Naoteru Miyata

CCL2-induced migration and SOCS3-mediated activation of macrophages are involved in cerulein-induced pancreatitis in mice

BACKGROUND & AIMS Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein- and L-arginine-induced acute pancreatitis in mice. METHODS Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages. RESULTS Almost all types of immune cells, except for CD11b(high)CD11c(-) cells, were detected in the pancreas of healthy mice. However, activated CD11b(high)CD11c(-) cells, including Gr-1(low) macrophages and Gr-1(high) cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2(-/-) mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b(high)CD11c(-)Gr-1(low) macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b(high)CD11c(-)Gr-1(low) macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1(low) macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b(high)CD11c(-)Gr-1(low) macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2. CONCLUSIONS Cerulein induction of pancreatitis in mice involves migration of CD11b(high)CD11c(-)Gr-1(low) macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3-dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis.

Transforming growth factor β and Ras/MEK/ERK signaling regulate the expression level of a novel tumor suppressor lefty

Objectives The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-&bgr;) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-&bgr;–dependent Lefty up-regulation. Methods Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-&bgr;–mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal–regulated kinase (ERK) inhibitor LY294002. Results Transforming growth factor &bgr; upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-&bgr;–Lefty pathway. Conclusions Our data suggest that Lefty is a novel TGF-&bgr; target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-&bgr;–Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-&bgr;.

Suppression of Lefty expression in induced pluripotent cancer cells

Cancer and stem cells share the ability to silence tumor suppressors. We focused on Lefty, which encodes one of the most abundant tumor suppressors in embryonic stem (ES) cells and is not expressed in somatic cancer cells. We found that transforming growth factor β (TGF‐β) induced demethylation of the Lefty B cytosine‐phosphate‐guanine (CpG) island and increased Lefty expression (10–200 times) in human pancreatic cancer cells and human liver cancer cells (PLC/PRF/5 and HLF). Expression of Cripto, another important factor in Nodal‐Lefty signaling, was not increased after adding TGF‐β. We generated reprogrammed cancer cells that revealed high expression of immature marker proteins, high proliferation, and the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, suggesting that these cells may have cancer stem cell‐like phenotypes. We investigated Lefty and found that reprogrammed human liver cancer cells (induced pluripotent cancer cells) displayed a much lower ability to express Lefty, although less Lefty B CpG methylation was also observed. We also found that a MEK inhibitor dramatically enhanced Lefty expression in human pancreatic cancers with mutated ras, whereas Lefty B CpG methylation was not decreased. These observations indicate that despite the demethylation of DNA strands in promoter regions of pluripotency‐associated genes, including Lefty gene, Lefty expression was not induced well in reprogrammed cells. Of note was the fact that Lefty is abundantly expressed in human ES cells but not in induced pluripotent stem (iPS) cells. We thus think that reprogrammed cancer cells share the mechanism for expression of Lefty with iPS cells. This shared mechanism may contribute to the cancerous transformation of iPS cells.—Saito, A., Ochiai, H., Okada, S., Miyata, N., Azuma, T. Suppression of Lefty expression in induced pluripotent cancer cells. FASEB J. 27, 2165–2174 (2013). www.fasebj.org

CCR2 knockout exacerbates cerulein-induced chronic pancreatitis with hyperglycemia via decreased GLP-1 receptor expression and insulin secretion.

