Hiroyuki Akatsuka

Receptor Activator of NF-κB Ligand (RANKL) Activates TAK1 Mitogen-Activated Protein Kinase Kinase Kinase through a Signaling Complex Containing RANK, TAB2, and TRAF6

ABSTRACT The receptor activator of NF-κB (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-κB through IκB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases. In the interleukin-1 signaling pathway, TAK1 MAPK kinase kinase (MAPKKK) mediates MAPK and IKK activation via interaction with TRAF6, and TAB2 acts as an adapter linking TAK1 and TRAF6. Here, we demonstrate that TAK1 and TAB2 participate in the RANK signaling pathway. Dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by overexpression of RANK. In 293 cells stably transfected with full-length RANK, RANKL stimulation facilitates the formation of a complex containing RANK, TRAF6, TAB2, and TAK1, leading to the activation of TAK1. Furthermore, in murine monocyte RAW 264.7 cells, dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by RANKL and endogenous TAK1 is activated in response to RANKL stimulation. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the development and function of osteoclasts.

Serratia marcescens S-layer protein is secreted extracellularly via an ATP-binding cassette exporter, the Lip system.

The Serratia marcescens Lip exporter belonging to the ATP‐binding cassette (ABC) exporter is known to be involved in signal peptide‐independent extracellular secretion of a lipase and a metalloprotease. Although the genes of secretory proteins and their ABC exporters are usually all reported to be linked in several Gram‐negative bacteria, neither the lipase nor the protease gene is located close to the Lip exporter genes, lipBCD. A gene (slaA) located upstream of the lipBCD genes was cloned, revealing that it encodes a polypeptide of 100 kDa and is partially similar to the Caulobacter crescentus paracrystalline cell surface layer (S‐layer) protein. The Lip exporter‐deficient mutants of S. marcescens failed to secrete the SlaA protein. Electron micrography demonstrated the cell surface layer of S. marcescens. The S‐layer protein was secreted to the cultured media in Escherichia coli cells carrying the Lip exporter. Three ABC exporters, Prt, Has and Hly systems, could not allow the S‐layer secretion, indicating that the S. marcescens S‐layer protein is strictly recognized by the Lip system. This is the first report concerning secretion of an S‐layer protein via its own secretion system.

Novel Alternative Splice Variants of cGMP-binding cGMP-specific Phosphodiesterase

After our recent findings that the amino-terminal portion of rat cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) differs from those of bovine and human cGB-PDEs, we found two forms of canine cGB-PDE cDNAs (CFPDE5A1 and CFPDE5A2) in canine lung. Each contained a distinct amino-terminal sequence, CFPDE5A1, possessing an amino-terminal portion with sequence similar to those of bovine and human, and CFPDE5A2, having one similar to that of rat. Other portions coding for the cGMP binding domains and the catalytic domain were conserved. Both CFPDE5A1 and CFPDE5A2 transcripts were detected in the cerebellum, hippocampus, retina, lung, heart, spleen, and thoracic artery. CFPDE5A1 transcripts were particularly abundant in the pylorus, whereas CFPDE5A2 transcripts were quite low in this tissue. CFPDE5A1 and CFPDE5A2 expressed in COS-7 cells had cGMP K m values of 2.68 and 1.97 μm, respectively, and both were inhibited by a low concentration of a cGB-PDE inhibitor, Zaprinast. Both CFPDE5A1 and CFPDE5A2 bound cGMP to their allosteric cGMP binding domains, and this cGMP binding was stimulated by 3-isobutyl-1-methylxanthine. Thus, two types of alternative splice variants of canine cGB-PDE have been identified and shown to have similar biological properties in vitro.

