PDCD10 promotes the tumor-supporting functions of TGF-ß in pancreatic cancer.

The progression of pancreatic ductal adenocarcinoma (PDAC) is significantly affected by transforming growth factor (TGF)-ß but targeting TGF-ß can also compromize physiological effects in patients. Our study examined the functions of the ubiquitously expressed protein, PDCD10, as a modulator of TGF-ß signaling in PDAC. Using in silico analyses we found that in patient samples, PDCD10 is significantly higher expressed in PDAC tumor tissue compared with normal pancreas and it is highly correlated with reduced survival. We created stable KO's of PDCD10 in two PDAC lines, PaTu 8902 (SMAD4 +/+) and PaTu 8988t (SMAD4 -/-), and found that KO lines are more sensitive to 5-FU and Gemcitabine treatment than their wild-type counterparts. Performing viability and wound closure assays we further found that PDCD10 promotes cell survival and proliferation by enhancing specifically the mitogenic functions of TGF-ß. The molecular mechanism underlying this effect was further investigated using Western blots and with primary organoid lines derived from patient PDAC tissue samples. The data imply that PDCD10 mediates an increase in p-ERK through a non-SMAD4 pathway, leading to EMT promotion. Furthermore, PDCD10 facilitates deactivation of RB via a SMAD4-dependent pathway, thereby counter-acting the anti-proliferative actions of TGF-ß. By performing proximity ligation assays (PLA) we found that PDCD10 associates with the kinase MST4, translocates it intracellularly and thereby facilitates phosphorylations of RB and ERK1/2. Our study indicates that PDCD10 promotes the proliferative function and EMT induction of TGF-ß in pancreatic cancer cells. Therefore, targeting PDCD10 in PDAC patients could represent a promising new strategy to optimize TGF-ß targeted therapies.

The drug-induced phenotypic landscape of colorectal cancer organoids.

Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression of LGR5, while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them.

Identification of liver-derived bone morphogenetic protein (BMP)-9 as a potential new candidate for treatment of colorectal cancer.

Colorectal cancer (CRC) is a high-incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)-9 and to find out whether the application of BMP-9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP-target gene ID1, especially in combination with low expression of the BMP-inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T-Orgs) and corresponding non-malignant areas (N-Orgs) of three patients. The N-Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP-9 stimulation of organoids promoted an enrichment of tumour-suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP-9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1. In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP-9 enhances this ratio, even in the presence of noggin. Thus, BMP-9 is identified as a novel target for the development of improved anti-cancer therapies of patients with CRC.

BMP9 promotes methionine- and choline-deficient diet-induced nonalcoholic steatohepatitis in non-obese mice by enhancing NF-κB dependent macrophage polarization.

Our previous study confirmed that bone morphogenetic protein 9 (BMP9) participated in the development of nonalcoholic steatohepatitis (NASH) by affecting macrophage polarization. The focus of this study was to further confirm the role of macrophages in BMP9-mediated NASH and to analyze the underlying mechanism. In vivo, mice that were administered adeno-associated viral (AAV) vectors containing a null transgene (AAV-null) or the BMP9 transgene (AAV-BMP9) were divided into methionine- and choline-deficient (MCD) and control diet (CD) groups, and they were administered either control liposomes or clodronate liposomes via tail vein injection, the latter to deplete macrophages. The mice were sacrificed after 4 weeks of MCD diet feeding. In vitro, RAW264.7 cells were pretreated with or without BAY11-7085 (an NF-κB inhibitor) and stimulated with recombinant human BMP9 (rh-BMP9). To explore the underlying mechanism of action of BMP9, primary human monocyte-derived macrophages were additionally investigated and immunohistochemistry, biochemical assays, qRT-PCR, and Western blotting were used. The characteristics of NASH-related inflammation were assessed by hepatic histological analysis. Serum AST and ALT and hepatic triglyceride were examined by biochemical assays. We found that the expression of M1 macrophage genes (including CD86, IL1ß, IL6, MCP-1 and TNFα) and the number of M1 macrophages (iNOS+ macrophages) in the liver were significantly elevated after BMP9 overexpression and BMP9 directly upregulated TLR4 expression in MCD-induced NASH. These effects were eliminated by macrophage depletion. In vitro, we discovered that BMP9 enhanced the nuclear translocation of NF-κB to induce macrophage M1 polarization in RAW264.7 cells and it promoted LPS-mediated activation of the NF-κB pathway in primary human macrophages. Taken together, this study demonstrates that BMP9 promotes NASH development by directly acting on macrophages.

