Smad3 Phospho-Isoform Signaling in Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis with insulin resistance, oxidative stress, lipotoxicity, adipokine secretion by fat cells, endotoxins (lipopolysaccharides) released by gut microbiota, and endoplasmic reticulum stress. Together, these factors promote NAFLD progression from steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and eventually end-stage liver diseases in a proportion of cases. Hepatic fibrosis and carcinogenesis often progress together, sharing inflammatory pathways. However, NASH can lead to hepatocarcinogenesis with minimal inflammation or fibrosis. In such instances, insulin resistance, oxidative stress, and lipotoxicity can directly lead to liver carcinogenesis through genetic and epigenetic alterations. Transforming growth factor (TGF)-ß signaling is implicated in hepatic fibrogenesis and carcinogenesis. TGF-ß type I receptor (TßRI) and activated-Ras/c-Jun-N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3 to create two phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TßRI/pSmad3C signaling terminates cell proliferation, while constitutive Ras activation and JNK-mediated pSmad3L promote hepatocyte proliferation and carcinogenesis. The pSmad3L signaling pathway also antagonizes cytostatic pSmad3C signaling. This review addresses TGF-ß/Smad signaling in hepatic carcinogenesis complicating NASH. We also discuss Smad phospho-isoforms as biomarkers predicting HCC in NASH patients with or without cirrhosis.

Phospho-Smad3 signaling is predictive biomarker for hepatocellular carcinoma risk assessment in primary biliary cholangitis patients.

INTRODUCTION: Patients with primary biliary cholangitis (PBC) are at increased risk for development of hepatocellular carcinoma (HCC), particularly in the presence of comorbidities such as excessive alcohol consumption. Although liver fibrosis is an important risk factor for HCC development, earlier predictors of future HCC development in livers with little fibrosis are needed but not well defined. The transforming growth factor (TGF)-ß/Smad signaling pathway participates importantly in hepatic carcinogenesis. Phosphorylated forms (phospho-isoforms) in Smad-related pathways can transmit opposing signals: cytostatic C-terminally-phosphorylated Smad3 (pSmad3C) and carcinogenic linker-phosphorylated Smad3 (pSmad3L) signals. METHODS AND RESULTS: To assess the balance between Smad signals as a biomarker of risk, we immunohistochemically compared Smad domain-specific Smad3 phosphorylation patterns among 52 PBC patients with various stages of fibrosis and 25 non-PBC patients with chronic hepatitis C virus infection. HCC developed in 7 of 11 PBC patients showing high pSmad3L immunoreactivity, but in only 2 of 41 PBC patients with low pSmad3L. In contrast, 9 of 20 PBC patients with minimal Smad3C phosphorylation developed HCC, while HCC did not occur during follow-up in 32 patients who retained hepatic tumor-suppressive pSmad3C. Further, PBC patients whose liver specimens showed high pSmad3L positivity were relatively likely to develop HCC even when little fibrosis was evident. CONCLUSION: In this study, Smad phospho-isoform status showed promise as a biomarker predicting likelihood of HCC occurrence in PBC. Eventually, therapies to shift favorably Smad phospho-isoforms might decrease likelihood of PBC-related HCC.

Role of phosphorylated Smad3 signal components in intraductal papillary mucinous neoplasm of pancreas.

BACKGROUND: Malignant intraductal papillary mucinous neoplasm (IPMN) has poor prognosis. The carcinogenesis of IPMN is not clear. The aim of this study was to clarify transitions in phosphorylated Smad3 signaling during IPMN carcinogenesis. METHODS: By using immunohistochemistry, we examined the expression of pSmad3C and pSmad3L from 51 IPMN surgical specimens resected at our institution between 2010 and 2013. We also examined the expression of Ki-67, c-Myc and p-JNK. RESULTS: The median immunostaining index of pSmad3C was 79.2% in low-grade dysplasia, 74.9% in high-grade dysplasia, and 42.0% in invasive carcinoma (P < 0.01), whereas that of pSmad3L was 3.4%, 4.3%, and 42.4%, respectively (P < 0.01). There was a negative relationship between the expression of pSmad3C and c-Myc (P < 0.001, r = -0.615) and a positive relationship between the expression of pSmad3L and c-Myc (P < 0.001, r = 0.696). Negative relationship between the expression of pSmad3C and Ki-67 (P < 0.01, r = -0.610) and positive relationship between the expression of pSmad3L and Ki-67 (P < 0.01, r = 0.731) were confirmed. p-JNK-positive cells were frequently observed among pSmad3L-positive cancer cells. The median of pSmad3L/pSmad3C ratio in the non-recurrence group and the recurrence group were 0.58 (range, 0.05-0.93), 3.83 (range, 0.85-5.96), respectively (P = 0.02). The median immunostaining index of c-Myc in the non-recurrence group and the recurrence group were 2.91 (range, 0-36.9) and 82.1 (range, 46.2-97.1), respectively (P = 0.02). The median immunostaining index of Ki-67 in the non-recurrence group and the recurrence group were 12.9 (range 5.7-30.8) and 90.9 (range 52.9-98.5), respectively (P = 0.02). CONCLUSIONS: pSmad3L was upregulated in malignant IPMN. pSmad3L/pSmad3C ratio may be a useful prognostic factor in IPMN.

