Biomedical subjects
Publications and source records attributed to A Ichikawa.
Mature 53 kDa histidine decarboxylase (HDC) peptide is produced from a precursor 74 kDa peptide. The mechanism of specific cleavage by processing enzyme is unknown. Using the recombinant mouse 74 kDa HDC, we found that porcine pancreatic elastase specifically converted the inactive 74 kDa HDC to its active form of 53 kDa HDC.
This review summarizes recent advances in the molecular characterization of prostanoid receptors. Prostanoids exert versatile actions in diverse tissues and cells through specific cell surface receptors. Molecular biological studies revealed the primary structure of eight types and subtypes of prostanoid receptor from various species. These include the thromboxane A2 receptor, prostacyclin receptor, prostaglandin (PG) F receptor, PGD receptor and four subtypes of PGE receptors. They are coupled to different signal transduction systems. In addition, multiple isoforms of PGE receptor EP3 subtype have been identified in various species. They are produced through alternative RNA splicing from a single gene and differ only in their carboxy-terminal tails. These isoforms differ in the efficiency of G protein activation, in the specificity of coupling to G proteins or in sensitivity to desensitization. This molecular characterization is useful for understanding the diverse physiological roles of prostanoids.
A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 >> iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.
We recently identified the cis-regulatory element and its specific nuclear binding factors for delta 12-prostaglandin (PG) J2-induced expression of the rat heme oxygenase, HO-1 [Koizumi, T., Odani, N., Okuyama, T., Ichikawa, A. and Negishi, M. (1995) J. Biol. Chem. 270, in press]. Here we further examined the molecular mechanism underlying the delta 12-PGJ2-induced HO-1 gene expression. Protein kinase inhibitors, 2-aminopurine and staurosporine, suppressed the delta 12-PGJ2-induced HO-1 mRNA and the nuclear protein binding to the delta 12-PGJ2-responsive cis-regulatory element in rat basophilic leukemia cells. Furthermore, the nuclear protein binding to the element was suppressed by in vitro phosphatase treatment of the nuclear proteins from delta 12-PGJ2-treated cells. These findings suggest that delta 12-PGJ2 induces the expression of the HO-1 gene through phosphorylation of the nuclear proteins which bind to the delta 12-PGJ2-responsive element.
We recently reported that delta 12-prostaglandin (PG) J2 caused various cells to synthesize heme oxygenase, HO-1 (Koizumi, T., Negishi, M., and Ichikawa, A. (1992) Prostaglandins 43, 121-131). Here we examined the molecular mechanism underlying the delta 12-PGJ2-induced HO-1 synthesis. delta 12-PGJ2 markedly stimulated the promoter activity of the 5'-flanking region of the rat HO-1 gene from -810 to +101 in rat basophilic leukemia cells. From functional analysis of various deletion mutant genes we found that the delta 12-PGJ2-responsive element was localized in a region from -690 to -660, containing an E-box motif, which was essential for the delta 12-PGJ2-stimulated promoter activity. When the region containing the delta 12-PGJ2-responsive element was combined with a heterologous promoter, SV40 promoter, in the sense and antisense direction, the element showed an enhancer activity in response to delta 12-PGJ2. Gel mobility shift assays demonstrated that delta 12-PGJ2 specifically stimulated the binding of two nuclear proteins to the E-box motif of this region. These results indicate that delta 12-PGJ2 induces the expression of the rat HO-1 gene through nuclear protein binding to a specific element having an E-box motif.
