Hayato Ihara

Molecular cloning of human platelet thromboxane A synthase

Complementary DNA coding for thromboxane A synthase was amplified by polymerase chain reaction using primers synthesized according to the partial amino acid sequences of human platelet thromboxane A synthase (Nüsing, R., Schneider-Voss, S., and Ullrich, V. (1990) Arch. Biochem. Biophys. 280, 325-330) and cloned into pBluescript SK II(-). The primary structure of human platelet enzyme was deduced from the nucleotide sequence of the cDNA. The enzyme is composed of 533 amino acids with a molecular weight of 60,487. The primary structure of the enzyme exhibited a 34-36% homology to the amino acid sequences of cytochrome P450s classified in the P450 III gene family. The highly conserved cysteine-containing sequence involved in the heme-binding site of P450 was found near the carboxyl terminus (residues 472-492). The size of the major thromboxane A synthase mRNA from human platelets and human erythroleukemia cells was estimated to be approximately 2.2 kilobases by RNA blot analysis.

The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.

In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1′: Arg346-Met347). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02–1.0 nm; half-maximal effect at ∼0.1 nm). Subtilisin NAT dose dependently (0.06–1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 ± 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 ± 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.

Rapid, specific and active site-catalyzed effect of tissue-plasminogen activator on hippocampus-dependent learning in mice.

In the present study we trained tissue-plasminogen activator (tPA)-knockout (tPA -/-) and wild-type (tPA +/+) male mice in step-down inhibitory avoidance learning, a hippocampus-dependent task. tPA -/- displayed significantly shorter latencies to step down at 90 min, one, two and seven days after training indicating the learning deficit in these animals (P < 0.05 vs tPA +/+). The locomotor activity, the level of anxiety in an elevated-plus maze, as well as the pain threshold did not differ between the two strains of mice. The learning disability of tPA -/- was overcome by more intense training. The learning deficit was also partially restored by limited intrahippocampal delivery of tPA (infused for 2 h before training; P < 0.05 vs control), but not by the delivery of urokinase plasminogen activator, indicating the acute need for tPA in learning. The beneficial effect of tPA was abolished by co-infusion of its inhibitor tPA-STOP, indicating that the facilitatory effect of tPA on learning requires a proteolytic step. However, tPA activity in the hippocampus was not indispensable for effective memory retrieval in tPA-infused tPA -/- mice. Thus, rapid, specific and proteolytic action of tPA facilitates hippocampus-dependent learning, but not retrieval of previously acquired information.

Cancer cells overexpress mRNA of urokinase-type plasminogen activator, its receptor and inhibitors in human non-small-cell lung cancer tissue: analysis by Northern blotting and in situ hybridization.

The transcriptional localizations of urokinase‐type plasminogen activator (uPA), its receptor (uPAR) and its inhibitors (PAI‐1 and PAI‐2), which are possibly involved in cancer metastasis, have not been determined in human lung cancer. To identify their regulation in primary non‐small‐cell lung cancer, we assayed mRNA levels by Northern blot analysis in 25 cases and determined the localizations of mRNA by in situ hybridization in 10 cases. The amounts of uPA and PAI‐2 mRNA were significantly higher in cancerous relative to normal lung tissues. However, no significant difference was observed in uPAR and PAI‐1 mRNA levels. All transcripts were present in cancer cells and were predominantly located in tumor edges in several cases. In addition, PAI‐1 transcripts were more abundant in poorly and moderately differentiated carcinomas relative to well‐differentiated carcinomas and PAI‐2 transcripts were more abundant in squamous cell carcinomas than in adenocarcinomas. Thus, PAIs may be involved in modulation of malignant potency. Our results indicate that human non‐small‐cell lung cancer cells can autonomously express the mRNAs of uPA, uPAR and PAIs, which are possibly involved in metastasis. Int. J. Cancer 78:286–292, 1998.© 1998 Wiley‐Liss, Inc.

Tissue-type plasminogen activator is involved in the process of neuronal death induced by oxygen-glucose deprivation in culture

Effect of tissue-type plasminogen activator (tPA) on oxygen–glucose deprivation (OGD) was studied in cultured cortical neurons prepared from tPA gene knockout (tPA-KO) and wild-type (Wt) mice. Three hours of OGD induced 45% and 23% of neuronal death in Wt and tPA-KO mice, respectively. Neuronal death in tPA-KO mice was increased to 42% by additional tPA. Six hours of OGD induced 80% and 40% of neuronal death in Wt and tPA-KO mice, respectively, whereas the addition of tPA increased to 62% in tPA-KO mice. These results suggest that tPA is directly involved in the process of neuronal death induced by ischemia-mimic stress without involving vascular or circulatory components.