Clinico-Pathological Importance of TGF-ß/Phospho-Smad Signaling during Human Hepatic Fibrocarcinogenesis.

Chronic viral hepatitis is a global public health problem, with approximately 570 million persons chronically infected. Hepatitis B and C viruses increase the risk of morbidity and mortality from liver cirrhosis, hepatocellular carcinoma (HCC), and extrahepatic complications that develop. Hepatitis virus infection induces transforming growth factor (TGF)-ß, which influences microenvironments within the infected liver. TGF-ß promotes liver fibrosis by up-regulating extracellular matrix production by hepatic stellate cells. TGF-ß is also up-regulated in patients with HCC, in whom it contributes importantly to bringing about a favorable microenvironment for tumor growth. Thus, TGF-ß is thought to be a major factor regulating liver fibrosis and carcinogenesis. Since TGF-ß carries out regulatory signaling by influencing the phosphorylation of Smads, we have generated several kinds of phospho-specific antibodies to Smad2/3. Using these, we have identified three types of phospohorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C), linker phosphorylated Smad2/3 (pSmad2L and pSmad3L), and dually phosphorylated Smad3 (pSmad2L/C and pSmad3L/C). TGF-ß-mediated pSmad2/3C signaling terminates cell proliferation; on the other hand, cytokine-induced pSmad3L signaling accelerates cell proliferation and promotes fibrogenesis. This review addresses TGF-ß/Smad signal transduction in chronic liver injuries and carcinogenic processes. We also discuss the reversibility of Smad signaling after antiviral therapy.

Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis.

OBJECTIVES: The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. METHODS: Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. RESULTS: pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. CONCLUSIONS: pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.

Reversible Human TGF-ß Signal Shifting between Tumor Suppression and Fibro-Carcinogenesis: Implications of Smad Phospho-Isoforms for Hepatic Epithelial-Mesenchymal Transitions.

Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are observed during both physiological liver wound healing and the pathological fibrotic/carcinogenic (fibro-carcinogenetic) process. TGF-ß and pro-inflammatory cytokine are considered to be the major factors accelerating liver fibrosis and promoting liver carcinogenesis. Smads, consisting of intermediate linker regions connecting Mad homology domains, act as the intracellular mediators of the TGF-ß signal transduction pathway. As the TGF-ß receptors, c-Jun N-terminal kinase and cyclin-dependent kinase, differentially phosphorylate Smad2/3, we have generated numerous antibodies against linker (L) and C-terminal (C) phosphorylation sites in Smad2/3 and identified four types of phosphorylated forms: cytostatic COOH-terminally-phosphorylated Smad3 (pSmad3C), mitogenic pSmad3L (Ser-213) signaling, fibrogenic pSmad2L (Ser-245/250/255)/C signaling and migratory pSmad2/3L (Thr-220/179)/C signaling. After acute liver injury, TGF-ß upregulates pSmad3C signaling and terminates pSmad3L (Ser-213)-mediated hepatocyte proliferation. TGF-ß and pro-inflammatory cytokines cooperatively enhance collagen synthesis by upregulating pSmad2L (Thr-220)/C and pSmad3L (Thr-179)/C pathways in activated hepatic stellate cells. During chronic liver injuries, hepatocytes persistently affected by TGF-ß and pro-inflammatory cytokines eventually become pre-neoplastic hepatocytes. Both myofibroblasts and pre-neoplastic hepatocyte exhibit the same carcinogenic (mitogenic) pSmad3L (Ser-213) and fibrogenic pSmad2L (Ser-245/250/255)/C signaling, with acquisition of fibro-carcinogenic properties and increasing risk of hepatocellular carcinoma (HCC). Firstly, we review phospho-Smad-isoform signalings in epithelial and mesenchymal cells in physiological and pathological conditions and then consider Smad linker phosphorylation as a potential target for pathological EMT during human fibro-carcinogenesis, because human Smad phospho-isoform signals can reverse from fibro-carcinogenesis to tumor-suppression in a process of MET after therapy.