Prostaglandin E receptor EP3D is coupled to stimulation and inhibition of adenylate cyclase and stimulation of phosphatidylinositol turnover. To examine the roles of the interaction of the carboxylic acid of an agonist and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine to the non-charged glutamine. TEI-3356, an EP3 agonist with a negatively charged the carboxylic acid, and TEI-4343, a non-charged methylester of TEI-3356, inhibited the forskolin-stimulated cAMP formation in the same concentration-dependent manner, but stimulation of basal cAMP formation and Ca2+ mobilization by TEI-4343 was much lower than that by TEI-3356. In the mutant receptor, both TEI-3356 and TEI-4343 showed the inhibition of forskolin-stimulated cAMP formation in the same profile, but did not stimulate basal cAMP formation or Ca2+ mobilization. These findings suggest that the interaction between the carboxylic acid of agonist and the arginine residue is important in signal transduction for adenylate cyclase stimulation and Ca2+ mobilization but not for adenylate cyclase inhibition.
Prostaglandin (PG) E receptor EP3D is coupled to both Gi and Gs. To examine the roles of the interaction of alpha-carboxylic acid of PGE2 and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine residue to the noncharged glutamine. PGE2 with a negatively charged alpha-carboxylic acid and sulprostone, an EP3 agonist with a noncharged modified alpha-carboxylic acid, inhibited the forskolin-stimulated adenylate cyclase activity via Gi activation in the EP3D receptor in the same concentration-dependent manner. In contrast, the adenylate cyclase stimulation via Gs activation by sulprostone was much lower than that by PGE2. On the other hand, both PGE2 and sulprostone showed potent Gi activity but failed to show Gs activity in the mutant receptor. EP3D receptor showed a high affinity binding for PGE2 in the form coupled to either Gi or Gs. Although the mutant receptor showed high affinity binding when coupled to Gi, it lost high affinity binding in the condition of Gs coupling. Furthermore, sulprostone bound to the Gi-coupled EP3D receptor with higher affinity than the Gs-coupled receptor. Among various EP3 agonists, alpha-carboxylic acid-unmodified agonists showed both Gi and Gs activities, but the modified agonists showed only Gi activity. These findings suggest that the interaction between the alpha-carboxylic acid of PGE2 and the arginine residue of the receptor regulates the selectivity of the G protein coupling.
We previously cloned a cDNA for a mouse PGE receptor positively coupled to adenylate cyclase from mouse mastocytoma cells, and reported it as EP2 subtype of PGE receptor [Honda, A., Sugimoto, Y., Namba, T., Watabe, A., Irie, A., Negishi, M., Narumiya, S. and Ichikawa, A. (1993) J. Biol. Chem. 268, 7759-7762]. However, it is not sensitive to one of the EP2 agonists, butaprost. Recently another subtype of PGE receptor coupled to adenylate cyclase has been identified pharmacologically and named EP4. These findings have led us to examine whether the cloned receptor is the EP4 subtype. AH23848B, a selective EP4 antagonist, not only displaced the [3H]PGE2 binding to the cloned receptor but antagonized the PGE2-stimulated cAMP formation in the receptor. In contrast, EP2 specific agonist, butaprost and 19(R)OH-PGE2 neither bound to the receptor nor stimulated the cAMP formation. These results suggest that this receptor previously reported as 'EP2' subtype is identical to the pharmacologically defined EP4 subtype and not of EP2 subtype.
We examined the signal transduction of mouse prostaglandin E receptor EP1 subtype using Chinese hamster ovary cells stably expressing the cloned EP1. Sulprostone, an EP1 agonist, induced a rapid increase in intracellular Ca2+ concentration in the EP1-expressing cells. Most of the increase was abolished by removal of extracellular Ca2+, and was insensitive to U-73122, a phospholipase C inhibitor. Sulprostone stimulated phosphatidylinositol hydrolysis, but this stimulation was abolished by removal of extracellular Ca2+, indicating that EP1-stimulated phosphatidylinositol hydrolysis is the result of extracellular Ca2+ influx. Thus, the signal transduction of EP1 is extracellular Ca2+ entry through a pathway independent of phospholipase C activation. We further examined the regulation of the signal transduction of EP1 having potential phosphorylation sites for either protein kinase C or protein kinase A. Short-term exposure of the cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) completely suppressed the sulprostone-induced increase in intracellular Ca2+ concentration, while forskolin or dibutyryl cAMP did not affect it, suggesting that protein kinase C but not protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 binding activity of EP1 due to the reduction of the EP1 mRNA level. Protein kinase C induces short- and long-term desensitization of EP1.