Adenovirus­mediated overexpression of bone morphogenetic protein­9 promotes methionine choline deficiency­induced non­alcoholic steatohepatitis in non­obese mice.

Liver inflammation and macrophage infiltration are critical steps in the progression of non­alcoholic fatty liver to the development of non­alcoholic steatohepatitis. Bone morphogenetic protein­9 is a cytokine involved in the regulation of chemokines and lipogenesis. However, the function of bone morphogenetic protein­9 in non­alcoholic steatohepatitis is still unknown. The present study hypothesized that bone morphogenetic protein­9 may contribute to steatohepatitis in mice fed a methionine choline deficiency diet (MCD). C57BL/6 mice overexpressing bone morphogenetic protein­9 and control mice were fed the MCD diet for 4 weeks. Liver tissue and serum samples were obtained for subsequent measurements. Bone morphogenetic protein­9 overexpression exacerbated steatohepatitis in mice on the MCD diet, as indicated by liver histopathology, increased serum alanine aminotransferase activity, aspartate transaminase activity, hepatic inflammatory gene expression and M1 macrophage recruitment. Although bone morphogenetic protein­9 overexpression did not affect the expression of pro­fibrogenic genes, including Collagen I (α)1 or matrix metalloproteinase (MMP) 9, it did upregulate the expression of transforming growth factor­ß and plasminogen activator inhibitor 1, and downregulated the expression of MMP2. The above results indicate that bone morphogenetic protein­9 exerts a pro­inflammatory role in MCD diet­induced non­alcoholic steatohepatitis.

Downregulation of SPARC Is Associated with Epithelial-Mesenchymal Transition and Low Differentiation State of Biliary Tract Cancer Cells.

BACKGROUND: Biliary tract cancers (BTCs) have a poor prognosis. BTCs are characterized by a prominent desmoplastic reaction which possibly contributes to the aggressive phenotype of this tumor. The desmoplastic reaction includes excessive production and deposition of extracellular matrix proteins such as periostin, secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1, as well as accumulation of α-smooth muscle actin-positive cancer-associated fibroblasts and immune cells, secreting growth factors and cytokines including transforming growth factor (TGF)-ß. In the present study, we investigated the expression of SPARC in BTC as well as its possible regulation by TGF-ß. METHODS: Expression levels of Sparc, TGF-ß1 and its receptor ALK5 were evaluated by quantitative real-time PCR in 6 biliary tract cell lines as well as 1 immortalized cholangiocyte cell line (MMNK-1). RNAs from tumor samples of 7 biliary tract cancer patients were analyzed for expression of Sparc, TGF-ß type II receptor (TbRII) as well as Twist and ZO-1. MMNK-1 cells were stimulated with TGF-ß for 24 h, and Sparc, ZO-1 and E-Cadherin expressions were determined. The presence of SPARC protein was analyzed by immunohistochemistry in tumor specimens from 10 patients. RESULTS: When comparing basal Sparc transcript levels in diverse BTC cell lines to MMNK-1 cells, we found that it was strongly downregulated in all cancer cell lines. The remaining expression levels were higher in highly differentiated cell lines (CCSW1, MZChA1, MZChA2 and TFK-1) than in less differentiated and undifferentiated ones (BDC, SKChA1). Expression of Sparc in BTC patient samples showed a significant positive correlation with expression of the epithelial marker ZO-1. In contrast, the mesenchymal marker Twist and the TbRII showed a trend of negative correlation with expression of Sparc in these samples. TGF-ß exposure significantly downregulated Sparc expression in MMNK-1 cholangiocytes in vitro in parallel to downregulation of epithelial markers (E-Cadherin and ZO-1). Finally, SPARC immunostaining was performed in 10 patient samples, and the correlation between absence of SPARC and survival times was analyzed. CONCLUSIONS: These data imply that a decrease in SPARC expression is correlated with dedifferentiation of BTC cells resulting in enhanced EMT being possibly mediated by TGF-ß. Thereby SPARC levels might be a marker for individual prognosis of a patient, and strategies aiming at inhibition of SPARC downregulation might have potential for new future therapies.

Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation.

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug-drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.

Epigenetic silencing of tumor suppressor candidate 3 confers adverse prognosis in early colorectal cancer.