S-1 monotherapy in a patient with cholangiolocellular carcinoma: A case report.

A 71-year-old man with alcoholic cirrhosis was found to have multiple hypervascular lesions in the liver on enhanced computed tomography. An ultrasound-guided biopsy of the lesion was performed. Immunohistochemical analysis for hepatocyte paraffin 1 expression was negative; cytokeratin (CK) 7, CK19, epithelial cell adhesion molecule and epithelial membrane antigens were positive; mucicarmine staining was negative. The tumor was thus histologically diagnosed as cholangiolocellular carcinoma (CoCC). The tumor was inoperable due to the associated advanced liver disease. In addition, the patient preferred systemic chemotherapy using only orally administered agents. Thus, S-1 monotherapy was recommended. S-1 was initially administered orally at a dose of 80 mg/day. Although the levels of tumor marker (prothrombin induced by vitamin K absence/antagonist-II and carbohydrate antigen 19-9) levels were marginally elevated, their values did not change over the entire course. The patient achieved a partial response according to the Response Evaluation Criteria In Solid Tumors (RECIST) and modified RECIST 1 year after chemotherapy initiation. In conclusion, S-1 monotherapy exhibited promising efficacy against unresectable CoCC.

Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation.

Transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-ß receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

Interstitial pneumonia induced by sorafenib in a patient with hepatocellular carcinoma: An autopsy case report.

Sorafenib is a multikinase inhibitor currently approved in Japan for the treatment of unresectable hepatocellular carcinoma. Interstitial pneumonia induced by sorafenib may have a fatal outcome, and therefore, has recently been the focus of many studies. The current report presents an autopsy case of diffuse alveolar damage (DAD) that occurred in a 59-year-old male, who had been treated with sorafenib. The patient had been given sorafenib for six months and had exhibited no respiratory symptoms during this time. However, 19 days after sorafenib treatment was resumed, acute interstitial pneumonia developed. In previously reported cases, the first symptoms of pulmonary toxicity appeared following a limited treatment duration with sorafenib; this was in contrast to the patient in the current study, who developed the first symptoms after eight months. We therefore conclude that physicians must be aware of interstitial pneumonia as a potential pulmonary toxicity associated with sorafenib treatment when treatment with sorafenib is resumed, even after prolonged use. In addition, to best of our knowledge, this is the first case of a postmortem examination reported in patient with interstitial pneumonia induced by sorafenib treatment.

Severe acute hepatitis in a printing company worker: a case study.

OBJECTIVES: It has been reported that chlorinated organic solvent is a cause of hepatitis. METHODS: we investigate clinical and pathological findings of a patient with severe acute hepatitis who was exposed to chlorinated organic solvents. RESULTS: A 34-year-old man who was exposed to chlorinated organic solvents including dichloromethane, 1,2-dichloropropane, and trichloroethylene, presented with general fatigue, vomiting, and diarrhea. At admission, his laboratory test results showed extremely elevated aspartate aminotransferase (4,872 IU/l), alanine aminotransferase (3,000 IU/l), and lactate dehydrogenase (11,600 IU/l) levels and a prothrombin level below normal (41%). No encephalopathy was noted. These findings were indicative of severe acute hepatitis. Viral hepatitis, autoimmune hepatitis, alcoholic disease, bile duct disease, and viral infection were excluded as causes of hepatitis by clinical, laboratory, and imaging findings. After diagnosis, the patient was administered fresh frozen plasma and glucagon-insulin therapy. Liver function recovered within a few weeks, and a liver biopsy performed 25 days after admission showed the recovery phase after acute liver damage. CONCLUSIONS: These clinical and pathological findings indicate that exposure to chlorinated organic solvents may have induced severe acute hepatitis in this patient.