A third type of nucleoside diphosphate kinase (NDP kinase III), distinct from the previously described NDP kinases I and II (Nomura et al. (1991) Biochim. Biophys. Acta 1077, 47-55), was purified from spinach leaves to electrophoretic homogeneity. NDP kinase III was judged by SDS-PAGE and by gel filtration to have molecular masses of 17 kDa and 102 kDa, respectively, suggesting that it is composed of six subunits similarly to the other spinach isoforms, NDP kinases I and II. Amino-acid sequence analysis revealed the primary structure of NDP kinase III to be comprised of 153 amino-acid residues, the sequence of which exhibited 61% and 53% homology with those of NDP kinases I and II, respectively. In the reaction catalyzed by the three isoforms, the order of Km as phosphate acceptor was determined as GDP << ADP for NDP kinase III, different from those observed for NDP kinase I (ADP << GDP) and for NDP kinase II (GDP = ADP). These results suggest that the three isoforms may have distinct roles in regulating intracellular 5'-di- and 5'-triphosphonucleotide levels in spinach leaves.
A cDNA clone for rat thromboxane (TX) A2 receptor was obtained from cultured astrocytes. The cDNA encodes a protein of 341 amino acids with seven putative transmembrane domains. The receptor is homologous with mouse and human TXA2 receptors at 92.7% and 71.8%, respectively. Expression of messenger RNA for the receptor was examined by reverse transcriptase-polymerase chain reaction, showing ubiquitous expression in brain glial cells such as astrocytes, oligodendrocytes and microglia.
The amino acid sequences of the receptors for various prostaglandins, thromboxane and lipoxin, which belong to the rhodopsin family, were aligned, and a phylogenetic tree was constructed to infer the evolutionary history of the arachidonic acid cascade. The obtained tree suggested that the origin of the cyclooxygenase pathway was different from that of the lipoxygenase pathway. The receptors involved in the cyclooxygenase pathway constructed an independent cluster, but the lipoxin A4 receptor, which is involved in the lipoxygenase pathway, belonged to the cluster of peptide receptors. The primitive form of the cyclooxygenase pathway had been a signal transduction system composed of prostaglandin E2 and its receptor associated with cAMP metabolism.
The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody.
BACKGROUND & AIMS: Prostaglandins (PGs) have important roles in the regulation of gastric acid secretion. The aim of this study was to examine the possible presence of PG receptors on the gastric enterochromaffin-like (ECL) carcinoid of Mastomys natalensis, which might be a useful model of normal ECL cells. METHODS: A [3H]PGE2 binding experiment was performed by using the ECL tumor membrane, and intracellular signal transduction was studied in the cells. In addition, Northern blot analysis using EP2 and EP3 receptor complementary DNAs was conducted. RESULTS: [3H]PGE2 specifically bound to the tumor cell membrane, and the binding was displaced by various PGs with a potency order of PGE1 = PGE2 > enprostil > PGF2 alpha. Although PGE1 and PGE2 stimulated 5'-cyclic adenosine monophosphate (cAMP) production, neither PGF2 alpha nor enprostil had any effect. On the other hand, all of PGE1, PGE2, PGF2 alpha, and enprostil attenuated the forskolin-induced cAMP production. Moreover, enprostil inhibited histamine release induced by forskolin. However, on pertussis toxin treatment, PGE2 paradoxically enhanced the forskolin-induced increase of cAMP production. Finally, the presence of EP2 and EP3 receptor messenger RNAs was confirmed by RNA blot analysis. CONCLUSIONS: The ECL carcinoid tumor cells of Mastomys seem to possess two subtypes of PGE receptor: EP2 linked to cAMP production and EP3 coupled with inhibitory guanosine 5'-triphosphate-binding proteins mediating the inhibition of cAMP production.