Colorectal cancer (CRC) is a biologically and clinically heterogeneous disease. Even though many recurrent genomic alterations have been identified that may characterize distinct subgroups, their biological impact and clinical significance as prognostic indicators remain to be defined. The tumor suppressor candidate-3 (TUSC3/N33) locates to a genomic region frequently deleted or silenced in cancers. TUSC3 is a subunit of the oligosaccharyltransferase (OST) complex at the endoplasmic reticulum (ER) which catalyzes bulk N-glycosylation of membrane and secretory proteins. However, the consequences of TUSC3 loss are largely unknown. Thus, the aim of the study was to characterize the functional and clinical relevance of TUSC3 expression in CRC patients' tissues (n=306 cases) and cell lines. TUSC3 mRNA expression was silenced by promoter methylation in 85 % of benign adenomas (n=46 cases) and 35 % of CRCs (n =74 cases). Epidermal growth factor receptor (EGFR) was selected as one exemplary ER-derived target protein of TUSC3-mediated posttranslational modification. We found that TUSC3 inhibited EGFR-signaling and promoted apoptosis in human CRC cells, whereas TUSC3 siRNA knock-down increased EGFR-signaling. Accordingly, in stage I/II node negative CRC patients (n=156 cases) loss of TUSC3 protein expression was associated with poor overall survival. In sum, our data suggested that epigenetic silencing of TUSC3 may be useful as a molecular marker for progression of early CRC.

BMP-9 interferes with liver regeneration and promotes liver fibrosis.

OBJECTIVE: Bone morphogenetic protein (BMP)-9, a member of the transforming growth factor-ß family of cytokines, is constitutively produced in the liver. Systemic levels act on many organs and tissues including bone and endothelium, but little is known about its hepatic functions in health and disease. DESIGN: Levels of BMP-9 and its receptors were analysed in primary liver cells. Direct effects of BMP-9 on hepatic stellate cells (HSCs) and hepatocytes were studied in vitro, and the role of BMP-9 was examined in acute and chronic liver injury models in mice. RESULTS: Quiescent and activated HSCs were identified as major BMP-9 producing liver cell type. BMP-9 stimulation of cultured hepatocytes inhibited proliferation, epithelial to mesenchymal transition and preserved expression of important metabolic enzymes such as cytochrome P450. Acute liver injury caused by partial hepatectomy or single injections of carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) into mice resulted in transient downregulation of hepatic BMP-9 mRNA expression. Correspondingly, LPS stimulation led to downregulation of BMP-9 expression in cultured HSCs. Application of BMP-9 after partial hepatectomy significantly enhanced liver damage and disturbed the proliferative response. Chronic liver damage in BMP-9-deficient mice or in mice adenovirally overexpressing the selective BMP-9 antagonist activin-like kinase 1-Fc resulted in reduced deposition of collagen and subsequent fibrosis. CONCLUSIONS: Constitutive expression of low levels of BMP-9 stabilises hepatocyte function in the healthy liver. Upon HSC activation, endogenous BMP-9 levels increase in vitro and in vivo and high levels of BMP-9 cause enhanced damage upon acute or chronic injury.

GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis.

Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis.

BMP9 a possible alternative drug for the recently withdrawn BMP7? New perspectives for (re-)implementation by personalized medicine.

Promotion of rhBMP2 and rhBMP7 for the routine use to support fracture healing has been hampered by high costs, safety concerns and reasonable failure rates, imposing restrictions in its clinical use. Since there is little debate regarding its treatment potential, there is rising need for a better understanding of the mode of action of these BMPs to overcome its drawbacks and promote more efficacious treatment strategies for bone regeneration. Recently, BMP9, owing to its improved osteogenic potential, is gaining attention as a promising therapeutic alternative. Our study aimed at identifying specific gene expression patterns which may predict and explain individual responses to rhBMP7 and rhBMP9 treatments. Therefore, we investigated the effect of rhBMP7 and rhBMP9 on primary human osteoblasts from 110 donors and corresponding THP-1-derived osteoclasts. This was further compared with each other and our reported data on rhBMP2 response. Based on the individual donor response, we found three donor groups profiting from rhBMP treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclasts. The response on rhBMP7 treatment correlated with expression levels of the genes BAMBI, SOST, Noggin, Smad4 and RANKL, while the response of rhBMP9 correlated to the expression levels of Alk6, Endoglin, Smurf1, Smurf2, SOST and RANKL in these donors. Noteworthy, rhBMP9 treatment showed significantly increased osteogenic activity (AP activity and Smad nuclear translocation) when compared to the two clinically used rhBMPs. Based on patient's respective expression profiles, clinical application of rhBMP9 either solely or in combination with rhBMP2 and/or rhBMP7 can become a promising new approach to fit the patient's needs to promote fracture healing.