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

BACKGROUND: Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. AIM: Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. METHODS: We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. RESULTS: In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. CONCLUSION: In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

Linker phosphorylation of Smad3 promotes fibro-carcinogenesis in chronic viral hepatitis of hepatocellular carcinoma.

Epidemiological and clinical data point to a close association between chronic hepatitis B virus infection or chronic hepatitis C virus infection and development of hepatocellular carcinoma (HCC). HCC develops over several decades and is associated with fibrosis. This sequence suggests that persistent viral infection and chronic inflammation can synergistically induce liver fibrosis and hepatocarcinogenesis. The transforming growth factor-ß (TGF-ß) signaling pathway plays a pivotal role in diverse cellular processes and contributes to hepatic fibro-carcinogenesis under inflammatory microenvironments during chronic liver diseases. The biological activities of TGF-ß are initiated by the binding of the ligand to TGF-ß receptors, which phosphorylate Smad proteins. TGF-ß type I receptor activates Smad3 to create COOH-terminally phosphorylated Smad3 (pSmad3C), while pro-inflammatory cytokine-activated kinases phosphorylates Smad3 to create the linker phosphorylated Smad3 (pSmad3L). During chronic liver disease progression, virus components, together with pro-inflammatory cytokines and somatic mutations, convert the Smad3 signal from tumor-suppressive pSmad3C to fibro-carcinogenic pSmad3L pathways, accelerating liver fibrosis and increasing the risk of HCC. The understanding of Smad3 phosphorylation profiles may provide new opportunities for effective chemoprevention and personalized therapy for patients with hepatitis virus-related HCC in the future.

Smad3 phospho-isoform signaling in hepatitis C virus-related chronic liver diseases.

The risk of hepatocellular carcinoma (HCC) development increases as hepatitis virus C (HCV)-related liver diseases progress, especially in patients with active inflammation. Insight into hepatic carcinogenesis have emerged from recent detailed analyses of transforming growth factor-ß and c-Jun-N-terminal kinase signaling processes directed by multiple phosphorylated (phospho)-isoforms of a Smad3 mediator. In the course of HCV-related chronic liver diseases, chronic inflammation and host genetic/epigenetic alterations additively shift the hepatocytic Smad3 phospho-isoform signaling from tumor suppression to carcinogenesis, increasing the risk of HCC. Chronic inflammation represents an early carcinogenic step that provides a nonmutagenic tumor-promoting stimulus. After undergoing successful antiviral therapy, patients with chronic hepatitis C could experience a lower risk of HCC as Smad3 phospho-isoform signaling reverses from potential carcinogenesis to tumor suppression. Even after HCV clearance, however, patients with cirrhosis could still develop HCC because of sustained, intense carcinogenic Smad3 phospho-isoform signaling that is possibly caused by genetic or epigenetic alterations. Smad3 phospho-isoforms should assist with evaluating the effectiveness of interventions aimed at reducing human HCC.

TGF-ß/Smad signaling during hepatic fibro-carcinogenesis (review).

After hepatitis virus infection, plasma transforming growth factor (TGF)-ß increases in either the acute or chronic inflammatory microenvironment. Although TGF-ß is upregulated in patients with hepatocellular carcinoma, it is one of the most potent growth inhibitors for hepatocytes. This cytokine also upregulates extracellular matrix (ECM) production of hepatic stellate cells. Therefore, TGF-ß is considered to be the major factor regulating liver carcinogenesis and accelerating liver fibrosis. Smad2 and Smad3 act as the intracellular mediators of TGF-ß signal transduction pathway. We have generated numerous antibodies against individual phosphorylation sites in Smad2/3, and identified 3 types of phosphorylated forms (phospho-isoforms): COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C), linker phosphorylated Smad2/3 (pSmad2L and pSmad3L) and dually phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). These Smad phospho-isoforms are categorized into 3 groups: cytostatic pSmad3C signaling, mitogenic pSmad3L signaling and invasive/fibrogenic pSmad2L/C signaling. In this review, we describe differential regulation of TGF-ß/Smad signaling after acute or chronic liver injuries. In addition, we consider how chronic inflammation associated with hepatitis virus infection promotes hepatic fibrosis and carcinogenesis (fibro-carcinogenesis), focusing on alteration of Smad phospho-isoform signaling. Finally, we show reversibility of Smad phospho-isoform signaling after therapy against hepatitis virus infection.

