Biomedical subjects

Masao Saitoh

Publications and source records attributed to Masao Saitoh.

11 recordsLinked to original sources

PubMed2007

Apoptosis signal-regulating kinase (ASK) 2 functions as a mitogen-activated protein kinase kinase kinase in a heteromeric complex with ASK1.

Apoptosis signal-regulating kinase (ASK) 1 is a mitogen-activated protein kinase kinase kinase (MAP3K) in the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways that play multiple important roles in cytokine and stress responses. Here we show that ASK2, a highly related serine/threonine kinase to ASK1, also functions as a MAP3K only in a heteromeric complex with ASK1. We found that endogenous ASK2 was constitutively degraded in ASK1-deficient cells, suggesting that ASK1 is required for the stability of ASK2. ASK2 in a heteromeric complex with a kinase-negative mutant of ASK1 (ASK1-KN) effectively activated MAP2K and was more competent to respond to oxidative stress than ASK2 alone. Knockdown of ASK2 revealed that ASK2 was required for oxidative stress-induced JNK activation. These results suggest that ASK2 forms a functional MAP3K complex with ASK1, in which ASK1 supports the stability and the active configuration of ASK2. Moreover, ASK2 was found to activate ASK1 by direct phosphorylation, suggesting that ASK1 and ASK2 in a heteromeric complex facilitate their activities to each other by distinct mechanisms. Such a formation of functional heteromeric complex between different MAP3Ks may be advantageous for cells to cope with a wide variety of stimuli by fine regulation of cellular responses.

Amino Acid Sequence↗

PubMed2006

Nuclear and cytoplasmic c-Ski differently modulate cellular functions.

c-Ski is a proto-oncogene product that induces morphologic transformation, anchorage independence, and myogenic differentiation when it is over-expressed in mesenchymal cells. c-Ski also inhibits signaling of transforming growth factor-beta (TGF-beta) superfamily members through interaction with Smad proteins. Although c-Ski is predominantly localized in the nucleus, aberrant cytoplasmic localization of it has also been reported in some tumor tissues and cell lines. In the present study, we identified the nuclear localization signal (NLS) in c-Ski. By introducing a mutation to abolish NLS activity, we examined the function of cytoplasmic c-Ski. Although cytoplasmic c-Ski suppressed TGF-beta superfamily-induced Smad signaling through sequestration of activated Smad complex to the cytoplasm, it failed to exhibit some of the activities that require nuclear localization of c-Ski, including suppression of basal transcription of the Smad7 gene. These findings indicate that subcellular localization of c-Ski affects its biologic activities. We also found that c-Ski accumulated in the cytoplasm when proteasome activity was inhibited. Mapping of the regions required for cytoplasmic accumulation by proteasome inhibitors suggests that subcellular localization of c-Ski may be regulated by proteasome-sensitive processes through amino acid residues 94-210 and 491-548.

Amino Acid Motifs↗

PubMed2005

The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta.

Transforming growth factor (TGF)-beta signaling facilitates tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for the treatment of patients with such cancer. In this study, we synthesized and characterized a small molecule inhibitor, A-83-01, which is structurally similar to previously reported ALK-5 inhibitors developed by Sawyer et al. (2003) and blocks signaling of type I serine/threonine kinase receptors for cytokines of the TGF-beta superfamily (known as activin receptor-like kinases; ALKs). Using a TGF-beta-responsive reporter construct in mammalian cells, we found that A-83-01 inhibited the transcriptional activity induced by TGF-beta type I receptor ALK-5 and that by activin type IB receptor ALK-4 and nodal type I receptor ALK-7, the kinase domains of which are structurally highly related to those of ALK-5. A-83-01 was found to be more potent in the inhibition of ALK5 than a previously described ALK-5 inhibitor, SB-431542, and also to prevent phosphorylation of Smad2/3 and the growth inhibition induced by TGF-beta. In contrast, A-83-01 had little or no effect on bone morphogenetic protein type I receptors, p38 mitogen-activated protein kinase, or extracellular regulated kinase. Consistent with these findings, A-83-01 inhibited the epithelial-to-mesenchymal transition induced by TGF-beta, suggesting that A-83-01 and related molecules may be useful for preventing the progression of advanced cancers.

Activin Receptors, Type I↗

PubMed2004

Negative regulation of transforming growth factor-beta (TGF-beta) signaling by WW domain-containing protein 1 (WWP1).