Angiocrine Bmp2 signaling in murine liver controls normal iron homeostasis.

Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

TGF-ß1 and TGF-ß2 abundance in liver diseases of mice and men.

TGF-ß1 is a major player in chronic liver diseases promoting fibrogenesis and tumorigenesis through various mechanisms. The expression and function of TGF-ß2 have not been investigated thoroughly in liver disease to date. In this paper, we provide evidence that TGF-ß2 expression correlates with fibrogenesis and liver cancer development.Using quantitative realtime PCR and ELISA, we show that TGF-ß2 mRNA expression and secretion increased in murine HSCs and hepatocytes over time in culture and were found in the human-derived HSC cell line LX-2. TGF-ß2 stimulation of the LX-2 cells led to upregulation of the TGF-ß receptors 1, 2, and 3, whereas TGF-ß1 treatment did not alter or decrease their expression. In liver regeneration and fibrosis upon CCl4 challenge, the transient increase of TGF-ß2 expression was accompanied by TGF-ß1 and collagen expression. In bile duct ligation-induced fibrosis, TGF-ß2 upregulation correlated with fibrotic markers and was more prominent than TGF-ß1 expression. Accordingly, MDR2-KO mice showed significant TGF-ß2 upregulation within 3 to 15 months but minor TGF-ß1 expression changes. In 5 of 8 hepatocellular carcinoma (HCC)/hepatoblastoma cell lines, relatively high TGF-ß2 expression and secretion were observed, with some cell lines even secreting more TGF-ß2 than TGF-ß1. TGF-ß2 was also upregulated in tumors of TGFα/cMyc and DEN-treated mice. The analysis of publically available microarray data of 13 human HCC collectives revealed considerable upregulation of TGF-ß2 as compared to normal liver.Our study demonstrates upregulation of TGF-ß2 in liver disease and suggests TGF-ß2 as a promising therapeutic target for tackling fibrosis and HCC.

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.

Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings.

Potential roles of bone morphogenetic protein (BMP)-9 in human liver diseases.

Bone morphogenetic proteins (BMP-2 to BMP-15) belong to the Transforming Growth Factor (TGF)-ß superfamily and, besides their well-documented roles during embryogenesis and bone formation, some of them have recently been described to be involved in the pathogenesis of different organs, including the liver. The role of BMPs in liver damage responses including hepatocellular carcinoma (HCC) development has only begun to be addressed and strong evidence supports the concept of a pro-tumorigenic role of BMP signaling in HCC cells. BMP-9 (also termed Growth and Differentiation Factor (GDF)-2) represents the most recently discovered member of the BMP family. We have previously demonstrated that in HCC patient samples BMP-9 expression was positively associated with the tumor seize ("T stage") and that it enhanced cell migration and induced epithelial to mesenchymal transition (EMT) in HCC cells in vitro. In another study we recently found that BMP-9 promotes growth in HCC cells, but not in non-transformed hepatocytes. Published as well as unpublished results obtained with primary hepatocytes support the concept of a dual function of BMP-9 in the liver: while in primary, non-malignant cells BMP-9 stabilizes the epithelial phenotype and inhibits proliferation, in HCC cells it induces cell growth and the acquisition of a migratory phenotype. In this review article we summarize current knowledge about BMPs in liver diseases, with special focus on the role of BMP-9 in HCC development and progression, that may provide new clues for a better understanding of the contribution of BMP-signaling to chronic liver diseases.

Rolipram attenuates bile duct ligation-induced liver injury in rats: a potential pathogenic role of PDE4.

Anti-inflammatory and antifibrotic effects of the broad spectrum phosphodiesterase (PDE) inhibitor pentoxifylline have suggested an important role for cyclic nucleotides in the pathogenesis of hepatic fibrosis; however, studies examining the role of specific PDEs are lacking. Endotoxemia and Toll-like receptor 4 (TLR4)-mediated inflammatory and profibrotic signaling play a major role in the development of hepatic fibrosis. Because cAMP-specific PDE4 critically regulates lipopolysaccharide (LPS)-TLR4-induced inflammatory cytokine expression, its pathogenic role in bile duct ligation-induced hepatic injury and fibrogenesis in Sprague-Dawley rats was examined. Initiation of cholestatic liver injury and fibrosis was accompanied by a significant induction of PDE4A, B, and D expression and activity. Treatment with the PDE4-specific inhibitor rolipram significantly decreased liver PDE4 activity, hepatic inflammatory and profibrotic cytokine expression, injury, and fibrosis. At the cellular level, in relevance to endotoxemia and inflammatory cytokine production, PDE4B was observed to play a major regulatory role in the LPS-inducible tumor necrosis factor (TNF) production by isolated Kupffer cells. Moreover, PDE4 expression was also involved in the in vitro activation and transdifferentiation of isolated hepatic stellate cells (HSCs). Particularly, PDE4A, B, and D upregulation preceded induction of the HSC activation marker α-smooth muscle actin (α-SMA). In vitro treatment of HSCs with rolipram effectively attenuated α-SMA, collagen expression, and accompanying morphologic changes. Overall, these data strongly suggest that upregulation of PDE4 expression during cholestatic liver injury plays a potential pathogenic role in the development of inflammation, injury, and fibrosis.

