Yoshiya Tahashi

TGF-β and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-β (TGF-β) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-β treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-β or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-β signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-β allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-β-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-β signals. Moreover, a combined treatment with HGF and TGF-β led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-β-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-β transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Transforming Growth Factor-β and Platelet-Derived Growth Factor Signal via c-Jun N-Terminal Kinase-Dependent Smad2/3 Phosphorylation in Rat Hepatic Stellate Cells after Acute Liver Injury

After liver injury, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-β-triggered cascade are not completely understood. TGF-β signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl4 administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of α-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-β and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-β and PDGF signals. Moreover, treatment of HSCs with both TGF-β and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-β and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-β signaling from tumor suppression to oncogenesis in early chronic hepatitis B†

Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor β (TGF‐β) signaling involves both tumor suppression and oncogenesis. TGF‐β activates TGF‐β type I receptor (TβRI) and c‐Jun N‐terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3‐mediated signaling between tumor suppression and oncogenesis in HBx‐expressing hepatocytes indicated that TβRI‐dependent pSmad3C transmitted a tumor‐suppressive TGF‐β signal, while JNK‐dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L‐ and pSmad3C‐mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx‐activated JNK/pSmad3L/c‐Myc oncogenic pathway was enhanced, while the TβRI/pSmad3C/p21WAF1 tumor‐suppressive pathway was impaired as human and mouse HBx‐associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor‐suppressive pSmad3C developed HCC within 12 years. Conclusion: HBx shifts hepatocytic TGF‐β signaling from the tumor‐suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV‐infected liver specimens should prove clinically useful for predicting risk of HCC. (HEPATOLOGY 2009.)

Differential regulation of activin A for hepatocyte growth and fibronectin synthesis in rat liver injury

BACKGROUND/AIMS Both hepatocyte growth and production of extracellular matrix such as fibronectin are essential for liver regeneration. Although activin A is reported to inhibit DNA replication in rat hepatocytes, the role of activin A for liver regeneration after acute injury has not been fully assessed. This study investigated the mechanism by which hepatocyte growth is regulated by activin A during liver regeneration and the effects of activin A on extracellular matrix production. METHODS The mRNA for betaA subunit of activin A and activin receptors in hepatocytes and hepatic stellate cells after CCl4 administration were studied by Northern blotting. Binding of 125I-activin A was tested in these cells. Effects of activin A were examined by DNA, collagen and fibronectin synthesis. RESULTS betaA mRNA was expressed in quiescent hepatocytes, and this expression peaked 12 h after CCl4 administration. Activin receptor mRNAs and cross-linked ligand/receptor complexes were expressed in hepatocytes and hepatic stellate cells However, these levels decreased specifically in hepatocytes at 24 h and had normalized by 72 h. The down-regulation of activin receptor was also observed after partial hepatectomy. Antiproliferative response to activin A decreased in hepatocytes at 24 h. Activin A stimulated production of fibronectin by hepatic stellate cells, but the synthesis of collagen was only slightly elevated in hepatic stellate cells following activin stimulation. CONCLUSIONS The down-regulation of activin receptors in hepatocytes may be partly responsible for these cells becoming responsive to mitogenic stimuli. The increase of activin A at the early stage of liver injury has the potential to contribute to the regulation of fibronectin production in hepatic stellate cells.

Distortion of autocrine transforming growth factor beta signal accelerates malignant potential by enhancing cell growth as well as PAI-1 and VEGF production in human hepatocellular carcinoma cells.

Resistance to growth inhibitory effects of transforming growth factor (TGF)-β is a frequent consequence of malignant transformation. On the other hand, serum concentrations of TGF-β, plasminogen activator inhibitor type 1 (PAI-1), and vascular endothelial growth factor (VEGF) are elevated as tumor progresses. The molecular mechanism of autocrine TGF-β signaling and its effects on PAI-1 and VEGF production in human hepatocellular carcinoma (HCC) is unknown. TGF-β signaling involves TGF-β type I receptor-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. To investigate the involvement of autocrine TGF-β signal in cell growth, PAI-1 and VEGF production of HCC, we made stable transfectants of human HCC line (HuH-7 cells) to express a mutant Smad2(3S-A), in which serine residues of SSXS motif were changed to alanine. The transfectants demonstrated an impaired Smad2 signaling. Along with the resistance to growth inhibition by TGF-β, forced expression of Smad2(3S-A) induced endogenous TGF-β secretion. Moreover, this increased TGF-β enhanced ligand-dependent signaling through the activated Smad3 and Smad4 complex, and transcriptional activities of PAI-1 and VEGF genes. In conclusion, distortion of autocrine TGF-β signals in human HCC accelerates their malignant potential by enhancing cell growth as well as PAI-1 and VEGF production.

Involvement of Smad3 phosphoisoform-mediated signaling in the development of colonic cancer in IL-10-deficient mice

Chronic inflammation predisposes to cancer. Transforming growth factor (TGF)-beta, a multifunctional protein, suppresses the growth of normal colonic epithelial cells, whereas it stimulates the proliferation of cancer cells. Interleukin (IL)-10-deficient mice, which develop colitis and colorectal cancer, show an increased level of plasma TGF-beta. Although TGF-beta may be a key molecule in the development of colon cancer arising from chronic colitis in IL-10-deficient mice, the role of TGF-beta still remains unclear. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which converts the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). We studied C57BL/6-IL-10-deficient mice (n=18) at 4 to 32 weeks of age. We investigated histology, and pSmad2/3L, pSmad2/3C, and p53 by immunohistochemistry. pSmad3L staining was detected in the cancer cells in all 10 mice with colonic cancer and in the epithelial cells in 7 of 12 mice with colonic dysplasia, but not in the normal or colitic mice. pSmad3c was detected without any significant difference between stages. p53 was weakly stained in a few cancer cells in 5 out of 10 mice. Smad3L signaling plays an important role in the carcinogenesis of chronic colitis in IL-10-deficient mice.