Smad7 negatively regulates transforming growth factor (TGF)-beta superfamily signaling by binding to activated type I receptors, thereby preventing the phosphorylation of receptor-regulated Smads (R-Smads), as well as by recruiting HECT-type E3 ubiquitin ligases to degrade type I receptors through a ubiquitin-dependent mechanism. To elucidate the regulatory mechanisms of TGF-beta signaling, we searched for novel members of proteins that interact with Smad7 using a yeast two-hybrid system. One of the proteins identified was the WW domain-containing protein 1 (WWP1) that is structurally related to Smad ubiquitin regulatory factors (Smurfs), E3 ubiquitin ligases for Smads and TGF-beta superfamily receptors. Using a TGF-beta-responsive reporter in mammalian cells, we found that WWP1 inhibited transcriptional activities induced by TGF-beta. Similar to Smurfs, WWP1 associated with Smad7 and induced its nuclear export, and enhanced binding of Smad7 to TGF-beta type I receptor to cause ubiquitination and degradation of the receptor. Consistent with these results, WWP1 inhibited phosphorylation of Smad2 induced by TGF-beta. WWP1 thus negatively regulates TGF-beta signaling in cooperation with Smad7. However, unlike Smurfs, WWP1 failed to ubiquitinate R-Smads and SnoN. Importantly, WWP1 and Smurfs were expressed in distinct patterns in human tissues and carcinoma cell lines, suggesting unique pathophysiological roles of WWP1 and Smurfs.

Active Transport, Cell Nucleus↗

PubMed2003

Arkadia amplifies TGF-beta superfamily signalling through degradation of Smad7.

Arkadia was originally identified as a protein that enhances signalling activity of Nodal and induces mammalian nodes during early embryogenesis; however, the mechanisms by which Arkadia affects transforming growth factor-beta (TGF-beta) superfamily signalling have not been determined. Here we show that Arkadia is widely expressed in mammalian tissues, and that it enhances both TGF-beta and bone morphogenetic protein (BMP) signalling. Arkadia physically interacts with inhibitory Smad, Smad7, and induces its poly-ubiquitination and degradation. In contrast to Smurf1, which interacts with TGF-beta receptor complexes through Smad7 and degrades them, Arkadia fails to associate with TGF-beta receptors. In contrast to Smad7, expression of Arkadia is down-regulated by TGF-beta. Silencing of the Arkadia gene resulted in repression of transcriptional activities induced by TGF-beta and BMP, and accumulation of the Smad7 protein. Arkadia may thus play an important role as an amplifier of TGF-beta superfamily signalling under both physiological and pathological conditions.

Animals↗

PubMed2003

SB-431542 and Gleevec inhibit transforming growth factor-beta-induced proliferation of human osteosarcoma cells.

Transforming growth factor-beta (TGF-beta) has growth-stimulating effects on mesenchymal cells and several tumor cell lines. The signaling pathway for this effect is, however, not well understood. We examined how TGF-beta stimulates proliferation of MG63 human osteosarcoma cells. Two distinct type I receptors for TGF-beta, ALK-1 and ALK-5, were expressed and functional in MG63 cells. Of these two receptors, ALK-5 appears to be responsible for the growth stimulation because expression of constitutively active ALK-5, but not ALK-1, stimulated proliferation of MG63 cells. SB-431542 (0.3 microM), a novel inhibitor of ALK4/5/7 kinase, suppressed TGF-beta-induced growth stimulation. DNA microarray analysis as well as quantitative real-time PCR analysis of RNAs from TGF-beta-treated cells demonstrated that several growth factors, including platelet-derived growth factor AA, were induced in response to TGF-beta in MG63 cells. Gleevec (1 microM) as well as AG1296 (5 microM) inhibited TGF-beta-induced growth stimulation of MG63 cells, suggesting that platelet-derived growth factor AA was mainly responsible for the growth-stimulatory effect of TGF-beta. We also examined the mechanisms of perturbation of growth-suppressing signaling in MG63 cells. We found that expression of c-Myc, which is down-regulated by TGF-beta in many other cells, was up-regulated in MG63 cells, suggesting that up-regulation of c-Myc expression may be the mechanism canceling growth-suppressing signaling of TGF-beta in MG63 cells.

Activin Receptors, Type I↗

PubMed2003

Successful treatment of ulcerative colitis with leukocytapheresis using non-woven polyester filter.

