Akio Saiura

Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts.

Signaling by RANKL is essential for terminal differentiation of monocytes/macrophages into osteoclasts. The TRAF6 and c-Fos signaling pathways both play important roles downstream of RANKL. We show here that RANKL selectively induces NFATc1 expression via these two pathways. RANKL also evokes Ca(2+) oscillations that lead to calcineurin-mediated activation of NFATc1, and therefore triggers a sustained NFATc1-dependent transcriptional program during osteoclast differentiation. We also show that NFATc1-deficient embryonic stem cells fail to differentiate into osteoclasts in response to RANKL stimulation, and that ectopic expression of NFATc1 causes precursor cells to undergo efficient differentiation without RANKL signaling. Thus, NFATc1 may represent a master switch for regulating terminal differentiation of osteoclasts, functioning downstream of RANKL.

Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate, proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs, there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis, bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably, purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs, and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization, homing, differentiation and proliferation of bone marrow-derived vascular progenitor cells.

Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma

We identified that suppressor of cytokine signaling-3 (SOCS-3) gene was aberrantly methylated in its CpG island in three of 10 human hepatocellular carcinoma (HCC) cell lines. SOCS-3 RNA was undetectable in five of the 10 HCC cell lines including the three methylated cell lines, and a demethylating agent, 5-aza-2′-deoxycytidine, reactivated SOCS-3 expression in three cell lines tested. The DNA region where we found aberrant DNA methylation includes a signal transducers and activators of transcription (STAT) binding consensus sequence. When the DNA region was used as a promoter, DNA methylation markedly reduced promoter activity. SOCS-3 was also aberrantly methylated in six of 18 primary HCC samples. SOCS-3 expression was reduced in three of the three methylated and one of the three unmethylated primary samples examined. Restoration of SOCS-3 in cells lacking SOCS-3 expression suppressed STAT3 phosphorylation and cell growth. We found that IL-6 acted as a growth factor in HCC cells. Inhibition of SOCS-3 expression in cells whose growth was induced by IL-6 enhanced STAT3 phosphorylation and cell growth. In addition, AG490, a chemical JAK2 inhibitor, suppressed cell growth and downregulated STAT3 phosphorylation, but not FAK phosphorylation. We also found that SOCS-3 physically interacted with phosphorylated FAK and Elongin B in HCC cells. Restoration of SOCS-3 decreased FAK phosphorylation as well as FAK protein level. Inhibition of SOCS-3 expression increased FAK phosphorylation, resulting in enhancement of cell migration. These data indicate that SOCS-3 negatively regulates cell growth and cell motility by inhibiting Janus kinase (JAK)/STAT and FAK signalings in HCC cells. Thus, loss of SOCS-3 by the associated DNA methylation confers cells advantage in growth and migration.

Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

A comparison of gene expression in murine cardiac allografts and isografts by means DNA microarray analysis.

BACKGROUND Acute rejection of allografts remains a significant problem in clinical transplantation, and the fundamental mechanism underlying this rejection are as yet only poorly elucidated. Recently, DNA microarrays have come into use for the study of gene expression profiles, and we have taken advantage of this new technology to investigate acute rejection. We compared mRNA profiles in murine cardiac allografts with isografts using DNA microarrays with probe sets corresponding to more than 11,000 mice genes. METHODS We screened for gene expression changes in murine cardiac allografts between fully incompatible mice strains (BALB/c H2d to C3H/He H2k) using a DNA microarray. The heart was heterotopically transplanted. Allografts (BALB/c to C3H/He) were removed on days 1, 3, and 5. As a control, isografts (C3H/He to C3H/He) harvested on days 1, 3, and 5 and native hearts of both strain mice (C3H/He and BALB/c) were obtained. RESULTS On day 5, interferon-gamma (IFN-gamma) and many IFN-gamma-inducible genes were profoundly induced in the allograft relative to isograft. Monokine induced by IFN-gamma was most profoundly induced followed by inducibly expressed GTPase and Lmp-2. IFN-gamma was also profoundly induced. The induction was detectable from day 3. In contrast, genes regulated by other cytokines exhibited only modest changes. CONCLUSION IFN-gamma-inducible genes are specifically up-regulated in murine cardiac allografts, suggesting that signaling mediated by IFN-gamma may play an important role in the late phase of acute rejection in vivo.

Requirement of the IFN-alpha/beta-induced CXCR3 chemokine signalling for CD8+ T cell activation.

Background: Activation of both CD4+ T and CD8+ T cells is triggered by the engagement of the T cell antigen receptor (TCR) with MHC/peptide complexes on antigen‐presenting cells. This process also requires other molecular interactions, which transmit co‐stimulatory signals to these T cells. To ensure an effective immune response, distinct T cell subsets may additionally employ unique mechanism(s) for efficient activation.

Pancreatoduodenectomy With Systematic Mesopancreas Dissection Using a Supracolic Anterior Artery-first Approach.