Tissue-type plasminogen activator has paradoxical roles in focal cerebral ischemic injury by thrombotic middle cerebral artery occlusion with mild or severe photochemical damage in mice

The role of endogenous tissue-type plasminogen activator (tPA) in focal cerebral ischemic injury (FCII) after middle cerebral artery occlusion was studied using tPA gene-deficient (KO) mice and their wild-type (WT) littermates. The middle cerebral artery was occluded by thrombi induced by three different intensities of photochemical damage, a method that was newly introduced in mice. In both WT and KO mice, the intensity-dependent increase of FCII size was observed. The FCII size in tPA WT mice was smaller than in KO mice in cases of mild damage, whereas the FCII size was larger in WT mice than in KO mice in cases of severe damage. There was no difference in FCII size between WT and KO mice in cases of moderate damage. The number of microthrombi also increased with damage intensity in both WT and KO mice, but was less in WT mice at all intensities of damage. The results support the validity of the model of thrombotic occlusion by photochemical damage in mice, and suggest that endogenous tPA protects FCII through thrombolytic action on transient occlusion of middle cerebral artery with mild damage, but deteriorates on persistent occlusion with severe damage.

Proteolysis of highly polysialylated NCAM by the tissue plasminogen activator-plasmin system in rats

Tissue-type plasminogen activator (tPA), a serine protease which converts the zymogen plasminogen to the active protease plasmin, is believed to regulate neurite extension and neural cell migration by modulating extracellular metabolism. The highly polysialylated form of the neural cell adhesion molecule (NCAM-H) is strongly expressed in the developing brain and is believed to play a role in organizing the neural network. In this report, we incubated neonatal rat brain homogenates with human tPA and rat plasminogen in order to determine whether NCAM-H would be degraded. NCAM-H was degraded by plasmin which was formed from rat plasminogen by human tPA. The degradation was inhibited by the addition of plasminogen activator inhibitor type 1 (PAI-1) or aprotinin. These results suggest a possible contribution of the tPA-plasmin system to NCAM-H turnover in the developing brain.

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.

Induction of plasminogen activator inhibitor 1 gene expression in adipocytes by thiazolidinediones

We studied the effect of thiazolidinediones (TZDs) on the regulation of plasminogen activator inhibitor 1 (PAI‐1) gene expression during 3T3‐L1 adipocyte differentiation. Treatment of the cells with pioglitazone and insulin shortened the time for differentiation from 9 d to 3 d, which resulted in a dramatic increase in PAI‐1 mRNA as well as PAI‐1 antigen secreted into the culture medium. Insulin or pioglitazone alone had relatively little effect on the rate of cell differentiation and did not alter PAI‐1 mRNA levels. A number of other TZDs (e.g., troglitazone, ciglitazone) also induced PAI‐1 gene expression in the presence of insulin, and the magnitude of induction was approximately proportional to the ability of these molecules to activate the nuclear receptor, peroxisome proliferator‐activated receptor‐γ. We also studied the mechanism of induction of PAI‐1 mRNA by pioglitazone/insulin. Although the half‐life of adipocyte PAI‐1 mRNA was not changed upon treatment with pioglitazone/insulin, this treatment induced PAI‐1 promoter activity 10‐fold. These results indicate that PAI‐1 mRNA and protein are up‐regulated by TZDs in an insulin‐dependent manner and that these effects result from an increase in the rate of transcription of the PAI‐1 gene. The increase in PAI‐1 occurs in parallel with the induction of adipogenesis, which suggests that the two processes are related.

Induction of thromboxane synthase and prostaglandin endoperoxide synthase mRNAs in human erythroleukemia cells by phorbol ester

The effects of 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) on the mRNA levels of two enzymes, thromboxane synthase (TXS) and prostaglandin endoperoxide synthase (PES), responsible for the synthesis of thromboxane A2 from arachidonic acid, were studied in human crythroleukemia (HEL) cells by RNA blot analysis. TPA induced both TXS and PES mRNAs in HEL cells in a dose‐dependent manner at 36 h. The half‐maximal and maximal effects for the induction of both mRNAs ware at approximately 3×10−9 M and at 10−8 M, respectively. TXS and PES mRNA levels increased in a time‐dependent fashion by TPA, and reached to 7‐ and 3.5‐fold of the control, respectively after 48 h of TPA treatment. These results suggest that expression of TXS and PES genes in HEL cells were simultaneously stimulated by TPA.