TGF-ß signal shifting between tumor suppression and fibro-carcinogenesis in human chronic liver diseases.

Perturbation of transforming growth factor (TGF)-ß signaling in hepatocytes persistently infected with hepatitis viruses promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into hepatocytic fibro-carcinogenesis have emerged from recent detailed analyses of context-dependent and cell type-specific TGF-ß signaling processes directed by multiple phosphorylated forms (phospho-isoforms) of Smad mediators. In the course of hepatitis virus-related chronic liver diseases, chronic inflammation, ongoing viral infection, and host genetic/epigenetic alterations additively shift hepatocytic Smad phospho-isoform signaling from tumor suppression to fibro-carcinogenesis, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma (HCC). After successful antiviral therapy, patients with chronic hepatitis can experience less risk of HCC occurrence by reversing Smad phospho-isoform signaling from fibro-carcinogenesis to tumor suppression. However, patients with cirrhosis can still develop HCC owing to sustained, intense fibro-carcinogenic signaling. Recent progress in understanding Smad phospho-isoform signaling should permit use of Smad phosphorylation as a tool predicting the likelihood of liver disease progression, and as a biomarker for assessing the effectiveness of interventions aimed at reducing fibrosis and cancer risk.

Smad phospho-isoforms direct context-dependent TGF-ß signaling.

Better understanding of TGF-ß signaling has deepened our appreciation of normal epithelial cell homeostasis and its dysfunction in such human disorders as cancer and fibrosis. Smad proteins, which convey signals from TGF-ß receptors to the nucleus, possess intermediate linker regions connecting Mad homology domains. Membrane-bound, cytoplasmic, and nuclear protein kinases differentially phosphorylate Smad2 and Smad3 to create C-tail (C), the linker (L), or dually (L/C) phosphorylated (p, phospho-) isoforms. According to domain-specific phosphorylation, distinct transcriptional responses, and selective metabolism, Smad phospho-isoform pathways can be grouped into 4 types: cytostatic pSmad3C signaling, mitogenic pSmad3L (Ser-213) signaling, invasive/fibrogenic pSmad2L (Ser-245/250/255)/C or pSmad3L (Ser-204)/C signaling, and mitogenic/migratory pSmad2/3L (Thr-220/179)/C signaling. We outline how responses to TGF-ß change through the multiple Smad phospho-isoforms as normal epithelial cells mature from stem cells through progenitors to differentiated cells, and further reflect upon how constitutive Ras-activating mutants favor the Smad phospho-isoform pathway promoting tumor progression. Finally, clinical analyses of reversible Smad phospho-isoform signaling during human carcinogenesis could assess effectiveness of interventions aimed at reducing human cancer risk. Spatiotemporally separate, functionally different Smad phospho-isoforms have been identified in specific cells and tissues, answering long-standing questions about context-dependent TGF-ß signaling.

Phosphorylated Smad2 and Smad3 signaling: Shifting between tumor suppression and fibro-carcinogenesis in chronic hepatitis C.

AIM: Insight into hepatic fibrogenesis and carcinogenesis (fibro-carcinogenesis) caused by hepatitis C virus (HCV) infection has come from recent analyses of transforming growth factor (TGF)-ß signaling. TGF-ß type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create C-terminally (C), linker (L) or dually (L/C) phosphorylated (p) isoforms. This study aimed to elucidate how HCV infection affected hepatic fibro-carcinogenesis, particularly via phospho-Smad signaling. METHODS: We first studied phospho-Smad2/3 positivity of 100 patients in different stages of HCV-related chronic liver disease. To examine changes in phospho-Smad2/3 after HCV clearance, we analyzed 32 paired liver biopsy samples obtained before and after sustained virological response (SVR), dividing patients into two groups: 20 patients not developing hepatocellular carcinoma (HCC) after attaining SVR (non-HCC group), and 12 patients who developed HCC despite SVR (HCC group). RESULTS: Hepatocytic tumor-suppressive pSmad3C signaling shifted to carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling as liver diseases progressed. In the non-HCC group, 13 patients (65%) displayed fibrotic regression and inflammation reduction after SVR. Interestingly, SVR restored cytostatic pSmad3C signaling in hepatocytes, while eliminating prior carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling. In the HCC group, seven patients (58%) displayed unchanged or even progressed fibrosis despite smoothened inflammatory activity, reflecting persistently high numbers of hepatocytes with pSmad3L- and pSmad2L/C-signaling and low pSmad3C-signaling. CONCLUSION: HCV clearance limits fibrosis and reduces HCC incidence by switching inflammation-dependent phospho-Smad signaling from fibro-carcinogenesis to tumor suppression. However, progression to HCC would occur in severely fibrotic livers if an inflammation-independent fibro-carcinogenic process has already begun before HCV clearance.