The Serratia marcescens bioH gene encodes an esterase

The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity. Interestingly, the ORF was 70% identical to a product of the E. coli bioH gene, which lies at a locus separated from the bioABFCD operon and acts in the early steps of the biotin synthetic pathway before pimeloyl-CoA synthesis. This gene complemented a bioH-deficient mutation of E. coli. From the sequence analysis, BioH is presumed to be a serine hydrolase, which belongs to the alpha/beta hydrolase-fold family comprising a wide variety of hydrolases including esterases. A catalytic triad composed of a nucleophilic residue (Ser80), an acidic residue (Asp206), and histidine (His234) was conserved in BioH, and the nucleophilic residue Ser, a catalytic center, was situated in the consensus sequence of G-X-S-X-G-G, a nucleophile elbow. Although the enzymatic function of BioH is not yet elucidated, the bioH gene products from S. marcescens and E. coli show esterase activity, which may imply the hydrolysis of a precursor leading to pimeloyl-CoA ester. The esterase activity of BioH and its CoA binding activity recently reported agree with a current hypothesis of pimeloyl-CoA ester synthesis from CoA and acylester derivatives including an acyl-carrier protein.

The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33.

In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently.

Novel Membrane Protein Containing Glycerophosphodiester Phosphodiesterase Motif Is Transiently Expressed during Osteoblast Differentiation

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation. Interestingly, the deduced amino acid sequence contains 539 amino acids including seven putative transmembrane domains and a glycerophosphodiester phosphodiesterase region in one of the extracellular loops. Northern blot analysis revealed that GDE3 was also expressed in spleen as well as primary calvarial osteoblasts and femur. We next transfected HEK293T cells with GDE3 with green fluorescent protein fused to the C terminus. The green fluorescent protein-fused protein accumulated at the cell periphery, and the transfected cells overexpressing the protein changed from a spread form to rounded form with disappearance of actin filaments. Immunofluorescence staining with GDE3 antibody and phalloidin in MC3T3-E1 cells indicated that endogenous GDE3 might be co-localized with the actin cytoskeleton. To identify a role for GDE3 in osteoblast differentiation, MC3T3-E1 cells stably expressing the full-length protein were constructed. Expression of GDE3 showed morphological changes, resulting in dramatic increases in alkaline phosphatase activity and calcium deposit. These results suggest that GDE3 might be a novel seven-transmembrane protein with a GP-PDE-like extracellular motif expressed during the osteoblast differentiation that dramatically accelerates the program of osteoblast differentiation and is involved in the morphological change of cells.

Enzymatic resolution of diltiazem intermediate by Serratia marcescens lipase: molecular mechanism of lipase secretion and its industrial application

Abstract A lipase from Serratia marcescens was selected as an asymmetric hydrolytic enzyme for trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride that is useful as a coronary vasodilator. This lipase has high enantioselectivity (E=135) and was applied to the industrial production of the optically active intermediate of diltiazem using two-phase reaction system of organic solvent–water. Introduction of enzymatic reaction into the chemical synthetic route of diltiazem reduces the number of processes from nine to five. Analyses of the secretion mechanism of the lipase from S. marcescens cell membrane revealed that lipase (LipA), metalloprotease (PrtA), cell surface protein (SlaA) and flagellin are secreted via ABC-transporter, which is a common secreting mechanism in Gram-negative bacteria other than N-terminal signal peptide-dependent secreting mechanism. Molecular cloning of both the lipA gene, which codes the lipase protein, and lipBCD genes, which code the secretion device proteins, enable the production of the lipase by the self-cloning strain 140-fold as compared to the wild type strain. Immobilization of the lipase on a hollow fiber type membrane reactor contributes to the repeated use of enzyme and to efficient separation of the reaction product. Thus, enzymatic reaction and product separation are achieved simultaneously.

Overproduction of the extracellular lipase is closely related to that of metalloprotease in Serratia marcescens

The recombinant strain of Serratia marcescens Sr41 carrying the lipA gene encoding the extracellular lipase on a high-copy-number plasmid, pUC19, overproduced lipase. The extracellular lipase activity of the hyperproducing strain reached 220,000 U/ml of the culture medium and was about 9-fold greater than that of the strain carrying pUC19. The extracellular protease activity of the lipase-hyperproducing recombinant strain was decreased by 90% compared with that of the strain carrying pUC19. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis revealed overproduction of the lipase and reduction of the extracellular metalloprotease production in the culture medium of the lipase-hyperproducing strain. The production of extracellular metalloprotease was impaired by the increase of lipA gene expression. Thus, construction of the lipase-hyperproducing strain which reduced extracellular metalloprotease production, was achieved by self-cloning of the lipA gene in S. marcescens. The mutants carrying the metalloprotease gene defects constructed by homologous recombination showed significantly elevated extracellular lipase activity. These results suggested that the production of extracellular lipase was closely related to the metalloprotease production and that the secretion of the two proteins may be competitive in S. marcescens.