Five distinct cDNA clones encoding four different isoforms of human prostaglandin (PG) E receptor EP3 subtype were isolated from a human kidney cDNA library. Two cDNA clones differed only in their 3'-untranslated regions. The four isoforms, tentatively named EP3-I, EP3-II, EP3-III, and EP3-IV, which were generated by alternative mRNA splicing, had identical amino acid sequences except for their different carboxyl-terminal tails. Transfection experiments revealed that all the four isoforms show high binding affinities to PGE2, PGE1, and M&B28767, an EP3-specific agonist, whereas their downstream signaling pathways are divergent. M&B28767 increased cAMP concentrations in cells expressing EP3-II and EP3-IV, whereas it inhibited forskolin-induced cAMP accumulations in cells expressing all EP3 isoforms. M&B28767 also stimulated phosphoinositide turnover in cells expressing EP3-I and EP3-II. Northern blot analysis revealed that the EP3 gene is expressed in a wide variety of human tissues. The human EP3 mRNA was present most abundantly in the kidney, pancreas, and uterus. A substantial expression was also detected in the heart, liver, skeletal muscle, small intestine, colon, prostate, ovary, and testis. Furthermore, reverse transcription-polymerase chain reaction analysis demonstrated tissue-specific expressions of the five different EP3 mRNA species. The present study suggests the presence of the multiple systems of PGE2/EP3 isoforms and leads to the better understanding of its physiological and pathophysiological implications in humans.
A 64-year-old woman was diagnosed as having myelodysplastic syndrome (MDS) at 45 months after receiving radiotherapy for advanced carcinoma of the uterine cervix. We chose low dose therapy of SPAC and ACR because of the diagnosis as therapy-related MDS and the existence of radiation colitis. She obtained minor response, but two months later she transformed to AML (M2). The interval between low dose therapies was getting shorter and shorter, so we tried intensive chemotherapy consisting of BHAC, ACR and 6MP. Blast numbers were reduced, but she died of sepsis and intestinal bleeding. The patients of MDS with t(8;21) and the patients of therapy-related AML (tAML) with t(8;21) are very rare. According to the literature, only karyotype is a prognostic factor in AML/MDS with t(8;21). And diagnosis by the criteria of FAB classification is of little value regarding clinical progress. That is to say, if the patient has only t(8;21) or karyotypic abnormalities which are of little value in prognosis, such as the loss of a sex chromosome, it must be treated as de novo AML, but if patient has karyotypic abnormalities such as -5, 5q-, -7, 7q-, and/or multiple (complicated) abnormalities, we must accept that the prognosis is poor and must treat it as ordinary MDS/tAML.
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The expression of the murine double minute-2 (MDM2) gene, the product of which binds to and inactivates p53, was studied in 60 patients with B-cell chronic lymphocytic leukemia (B-CLL) or non-Hodgkin's lymphoma (B-NHL). Northern blot analysis showed that the level of MDM2 gene expression was low in normal human B-cells, whereas 17 of the patients (28.3%) with B-CLL or NHL had more than 10-fold higher levels of MDM2 gene expression than that observed in normal B cells. Immunohistochemical analysis confirmed MDM2 overexpression at the cellular protein level. MDM2 gene overexpression was found more frequently in patients with the low-grade type of lymphoma (56.5%) than in those with intermediate-/high-grade types (10.8%) (P = .001). Moreover, MDM2 overexpression was found significantly more frequently in patients at advanced clinical stages. Simultaneous analysis of p53 gene mutation showed that three patients had both MDM2 gene overexpression and p53 gene mutation. The results of the present study suggest that MDM2 gene overexpression may play an important role in the tumorigenicity and/or disease progression of CLL and low-grade lymphomas of B-cell origin.