Glucagon-like peptide-1 (GLP-1) promotes insulin release; however, the relationship between the GLP-1 signal and chronic pancreatitis is not well understood. Here we focus on chemokine (C-C motif) ligand 2 (CCL2) and its receptor (CCR2) axis, which regulates various immune cells, including macrophages, to clarify the mechanism of GLP-1-mediated insulin secretion in chronic pancreatitis in mice. One and multiple series of repetitive cerulein administrations were used to induce acute and chronic cerulein pancreatitis, respectively. Acute cerulein-administered CCR2-knockout (KO) mice showed suppressed infiltration of CD11b(+)Gr-1(low) macrophages and pancreatic inflammation and significantly upregulated insulin secretion compared with paired wild-type (WT) mice. However, chronic cerulein-administered CCR2-KO mice showed significantly increased infiltration of CD11b(+)/Gr-1(-) and CD11b(+)/Gr-1(high) cells, but not CD11b(+)/Gr-1(low) cells, in pancreas with severe inflammation and significantly decreased insulin secretion compared with their WT counterparts. Furthermore, although serum GLP-1 levels in chronic cerulein-administered WT and CCR2-KO mice were comparably upregulated after cerulein administrations, GLP-1 receptor levels in pancreases of chronic cerulein-administered CCR2-KO mice were significantly lower than in paired WT mice. Nevertheless, a significantly higher hyperglycemia level in chronic cerulein-administered CCR2-KO mice was markedly restored by treatment with a GLP-1 analog to a level comparable to the paired WT mice. Collectively, the CCR2/CCL2 axis-mediated CD11b(+)-cell migration to the pancreas is critically involved in chronic pancreatitis-mediated hyperglycemia through the modulation of GLP-1 receptor expression and insulin secretion.

Induction of matrix metalloproteinase-1 gene transcription by tumour necrosis factor α via the p50/p50 homodimer of nuclear factor-κ B in activated human hepatic stellate cells

Background/Aims: Liver injury results in the activation of hepatic stellate cells (HSCs), which in turn produce matrix metalloproteinase (MMP) in response to pro‐inflammatory cytokines for tissue remodelling. This study explored the transcriptional induction of the MMP‐1 gene by tumour necrosis factor‐α (TNF‐α) in HSCs.

Ampullary Adenoma Treated by Endoscopic Double-Snare Retracting Papillectomy.

We report herein improved methods for the safe and successful completion of endoscopic papillectomy (EP). Between January 2008 and November 2011, 12 patients underwent double-snare retracting papillectomy for the treatment of lesions of the major duodenal papilla. The main outcomes were en bloc resection rates, pathological findings, and adverse events. All of the patients (mean age, 60.1 years; range, 38 to 80 years) were diagnosed with ampullary adenoma by endoscopic forceps biopsies prior to endoscopic snare papillectomy. En bloc resection by double-snare retracting papillectomy was successfully performed for all lesions (median size, 12.3 mm), comprising six tubular adenomas, one tubulovillous adenoma, three cases of epithelial atypia, one hamartomatous polyp, and one case of duodenitis with regenerative change. Significant hemorrhage and pancreatitis were observed in one case after EP. Adenoma recurrence occurred in three patients during follow-up (median, 28.5 months) at a mean interval of 2 months postoperatively (range, 1 to 3 months). No serious adverse events were observed. Double-snare retracting papillectomy is effective and feasible for treating lesions of the major duodenal papilla. Further treatment experience, including a single-arm phase II study, needs to be accumulated before conducting a randomized controlled study.