TGF-ß signaling in onset and progression of hepatocellular carcinoma.

Transforming growth factor (TGF)-ß is a central regulator in chronic liver disease, contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver damage-induced levels of active TGF-ß enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Further evidence points to a decisive role of cytostatic and apoptotic functions mediated on hepatocytes, which is critical for the control of liver mass, with loss of TGF-ß activities resulting in hyperproliferative disorders and cancer. This concept is based on studies that describe a bipartite role of TGF-ß with tumor suppressor functions at early stages of liver damage and regeneration, whereas during cancer progression TGF-ß may turn from a tumor suppressor into a tumor promoter that exacerbates invasive and metastatic behavior. We have delineated this molecular switch of the pathway from cytostatic to tumor promoting in further detail and identify activation of survival signaling pathways in hepatocytes as a most critical requirement. Targeting the TGF-ß signaling pathway has been explored to inhibit liver disease progression. While interfering with TGF-ß signaling in various short-term animal models has demonstrated promising results, liver disease progression in humans is a process of decades with different phases in which TGF-ß or its targeting may have both beneficial and adverse outcomes. We emphasize that, in order to achieve therapeutic effects, targeting TGF-ß signaling in the right cell type at the right time is required.

Gastrointestinal bleeding and blood transfusion in the elderly in Japan.

BACKGROUND/AIMS: The incidence of gastrointestinal bleeding (GIB) increases with age and blood transfusion is frequently given for the management of GIB. In this report, we summarized our data of the patients with GIB and discussed the relationship between blood transfusion and age in patients with GIB. METHODOLOGY: The patients were divided into two groups according to age, following elderly (≥75 years old) and younger (<75 years old) group. The causes and clinical outcome (blood transfusions, management) of each group were compared. RESULTS: One-hundred and twenty patients with GIB were hospitalized (59 men, 61 women) with a mean age of 72.0±15.8 years (range 16-96 years old). Thirty-one patients (25.8%) received blood transfusion. The mean pre-transfusion hemoglobin was 6.4±1.2g/dL (elderly 6.3±1.4, younger 6.6±1.0g/dL) and the mean amount of blood transfusion was 2.8±1.6U (elderly 3.2±1.8, younger 2.3±0.9U). The elderly patients using antithrombotic drugs need greater amounts of blood transfusion than younger patients using antithrombotic drugs. The hemoglobin level of the elder patients without antithrombotic drugs was significantly lower than that of younger patients without antithrombotic drugs. CONCLUSIONS: Our data suggest that our blood transfusion strategy seems to be in tolerance level with restrictive blood transfusion strategy.

Gastrointestinal bleeding associated with antithrombotic therapy in the elderly in Japan.

BACKGROUND/AIMS: We summarize data of patients with gastrointestinal bleeding (GIB) and discuss the relationship between antithrombotic drug use and age in patients with GIB. METHODOLOGY: One-hundred and twenty patients with GIB were divided into two groups according to age (=75 years old and <75 years old). The causes and clinical outcome of each group were compared. RESULTS: Forty-two patients received antithrombotic therapy. The main antithrombotic drugs were low dose aspirin (38 patients), ticlopidine (5 patients) and warfarin (3 patients). Compared with younger GIB patients, elderly patients had more coexisting illness and antithrombotic drugs. In patients taking antithrombotic drugs, upper GIB is more frequent than those not taking antithrombotic drugs (p<0.05) and antithrombotic drugs were the risk for GIB from erosive lesions of the esophagus or stomach. In the lower gastrointestinal tract, there was no difference of incidence related to antithrombotic use. The initial endoscopic hemostasis was performed in 14 patients. Eight varices patients received endoscopic vanding and 6 of 43 gastroduodenal ulcer patients had mechanical clip hemostasis. CONCLUSIONS: From our findings, antithrombotic drugs were considered to be a risk for GIB. It might be important to prevent or minimize GIB in elderly patients prescribed antithrombotic drugs.