Koichi Matsuzaki

Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor β signaling, promoting cirrhosis and hepatocellular carcinoma†

Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF‐β) activates not only TGF‐β type I receptor (TβRI) but also c‐Jun N‐terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). Whereas the TβRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21WAF1 transcription, JNK/pSmad3L‐mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI‐1). We studied the domain‐specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV‐infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI‐1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21WAF1 decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro‐inflammatory cytokine interleukin‐1β stimulated the pSmad3L/PAI‐1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF‐β‐dependent tumor‐suppressive activity by the pSmad3C/p21WAF1 pathway. Conclusion: These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF‐β signaling from tumor‐suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC. (HEPATOLOGY 2007;46:48–57.)

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.)

Reversible Smad-Dependent Signaling between Tumor Suppression and Oncogenesis

Cancer cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor-beta (TGF-beta) together with stimulation of its oncogenic activity as in Ras-transformed cells; however, molecular mechanisms remain largely unknown. TGF-beta activates both its type I receptor (TbetaRI) and c-Jun NH2-terminal kinase (JNK), which phosphorylate Smad2 and Smad3 at the COOH-terminal (pSmad2/3C) and linker regions (pSmad2/3L). Here, we report that Ras transformation suppresses TbetaRI-mediated pSmad3C signaling, which involves growth inhibition by down-regulating c-Myc. Instead, hyperactive Ras constitutively stimulates JNK-mediated pSmad2/3L signaling, which fosters tumor invasion by up-regulating plasminogen activator inhibitor-1 and matrix metalloproteinase-1 (MMP-1), MMP-2, and MMP-9. Conversely, selective blockade of linker phosphorylation by a mutant Smad3 lacking JNK-dependent phosphorylation sites results in preserved tumor-suppressive function via pSmad3C in Ras-transformed cells while eliminating pSmad2/3L-mediated invasive capacity. Thus, specific inhibition of the JNK/pSmad2/3L pathway should suppress cancer progression by shifting Smad-dependent signaling from oncogenesis to tumor suppression.

Smad2 and Smad3 Phosphorylated at Both Linker and COOH-Terminal Regions Transmit Malignant TGF-β Signal in Later Stages of Human Colorectal Cancer

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.

Smad phosphoisoform signaling specificity: the right place at the right time

Transforming growth factor (TGF)-β antagonizes mitogenic Ras signaling during epithelial regeneration, but TGF-β and Ras act synergistically in driving tumor progression. Insights into these apparently contradictory effects have come from recent detailed analyses of the TGF-β signaling process. Here, we summarize the different modes of TGF-β/Ras signaling in normal epithelium and neoplasms and show how perturbation of TGF-β signaling by Ras may contribute to a shift from tumor-suppressive to protumorigenic TGF-β activity during tumor progression. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β Type I receptor and Ras-associated kinases differentially phosphorylate Smad2 and Smad3 to create C-terminally (C), linker (L) or dually (L/C) phosphorylated (p) isoforms. In epithelial homeostasis, TGF-β-mediated pSmad3C signaling opposes proliferative responses induced by mitogenic signals. During carcinogenesis, activation of cytoplasmic Ras-associated kinases including mitogen-activated protein kinase confers a selective advantage on benign tumors by shifting Smad3 signaling from a tumor-suppressive pSmad3C to an oncogenic pSmad3L pathway, leading to carcinoma in situ. Finally, at the edges of advanced carcinomas invading adjacent tissues, nuclear Ras-associated kinases such as cyclin-dependent kinases, together with cytoplasmic kinases, alter TGF-β signals to more invasive and proliferative pSmad2L/C and pSmad3L/C signaling. Taken together, TGF-β signaling specificity arises from spatiotemporal dynamics of Smad phosphoisoforms. Based on these findings, we have reason to hope that pharmacologic inhibition of linker phosphorylation might suppress progression to human advanced carcinomas by switching from protumorigenic to tumor-suppressive TGF-β signaling.

Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor- suppression in rat hepatocellular carcinoma†

Transforming growth factor beta (TGF‐β) signaling involves both tumor‐suppression and oncogenesis. TGF‐β activates the TGF‐β type I receptor (TβRI) and c‐Jun N‐terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become COOH‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). TβRI‐dependent pSmad3C transmits a tumor‐suppressive TGF‐β signal, while JNK‐dependent pSmad3L promotes carcinogenesis in human chronic liver disorders. The aim of this study is to elucidate how SP600125, a JNK inhibitor, affected rat hepatocellular carcinoma (HCC) development, while focusing on the domain‐specific phosphorylation of Smad3. The rats received subcutaneous injections of either SP600125 or vehicle 11 times weekly together with 100 ppm N‐diethylnitrosamine (DEN) administration for 56 days and were sacrificed in order to evaluate HCC development 28 days after the last DEN administration. The number of tumor nodules greater than 3 mm in diameter and the liver weight/body weight ratio were significantly lower in the SP600125‐treated rats than those in the vehicle‐treated rats (7.9 ± 0.8 versus 17.7 ± 0.9: P < 0.001; 6.3 ± 1.2 versus 7.1 ± 0.2%: P < 0.05). SP600125 significantly prolonged the median survival time in rats with DEN‐induced HCC (113 versus 97 days: log‐rank P = 0.0018). JNK/pSmad3L/c‐Myc was enhanced in the rat hepatocytes exposed to DEN. However, TβRI/pSmad3C/p21WAF1 was impaired as DEN‐induced HCC developed and progressed. The specific inhibition of JNK activity by SP600125 suppressed pSmad3L/c‐Myc in the damaged hepatocytes and enhanced pSmad3C/p21WAF1, acting as a tumor suppressor in normal hepatocytes. Conclusion: Administration of SP600125 to DEN‐treated rats shifted hepatocytic Smad3‐mediated signal from oncogenesis to tumor suppression, thus suggesting that JNK could be a therapeutic target of human HCC development and progression. (HEPATOLOGY 2009.)

Differential Regulation of TGF-β/Smad Signaling in Hepatic Stellate Cells between Acute and Chronic Liver Injuries

Current evidence suggests that regulation of extracellular matrix (ECM) accumulation by fibrogenic transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) signals involves different mechanisms in acute and chronic liver injuries, even though hepatic stellate cells (HSC) are the principal effecter in both cases. As a result of chronic liver damage, HSC undergo progressive activation to become myofibroblasts (MFB)-like cells. Our current review will discuss the differential regulation of TGF-β signaling between HSC and MFB in vitro and in vivo. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad-homology (MH) 1 and MH2 domains. TGF-β type I receptor and Ras-associated kinases differentially phosphorylate Smad2 and Smad3 to create COOH-terminally (C), linker (L), or dually (L/C) phosphorylated (p) isoforms. After acute liver injury, TGF-β and PDGF synergistically promote collagen synthesis in the activated HSC via pSmad2L/C and pSmad3L/C pathways. To avoid unlimited ECM deposition, Smad7 induced by TGF-β negatively regulates the fibrogenic TGF-β signaling. In contrast, TGF-β and PDGF can transmit the fibrogenic pSmad2L/C and mitogenic pSmad3L signals in MFB throughout chronic liver injury, because Smad7 cannot be induced by the pSmad3L pathway. This lack of Smad7 induction might lead to constitutive fibrogenesis in MFB, which eventually develop into accelerated liver fibrosis.

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.