Objective: To describe the details of the surgical technique of pancreatoduodenectomy (PD) with systematic mesopancreas dissection (SMD-PD), using a supracolic anterior artery-first approach. Background: An artery-first approach in PD has been advocated in pancreatic cancer to judge resectability, clear the superior mesenteric artery margin from invasion, or reduce blood loss. However, the efficacy of an artery-first approach in mesopancreas dissection remains unclear. Methods: This study involved 162 consecutive patients who underwent PD with curative intent. The patients were divided into 82 SMD-PDs and 80 conventional PDs (CoPD) and then stratified further according to the dissection level, that is, level 1 was applied to 24 simple mesopancreas divisions for early inflow occlusion including 11 SMD-PDs, level 2 for 63 en bloc mesopancreas resections (26 SMD-PDs), and level 3 for 75 patients who underwent a hemicircumferential superior mesenteric artery plexus resection to keep the margin free from cancer invasion (45 SMD-PDs). The clinical and imaging results were collected to assess the feasibility and validity of SMD-PD with an artery-first approach. Results: Blood loss and operation duration were significantly less in the SMD-PD group than in the CoPD group among the total 162 patients. The imaging analysis showed that four fifths of pancreatic arterial branches came from the right dorsal aspect of the superior mesenteric artery and cancer abutment occurred exclusively from the same direction indicating the validity of an artery-first approach. Conclusions: SMD-PD using an SAA is feasible across PD cases, with acceptable short-term outcomes, and we propose this procedure as a promising option for PD.

Detection of an up-regulation of a group of chemokine genes in murine cardiac allograft in the absence of interferon-gamma by means of DNA microarray.

Backgound. Interferon (IFN)-&ggr; and the IFN-&ggr;-dependent pathway are prominent in vascularized allograft during acute rejection. However, IFN-&ggr; deficient (IFN-&ggr;−/−) mice can rapidly reject cardiac allografts. To bring the alternative pathway during allograft rejection into more precise focus, we investigated the gene expression profile in murine cardiac allografts in IFN-&ggr;−/− mice by means of DNA microarray. Material and Method. We screened for gene expression changes in murine cardiac allografts of BALB/c H-2d into both wild-type C57BL/6 H-2b (n=3) and IFN-&ggr;−/− C57BL/6 H-2b(IFN-&ggr;−/−, n=4) using Affymetrix oligonucleotide arrays to monitor more than 11,000 genes and expressed sequence tag (ESTs). The heart was heterotopically transplanted. Transplanted hearts were harvested on day 5. As a control, isografts (C57BL/6 to C57BL/6) were also harvested on day 5. Results. On day 5, 64 of the 84 genes induced in the allografts in wild-type mice were not up-regulated in IFN-&ggr;−/− mice. We identified a group of 54 genes that were up-regulated in allografts in IFN-&ggr;−/− mice. Several chemokine genes, including monocyte chemoattractant protein=1 and macrophage inflammatory protein, were induced in the allografts in both wild-type and IFN-&ggr;−/− mice. Interestingly, a group of genes, including C10-like chemokine and platelet factor 4, were specifically induced in the IFN-&ggr;−/− mice. Conclusion. DNA microarray analysis reveals a unique pattern of mRNA expression in allografts in IFN-&ggr;−/− mice as well as a group of genes induced in cardiac allografts in both wild-type and IFN-&ggr;−/− mice, including monocyte chemoattractant protein-1 and monocyte chemoattractant protein-1.

Antimonocyte Chemoattractant Protein-1 Gene Therapy Attenuates Graft Vasculopathy

Objective—Accelerated coronary arteriosclerosis remains a major problem in the long-term survival of cardiac transplant recipients. However, the pathogenesis of graft vasculopathy is poorly understood, and there is no effective therapy. Transplant arteriosclerosis is characterized by early mononuclear cell attachment on the transplanted vessel followed by development of concentric neointimal hyperplasia. Early and persistent expression of monocyte chemoattractant protein-1 (MCP-1) in cardiac allografts has been implicated for the pathogenesis of transplant arteriosclerosis. Methods and Results—We investigated whether anti-MCP-1 gene therapy can inhibit the development of intima hyperplasia in a mouse model of cardiac transplantation. Either the dominant-negative form of MCP-1 (7ND) or control vector was transfected into the skeletal muscles of B10.D2 mice. Cardiac allografts from DBA/2 mice were transplanted heterotopically into B10.D2 mice. 7ND gene transfer was associated with a significant reduction of the number of mononuclear cells accumulating in the lumen of the graft coronary arteries at 1 week and an attenuation of the development of the lesion at 8 weeks (intima/media ratio 0.79±0.05 versus 0.48±0.04). Conclusions—The MCP-1/chemokine receptor 2 (CCR2) signaling pathway plays a critical role in the pathogenesis of graft vasculopathy. This new anti-MCP-1 gene therapy might be useful to treat graft vascular disease.

Multicentre analysis of long-term outcome after surgical resection for gastric cancer liver metastases.

The efficacy of surgical resection for gastric cancer liver metastases (GCLMs) is currently debated. Hitherto, no large‐scale clinical studies have been conducted.