171 COMMD1 Restrains Tumor Promoting Activities of Myeloid Cells in Colon Cancer

G A A b st ra ct s primary resistance to imatinib. Amongst c-KIT/PDGFR wild type GIST, the most frequent driver mutation is BRAF-V600E, especially in small intestinal GIST. Thus, a mouse model of mutant Braf GIST might allow for development of more effective therapeutic strategies for these less common forms of GIST. Somatic activating mutations in c-Kit induce ICC hyperplasia and GIST in mouse models. However, whether GIST can arise from other cell types is unknown. In this study, we sought to generate a mouse model for Braf-mutant GIST from intestinal smooth muscle. Methods: Mice with a conditionally active (CA) mutant Braf allele, Braf-lox-stop-lox-V600E (Braf-CA), were crossed to mice carrying the tamoxifeninducible smooth muscle driver (Myh11-CreERT2). Adult mice received a single intraperitoneal injection of tamoxifen, ranging from 2.5-25mg/kg. The stomach, small intestine and colon were evaluated by H&E staining and immunohistochemistry. Results: Three weeks after tamoxifen injection, clusters of dysplastic cells with high nuclear-cytoplasmic ratio were observed in the intestinal muscular layer. The phenotype was most striking at the outer longitudinal layer of the greater curvature of stomach, but was also seen in the small intestinal and colonic muscle layer. The dysplastic cells were spindle-shaped and surrounded ganglion cells. CD34 immunoreactivity decorated, the dysplastic cells, a common finding in human GIST. Some of the clusters of dysplastic cells exhibited c-Kit-positivity and decreased a-SMA staining. c-Kit-positive cell clusters were also positive for anoctamin-1 (Ano1), an additional marker for human c-Kit-positive GIST. Conclusions: By conditionally activating mutant Braf in smooth muscle cells, we have developed a new mouse model for Braf-mutant GIST, as determined by characteristic histological features and markers. These results suggest that smooth muscle, rather than ICC, may be the cell-of-origin for Brafmutant GIST.

SOCS3 Promotes the Activation of CCR2-Dependent Migratory Cd11b+GR-1lowly6chigh Macrophages in Murine Cerulein Pancreatitis

mice followed by comparison of MFG-E8+/+ and MFG-E8-/mice for pathological changes in the pancreas. Typically, MFG-E8+/+ and MFG-E8-/mice (7 weeks, male) were subjected to ten injections of cerulein (50 μg/kg, i.p.) at hourly intervals. They were sacrificed at various time points after the final cerulein administration. We found that MFG-E8 deficiency did not affect the severity of acute phase of cerulein-induced pancreatitis. However, pancreatic recovery following cerulein-induced pancreatitis was markedly delayed in MFG-E8-/mice comparing to wild-type controls. Furthermore, we examined whether lack of MFG-E8 leads to progression of recurrent pancreatic injury to chronic inflammation. Briefly, MFG-E8-/and MFG-E8+/+ mice were respectively subjected to repeated courses of acute pancreatitis for two weeks. Control mice were treated with saline. Using histological examination, we revealed that wild-type mice recovered from repeated acute pancreatitis within 7 days after the final cerulein administration. In contrast, pancreas from MFG-E8-/displayed persistent inflammatory cell infiltration, disruption of acinar cells, and replacement of parenchymal tissues with collagen fibers and stromal cells. Together, the data suggest a central role for MFG-E8 in pancreatic recovery from pancreatitis. In conclusion, MFG-E8 deficiency impairs pancreatic recovery. This novel finding may provide a potential therapeutic target in the further development of novel molecular agents for patients with pancreatitis and pancreatic fibrosis.

S1998 Correlation of TGF-β Induced Tumor Suppressive Effect and Left-Right Determination Factor (Lefty) in Side Population Cell of Pancreatic Cancer Cells

biosystems) was performed. The siRNA targeting of Smad4 (Ambion:ID5365) was performed. SP cell were isolated from each pancreatic cancer cell line. TGF-β treatment significantly reduced SP cell frequencies and enhanced the effects of Gemcitabine. SP cell with TGF-β treatment survived better than non-SP cell. KP1N and KP1NL cell were derived from the same parental cell line and their responses to TGF-β treatment were opposite. It revealed growth inhibition in SP cell of KP1NL, whereas growth-promotion in KP1N cell. Using DNA-microarray analysis we identified the 50 genes differentially expressed gene between KP1N and KP1NL. Quantitative Real-time PCR revealed obvious change in. Leftright-axis determination gene Lefty. DNA array analysis and real-time PCR analysis revealed more than 50 times higher expressions in TGF-β treated group. We found that Smad4 downregulation by siRNA administration un-expectedly increased Lefty expressions. Lefty was induced by TGF-β in SP cell of Pancreatic cancer cell. This up-regulation of Lefty was not directly controlled by Smad4 cascade. Lefty could work as an effecter of TGF-β and as a tumor suppressor in pancreatic cancer.