Ulcerative colitis is a chronic inflammatory disease of the rectum and colon. Although the pathogenesis of ulcerative colitis is not fully elucidated, cell-mediated immunity plays an important role in disease pathogenesis. Leukocytapheresis is a newly emerging therapy to eliminate activated leukocyte from systemic circulation. We have studied the effects of leukocytapheresis on patients with ulcerative colitis who had failed to respond to conventional therapy. A total of 51 patients with ulcerative colitis were treated with apheresis using a non-woven polyester fiber filter (Finecell, Asahi Medical Co.,Tokyo, Japan) originally developed as a microcoagulation elimination filter for massive transfusion. Of the 51 patients, 33 (64.7%) achieved clinical remission manifested by clinical activity and colonoscopic findings without any adverse effects. This result suggested that leukocytapheresis using Finecell might serve as an alternative therapy for ulcerative colitis as other leukocytapheresis using centrifugation or column.

Adolescent↗

PubMed2002

Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent.

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81-93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053-1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345-355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357-368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-DeltaC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site.

A critical step in S6 kinase 1 (S6K1) activation is Thr(229) phosphorylation in the activation loop by the phosphoinositide-dependent protein kinase (PDK1). Thr(229) phosphorylation requires prior phosphorylation of the Ser/Thr-Pro sites in the autoinhibitory domain and Thr(389) in the linker domain, consistent with PDK1 more effectively catalyzing Thr(229) phosphorylation in a variant harboring acidic residues in these positions (S6K1-E389D(3)E). S6K1-E389D(3)E has high basal activity and exhibits partial resistance to rapamycin and wortmannin, and its activity can be further augmented by mitogens, effects presumably mediated by Thr(229) phosphorylation. However, PDK1-induced Thr(229) phosphorylation is reported to be constitutive rather than phosphatidylinositide 3,4,5-trisphosphate-dependent, suggesting that S6K1-E389D(3)E activity is mediated through a distinct site. Here we use phosphospecific antibodies to show that Thr(229) is fully phosphorylated in S6K1-E389D(3)E in the absence of mitogens and that regulation of S6K1-E389D(3)E activity by mitogens, rapamycin, or wortmannin parallels Ser(371) phosphorylation. Consistent with this observation, a dominant interfering allele of the mammalian target of rapamycin, mTOR, inhibits mitogen-induced Ser(371) phosphorylation and activation of S6K1-E389D(3)E, whereas wild type mTOR stimulates both responses. Moreover, in vitro mTOR directly phosphorylates Ser(371), and this event modulates Thr(389) phosphorylation by mTOR, compatible with earlier in vivo findings.

Androstadienes↗

PubMed2002

Activation of apoptosis signal-regulating kinase 1 by the stress-induced activating phosphorylation of pre-formed oligomer.

Apoptosis signal-regulating kinase 1 (ASK1) is a MAPKKK family member which activates c-Jun N-terminal kinase (JNK) and p38. In non-stressed cells, ASK1 exists as an inactive complex with the reduced form of thioredoxin. Oxidative stress such as hydrogen peroxide (H2O2) disrupts the ASK1-thioredoxin complex by oxidization of thioredoxin and thereby activates ASK1. The precise mechanism by which ASK1 is activated after its release from thioredoxin is unknown. Here we show that phosphorylation of Thr845 at the activation loop is essential for ASK1 to be activated by H2O2. ASK1 appears to form a silent homo-oligomer through its C-terminal coiled-coil region in non-stressed cells. Following H2O2 treatment, pre-existing ASK1 oligomer undergoes conformational change and creates a new interface within an oligomer, which ultimately leads to trans-autophosphorylation of Thr845. Thus, direct interaction via the coiled-coil region is required for self-scaffolding but not sufficient for activation of ASK1. Importantly, Thr845 of ASK1 can also be trans-phosphorylated by an unidentified Thr845 kinase in response to H2O2 treatment. We propose that this potential Thr845 kinase may be an ignition kinase that triggers Thr845 phosphorylation in oligomerized and activation-competent forms of ASK1.

Amino Acid Sequence↗

PubMed2002

Critical care by cytapheresis.

We report our experience of cytapheresis using a nonwoven polyester fiber filter to treat critical states of immune diseases. In 7 critical states of ulcerative colitis (UC), cytapheresis was effective in improving symptoms of UC. Administration of steroids was important in some cases. In 3 cases of renal transplantation, cytapheresis was also effective in controlling rejection. IgA nephropathy of transplanted cases was well controlled. Furthermore, an original disease such as focal segmental glomerulosclerosis (FCGS) in a transplant patient was well treated by extracorporeal immune modulation of the cytapheresis.

Adult↗