Localization of clearance receptor in rat lung and trachea: association with chondrogenic differentiation

The lung is rich in atrial natriuretic peptide binding sites, and the majority of them are considered to be the natriuretic peptide clearance receptor (NPR-C). In this study, localization of NPR-C in the rat lung and trachea was investigated by immunohistochemical analysis with the specific antibody. Positive staining was observed in the epithelial cell layers of the trachea and bronchiole and the myocardium surrounding the pulmonary vein. Moreover, expression of NPR-C was seen in mesenchymal cells; it was especially strong in cells in the perichondrium and decreased in chondrocytes in the cartilage. Because mesenchymal cells in the perichondrium differentiate to chondrocytes, NPR-C expression is suggested to be associated with chondrogenic differentiation. The chondrogenic cell line ATDC5 was used to study NPR-C expression during chondrogenic differentiation in vitro. The undifferentiated ATDC5 cells expressed NPR-C at a much higher level than the differentiated ATDC5 cells, in accordance with the observation of the immunohistochemical analysis in the cartilage. These findings suggest that NPR-C expression is differentially regulated in chondrocytes and that the natriuretic peptides may play a role in regulating chondrocyte development in the lung.The lung is rich in atrial natriuretic peptide binding sites, and the majority of them are considered to be the natriuretic peptide clearance receptor (NPR-C). In this study, localization of NPR-C in the rat lung and trachea was investigated by immunohistochemical analysis with the specific antibody. Positive staining was observed in the epithelial cell layers of the trachea and bronchiole and the myocardium surrounding the pulmonary vein. Moreover, expression of NPR-C was seen in mesenchymal cells; it was especially strong in cells in the perichondrium and decreased in chondrocytes in the cartilage. Because mesenchymal cells in the perichondrium differentiate to chondrocytes, NPR-C expression is suggested to be associated with chondrogenic differentiation. The chondrogenic cell line ATDC5 was used to study NPR-C expression during chondrogenic differentiation in vitro. The undifferentiated ATDC5 cells expressed NPR-C at a much higher level than the differentiated ATDC5 cells, in accordance with the observation of the immunohistochemical analysis in the cartilage. These findings suggest that NPR-C expression is differentially regulated in chondrocytes and that the natriuretic peptides may play a role in regulating chondrocyte development in the lung.

Protein kinase C activation down-regulates natriuretic peptide receptor C expression via transcriptional and post-translational pathways

Natriuretic peptide receptor C (NPR‐C) mRNA expression and ANP‐binding activity via NPR‐C are significantly down‐regulated in HeLa cells with phorbol myristate acetate (PMA) treatment. Stabilization of the NPR‐C mRNA by PMA indicated that down‐regulation of its mRNA was mediated through negative transcriptional regulation. Despite the significant loss of the mRNA, reduction of NPR‐C‐specific ANP‐binding activity after PMA exposure (4 h) was accompanied by a slight decrease in total NPR‐C protein (with a 5% loss) and was also produced in the presence of actinomycin D or cycloheximide. The inhibitory effect of a long PMA exposure (18 h) paralleled with a decrease in total NPR‐C protein is suggested to be dependent on reduction of de novo NPR‐C synthesis. PMA‐induced transcriptional and post‐translational down‐regulation of NPR‐C was effectively reversible in the presence of the protein kinase C inhibitor GF109203X. These findings demonstrate that protein kinase C activation down‐regulated NPR‐C expression through transcriptional and post‐translational pathways and that immediate functional receptor loss was mediated via a post‐translational mechanism, such as enhanced receptor internalization.