Seikichi Toku

Selective cleavage of ErbB4 by G‐protein‐coupled Gonadotropin‐Releasing Hormone Receptor in Cultured Hypothalamic Neurons

Gonadotropin‐releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). GnRH neurons have a GnRH receptor belonging to the G‐protein‐coupled receptors. The stimulation of this receptor activates extracellular signal‐regulated kinase (ERK). In the present study, we found that epidermal growth factor receptor (EGFR) and ErbB4 were expressed in immortalized GnRH neurons (GT1‐7 cells). AG1478, a relatively specific inhibitor of the ErbB family, and small interfering RNA (siRNA) for ErbB4 inhibited the GnRH‐induced activation of ERK in GT1‐7 cells, suggesting that EGFR and ErbB4 were necessary for the activation. In addition, GnRH induced the cleavage of ErbB4 and accumulation of an 80‐kDa fragment. After treatment of the cells with 50 nM GnRH for 5 min, about 80% of ErbB4 was cleaved. Biotinylation of cell surface proteins revealed that more than 70% of the cell surface ErbB4 was cleaved by GnRH treatment. A higher concentration and longer treatment were necessary for GnRH to induce ErbB4 cleavage than ERK activation. TAPI‐2, an inhibitor of tumor necrosis factor‐α‐converting enzyme (TACE), and siRNA for TACE inhibited the cleavage of ErbB4, suggesting that TACE was involved. After ErbB4 cleavage, the activation of ERK by neuregulin 1 was almost completely inhibited. These results suggest that the down‐regulation of ErbB4 expression is induced by G‐protein‐coupled receptor stimulation. J. Cell. Physiol. 227: 2492–2501, 2012.

Inhibition by ethyl pyruvate of the nuclear translocation of nuclear factor-κB in cultured lung epithelial cells

Tumor necrosis factor alpha (TNFalpha) is a cytokine inducing inflammatory responses. It has been reported that ethyl pyruvate has anti-inflammatory actions through inhibition of the transcription mediated by nuclear factor-kappa B (NF-kappaB). By reporter gene assay, we first confirmed that TNFalpha activated the NF-kappaB pathway in cultured alveolar epithelial cells, A549 cells. This activation was strongly inhibited by ethyl pyruvate in a concentration-dependent manner. Treatment of the cells with TNFalpha-induced phosphorylation and degradation of IkappaBalpha within 15 min. The level of IkappaBalpha protein was increased from 30 min, suggesting an increase in the NF-kappaB-mediated transcription of IkappaBalpha. Ethyl pyruvate did not affect the changes in IkappaBalpha within 15 min, but strongly inhibited the increase in the IkappaBalpha protein level from 30 min. An immunoblot analysis revealed that ethyl pyruvate inhibited the nuclear translocation of RelA from 5 min of the treatment with TNFalpha. These results strongly suggested that ethyl pyruvate inhibited the NF-kappaB pathway through inhibition of the nuclear translocation of RelA. Ethyl pyruvate may be a good therapeutic drug for inflammation in which activation of the NF-kappaB pathway is involved.

Phosphorylation of ribosomal protein S19 at Ser59 by CaM Kinase Iα

In order to examine the possible involvements of Ca2+/calmodulin‐dependent protein kinases (CaM kinases) in the regulation of ribosomal functions, we tested the phosphorylation of rat ribosomal protein S19 (RPS19) by various CaM kinases in vitro. We found that CaM kinase Iα, but not CaM kinase Iβ1, Iβ2, II, or IV, robustly phosphorylated RPS19. From the consensus phosphorylation site sequence, Ser59, Ser90, and Thr124 were likely to be phosphorylated; therefore, we mutated each amino acid to alanine and found that the mutation of Ser59 to alanine strongly attenuated phosphorylation by CaM kinase Iα, suggesting that Ser59 was a major phosphorylation site. Furthermore, we produced a specific antibody against RPS19 phosphorylated at Ser59, and found that Ser59 was phosphorylated both in GT1‐7 cells and rat brain. Phosphorylation of RPS19 in GT1‐7 cells was inhibited by KN93, an inhibitor of CaM kinases. Immunoblot analysis after subcellular fractionation of rat brain demonstrated that phosphorylated RPS19 was present in 80S ribosomes. Phosphorylation of RPS19 by CaM kinase Iα augmented the interaction of RPS19 with the previously identified S19 binding protein. These results suggest that CaM kinase Iα regulates the functions of RPS19 through phosphorylation of Ser59.

Induction of epithelial-mesenchymal transition by flagellin in cultured lung epithelial cells.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and activates host inflammatory responses, mainly through activation of the NF-κB pathway. Although pulmonary fibrosis occurs in some cases of lung infection by flagellated bacteria, the pathological roles of TLR5 stimulation in pulmonary fibrosis have yet to be elucidated. In the present study, we first confirmed that flagellin activated the NF-κB pathway in cultured A549 alveolar epithelial cells. Next, we examined the types of genes whose expression was modulated by flagellin in the cells. Microarray analysis of gene expression indicated that flagellin induced a change in gene expression that had a similar trend to transforming growth factor-β1 (TGF-β(1)), a key factor in the induction of epithelial-mesenchymal transition (EMT). Biochemical analysis revealed that TGF-β(1) and flagellin increased the level of fibronectin protein, while they reduced the level of E-cadherin protein after 30 h of treatment. Interestingly, simultaneous treatment with TGF-β(1) and flagellin significantly augmented these EMT-related changes. Flagellin strongly activated p38 MAP kinase, and the activation was sustained for longer than 30 h. SB203580, an inhibitor of p38 MAP kinase, inhibited the upregulation of fibronectin by both flagellin and TGF-β(1). Simultaneous treatment with TGF-β(1) and flagellin augmented the activation of p38 MAP kinase by TGF-β(1) or flagellin alone. These results strongly suggest that flagellin cooperates with TGF-β(1) in the induction of EMT in alveolar epithelial cells.