DIAGNOSTIC YIELD OF DOUBLE-BALLOON ENTEROSCOPY WITH INTESTINAL JUICE ANALYSIS FOR INTESTINAL STRONGYLOIDIASIS

Strongyloidiasis is relatively common in tropical and subtropical areas. Most patients with Strongyloides stercoralis hyperinfection are immunocompromised, most commonly from corticosteroids or human T‐cell lymphoma virus type 1 (HTLV‐1) infection. We encountered a patient with HTLV‐1 infection accompanied by paralytic ileus, in whom strongyloidiasis in the duodenum and jejunum was disclosed by esophagogastroduodenoscopy (EGD) and double‐balloon enteroscopy (DBE). Until the age of 7 years, he lived on Amami‐Oshima Island, Japan, where both S. stercoralis and HTLV‐1 are endemic. EGD and peroral DBE disclosed white villi, edematous mucosa, and the disappearance of folds in the duodenum and jejunum. Biopsy specimens from the white villi in the duodenum and jejunum revealed S. stercoralis larvae. In both duodenal and jejunal juice, the rhabditiform larvae moved around. Because the larvae invade the lymph vessels, resulting in lymphangiectasia in edematous enteritis, the appearance of white villi may reflect villous atrophy/destruction and mucosal edema. Although our patient revealed no eosinophilia and negative stool specimens for parasites or ova, EGD and DBE with multiple biopsies and intestinal juice analysis are valuable diagnostic tools for strongyloidiasis.

Sclerosing Esophagitis with IgG4-positive Plasma Cell Infiltration: A Case Report

The patient was a 76-year-old woman who had noticed slight difficulty in swallowing in the 3 years prior to this presentation. Her dysphagia progressed while she was hospitalized following cervical cancer surgery. Esophagogastroduodenoscopy and an esophagram showed circumferential erosion and a stricture of the thoracic esophagus. Esophageal resection was performed; the resected specimens showed a stricture and wall thickening. Histologically, transmural hyperplasia, which consisted of inflammatory granulation tissue with the abundant infiltration of IgG4-positive plasma cells and lymphocytes, was observed. The patient was diagnosed with probable IgG4-related disease. IgG4-related esophageal disease presenting as esophageal lesions alone is a very rare condition.

Excessive length of a tube in the stomach: a risk factor for a tangled or knotted tube.

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The site of delayed bleeding suggests the cause of bleeding after endoscopic submucosal dissection

TO THE EDITOR: We read with interest the article by Jang et al. [1] on risk factors for bleeding associated with endoscopic submucosal dissection (ESD) of gastric neoplastic lesions. Bleeding occurred in 32 (22.2%) of the 144 patients: immediate in 29 and delayed in three. The malignant histology of lesions was the only significant risk factor associated with bleeding (p = 0.004). We would like to know the site of the delayed bleeding, the post-ESD ulcer center or edge, which might disclose the cause of delayed bleeding. Endoscopic therapy, endoscopic mucosal resection (EMR) and ESD, is acknowledged as a useful alternative technique to surgery for gastric mucosal lesions including early gastric cancers and adenomas because of its minimum invasiveness, cost effectiveness, and excellent quality of life in the patients [2–6]. Although EMR is not a reliable method for lesions larger than 20 mm in diameter because of poor rate en bloc resection, ESD has facilitated en bloc resection of large lesions, resulting in accurate histological evaluation and prevention of local recurrence [3–5,7]. Despite its advantage, ESD technique requires higher technical skills and a longer procedure time, and is associated with more complications, such as bleeding and perforation, than EMR [2,4,5,8]. Bleeding is the most common complication; the rate is reported to be 7–38% [4]. Most bleeding occurs during the procedure, although delayed bleeding occurs in up to 10% of the patients. Several investigators reported various risk factors associated with bleeding during and after EMR or ESD, such as young patient age, inexperienced endoscopist skills, cutting method, and characteristics of lesions; large, depressed type, proximal location, and malignant histology [2–6]. We previously evaluated risk factors for delayed bleeding associated with EMR [9]. In 503 gastric lesions, immediate bleeding occurred in 73 (14.5%) and delayed bleeding in 25 (5.0%). Immediate bleeding was observed in 14 (56.0%) of the 25 lesions with delayed bleeding, and in 59 (12.3%) of the 478 lesions without delayed bleeding. The only significant risk factor for delayed bleeding was the occurrence of immediate bleeding (p = 0.001). Although most immediate bleeding (66/73) was characterized as oozing from the post-EMR ulcer edge without visible vessel, most delayed bleeding (23/25) arose from exposed vessels in the post-EMR ulcer center. The delayed bleeding may be caused by insufficient coagulation during resection, but not by insufficient initial hemostasis. If the site of delayed bleeding was the post-ESD ulcer center of the study of Jang et al. [1], the same cause would be suspected for the occurrence of delayed bleeding in EMR and ESD.