Bone morphogenetic protein-9 induces epithelial to mesenchymal transition in hepatocellular carcinoma cells.

Epithelial-mesenchymal transition (EMT) is an important mechanism to initiate cancer invasion and metastasis. Bone morphogenetic protein (BMP)-9 is a member of the transforming growth factor (TGF)-ß superfamily. It has been suggested to play a role in cancer development in some non-hepatic tumors. In the present study, two hepatocellular carcinoma (HCC) lines, HLE and HepG2, were treated with BMP-9 in vitro, and phenotypic changes and cell motility were analyzed. In situ hybridization (ISH) and immunohistochemical analyses were performed with human HCC tissue samples in order to assess expression levels of BMP-9. In vivo, BMP-9 protein and mRNA were expressed in all the tested patients to diverse degrees. At the protein level, mildly positive (1 + ) BMP-9 staining could be observed in 25/41 (61%), and moderately to strongly positive (2 + ) in 16/41 (39%) of the patients. In 27/41 (65%) patients, the BMP-9 protein expression level was consistent with the mRNA expression level as measured by ISH. In those patients with 2 + protein level, nuclear pSmad1 expression in cancer cells was also significantly increased. Expression of BMP-9 was positively related to nuclear Snail expression and reversely correlated to cell surface E-cadherin expression, although this did not reach statistical significance. Expression levels of BMP-9 were significantly associated with the T stages of the investigated tumors and high levels of BMP-9 were detected by immunofluorescence especially at the tumor borders in samples from an HCC mouse model. In vitro, BMP-9 treatment caused a reduction of E-cadherin and ZO-1 and an induction of Vimentin and Snail expression. Furthermore, cell migration was enhanced by BMP-9 in both HCC cell lines. These results imply that EMT induced by BMP-9 is related to invasiveness of HCC.

Smad6 and Smad7 are co-regulated with hepcidin in mouse models of iron overload.

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe-/- and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.

Sustained submicromolar H2O2 levels induce hepcidin via signal transducer and activator of transcription 3 (STAT3).

The peptide hormone hepcidin regulates mammalian iron homeostasis by blocking ferroportin-mediated iron export from macrophages and the duodenum. During inflammation, hepcidin is strongly induced by interleukin 6, eventually leading to the anemia of chronic disease. Here we show that hepatoma cells and primary hepatocytes strongly up-regulate hepcidin when exposed to low concentrations of H(2)O(2) (0.3-6 µM), concentrations that are comparable with levels of H(2)O(2) released by inflammatory cells. In contrast, bolus treatment of H(2)O(2) has no effect at low concentrations and even suppresses hepcidin at concentrations of >50 µM. H(2)O(2) treatment synergistically stimulates hepcidin promoter activity in combination with recombinant interleukin-6 or bone morphogenetic protein-6 and in a manner that requires a functional STAT3-responsive element. The H(2)O(2)-mediated hepcidin induction requires STAT3 phosphorylation and is effectively blocked by siRNA-mediated STAT3 silencing, overexpression of SOCS3 (suppressor of cytokine signaling 3), and antioxidants such as N-acetylcysteine. Glycoprotein 130 (gp130) is required for H(2)O(2) responsiveness, and Janus kinase 1 (JAK1) is required for adequate basal signaling, whereas Janus kinase 2 (JAK2) is dispensable upstream of STAT3. Importantly, hepcidin levels are also increased by intracellular H(2)O(2) released from the respiratory chain in the presence of rotenone or antimycin A. Our results suggest a novel mechanism of hepcidin regulation by nanomolar levels of sustained H(2)O(2). Thus, similar to cytokines, H(2)O(2) provides an important regulatory link between inflammation and iron metabolism.