The primary structure of chicken ribosomal protein L37a

The amino acid sequence of chicken ribosomal protein L37a was deduced from the nucleotide sequence of a recombinant cDNA and its genomic DNA. Chicken ribosomal protein L37a has 92 amino acids and a molecular mass of 10,247 Da including the initiator methionine. The protein contains a typical Cys2Cys2 zinc finger motif, which may be involved in protein-RNA interaction.

Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.

In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms.

Phosphorylation of epidermal growth factor receptor at serine 1047 by MAP kinase-activated protein kinase-2 in cultured lung epithelial cells treated with flagellin

It has been reported that tumor necrosis factor α (TNFα) activated the p38 MAP kinase pathway, followed by phosphorylation of epidermal growth factor receptor (EGFR) at serine 1047 (Ser1047). Although the phosphorylation of Ser1047 reportedly induced an internalization of EGFR, a protein kinase responsible for the phosphorylation has not been elucidated. In the present study, we found that treatment with flagellin of A549 cells, an alveolar epithelial cell line, induced the activation of p38 MAP kinase, followed by phosphorylation of EGFR at Ser1047. The phosphorylation was strongly inhibited by SB203580, an inhibitor of p38 MAP kinase. The flagellin treatment activated MAP kinase-activated protein kinase-2 (MAPKAPK-2), a protein kinase downstream of p38 MAP kinase, and MK2a inhibitor, an inhibitor of MAPKAPK-2, inhibited the flagellin-induced phosphorylation of EGFR at Ser1047. Unlike the flagellin treatment, the TNFα treatment induced the phosphorylation of EGFR at both Ser1047 and Tyr1173. SB203580 and MK2a inhibitor strongly inhibited the phosphorylation of Ser1047 but not Tyr1173 in EGFR. Finally, bacterially expressed and activated MAPKAPK-2 phosphorylated EGFR at Ser1047 in vitro. These results suggest that flagellin regulates the residence time of EGFR on the plasma membrane and thus the signaling of EGFR through phosphorylation of Ser1047 by MAPKAPK-2.

Interaction of ethyl pyruvate in vitro with NF-κB subunits, RelA and p50

Ethyl pyruvate, an aliphatic ester derived from pyruvate, reportedly has anti-inflammatory actions through inhibition of the transcription mediated by nuclear factor-kappa B (NF-κB). It was suggested that ethyl pyruvate inhibited NF-κB/DNA-binding activity through the covalent modification of RelA. However, the interaction of ethyl pyruvate with RelA in vitro has not been reported. In the present study, we confirmed that treatment of cultured alveolar epithelial cells, A549 cells, with tumor necrosis factor α (TNFα) increased the NF-κB/DNA-binding activity. When the nuclear extract of the cells was incubated with ethyl pyruvate, the NF-κB/DNA-binding activity was strongly inhibited. Because we previously found that the NF-κB/DNA complex included RelA and p50, we bacterially expressed a deletion mutant of RelA, RelA (1-220), and a full-length form of p50. Incubation of RelA (1-220) or p50 with ethyl pyruvate induced dramatic changes in mobility in two types of nondenaturing gel electrophoresis. Electrophoretic mobility shift assays revealed that incubation of RelA (1-220) or p50 with ethyl pyruvate inhibited the DNA-binding activity. Furthermore, immunostaining of A549 cells revealed that ethyl pyruvate inhibited the nuclear association of RelA after TNFα treatment. These results suggest that ethyl pyruvate interacts with RelA and p50 to inhibit their functions at multiple points.

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-Releasing Hormone

Background: Gonadotropin-releasing hormone (GnRH) plays critical roles in the progression of sex hormone-dependent cancers. Results: Activation of protein kinase D1 (PKD1) by protein kinase C was necessary for activation of the tyrosine kinase pathway. Conclusion: PKD1 is involved in signal transduction in GnRH-induced activation of extracellular signal-regulated protein kinase. Significance: Modification of PKD1 activity may be a new strategy for a therapy of sex hormone-dependent cancers. The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.

A Serine Proteinase from Cow Snout Epidermis Degrades High Molecular Weight Keratins

This study describes a serine proteinase which degrades high molecular weight polypeptides of keratins. The enzyme was coextracted with 8M urea together with keratins and separated from the keratins by gel filtration chromatography. Molecular weight of the enzyme was estimated at 25,000 daltons by gel filtration. Revealed characteristics of the enzyme were as follows; 1) maximally active at pH 8.5–9.0, 2) requires 0.04% SDS for maximum activity, 3) heat stable in the absence of SDS up to 65°C for 2 min, 4) sensitive against serine protease inhibitor, phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, 5) highly specific for natural protein substrates, such as keratin polypeptides.