion strength and proteolytic activity.

A member of tetraspanin CD151 is a scaffold protein of laminin-binding integrins and it plays an important role in stable interaction between cells and basement membrane. Although the upregulation of CD151 in tumor cells is thought to accelerate tumor invasion and metastasis, detailed pathological investigation on CD151 and its association with integrins has not been well documented, yet. In the present study, we showed that the expression levels of CD151 and its associated integrin subunits in epidermal carcinoma cell HSC5 were higher than those in immortalized epidermal cell HaCaT. By the stimulation of epidermal growth factor, CD151 was dissociated from cell surface and dispersed in the cytoplasm, and α3β1 integrin was concomitantly internalized. To understand the significance of CD151 in tumor cell dynamics, CD151 in HSC5 was knocked down (HSC5CD151−), and the expression of integrin subunits and matrix metalloproteinases (MMPs) were investigated. In HSC5CD151−, striking morphological alteration on Matrigel and laminin, and cytoskeletal rearrangements were demonstrated. α3β1 integrin was internalized in part, and α6β4 integrin was re-distributed from basal site to cell periphery. Quantitative RT-PCR, Western blot and zymography revealed that the expression levels of MMP2, MMP7 and MMP9 were markedly downregulated in HSC5CD151−. Immunoprecipitation assay demonstrated that MMP7 was co-immunoprecipitated with CD151. In double stainings, MMP7 was colocalized with CD151 at the leading edge of lamellipodia under migratory status. These results elucidated the importance of CD151 as one of the key molecules for integrin-dependent carcinoma–stroma interaction. It is indicated that CD151 might contribute not only to cell stabilization by associating with adhesion complexes but also to cell migration by inducing integrins re-localization and MMPs production.

Involvement of p38α Mitogen-activated Protein Kinase in Lung Metastasis of Tumor Cells

To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38α+/- mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38α+/- mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38α+/- mice. Platelets of p38α+/- mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38α+/- mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38α+/- mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38α+/- mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38α+/- mice was similar to that of WT mice. These results suggest that p38α plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis.

Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: Roles of its N-terminal domain

Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca(2+) responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1-71 of RGS2 with the corresponding residues (1-51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1-51 of RGS5 with the residues 1-71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16-55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56-71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1-33 or 34-52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1-55 or 56-71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.

Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Gαq subunit

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus.

Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells Analysis of endothelin-2 biosynthetic pathway

The biosynthetic pathway of endothelin (ET)‐2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET‐2‐related peptides, ET‐2, big ET‐2(1–38) and big ET‐2(22–28). Phosphoramidon decreased the amount of ET‐2 and increased that of big ET‐2(13̄8)dose‐dependently. The amount of big ET‐2(1–37) did not significantly change. These results suggest that big ET‐2 is composed of 38 and not 37 amino acid residues, and that a putative ET‐2‐converting enzyme (ECE‐2) should be classified as a phosphoramidon‐sensitive neutral metalloprotease, bearing a resemblance to the putative ET‐1‐converting enzyme (ECE‐1) in endothelial cells.