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.

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.

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.

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.

Di (2-Ethylhexyl) Phthalate and Its Role in Developing Cholestasis: An In Vitro Study on Different Liver Cell Types.

OBJECTIVES: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer used in many polyvinylchloride medical devices and is washed out easily. Thereby critically ill infants can become exposed to DEHP concentrations significantly exceeding the recommended threshold. We suspect DEHP to play an important role in the development of intestinal failure-associated liver disease. The aim of this study was therefore to determine the direct influence of DEHP on different liver cell types. METHODS: HepG2, human upcyte hepatocytes, primary murine hepatocytes, LX-2, human upcyte hepatic stellate cells, and liver organoids were cultured with DEHP (0.5-500 µmol/L) and parameters including cytotoxicity, cell-cell interactions, and expression of metabolizing enzymes were investigated. RESULTS: DEHP modulated the expression of xenobiotic metabolizing enzymes, reduced the formation of bile canaliculi and cell polarity, and inhibited Cyp-activity in hepatocytes. DEHP had a toxic effect on LX-2 and induced the fibrogenic activation of hepatic stellate cells. The mode of action of DEHP was different in monolayer cultures compared to 3D-liver organoids, which were more sensitive to DEHP. CONCLUSIONS: This study suggests that DEHP modulates expression and activity of drug-detoxifying liver enzymes in humans at a clinically relevant concentration. Furthermore, it may contribute to the development of cholestasis and fibrosis. These findings strongly support the opinion, that there is a significant potential for serious adverse effects of DEHP derived from medical devices on human health, especially in very young infants with immature livers.

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.

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.

Transforming Growth Factor ß1 (TGF-ß1) Activates Hepcidin mRNA Expression in Hepatocytes.

The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-ß1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-ß1-induced hepcidin expression are still unclear. Here we show that TGF-ß1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-ß1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-ß1 depends on functional TGF-ß1 type I receptor (ALK5) and TGF-ß1 type II receptor (TßRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-ß1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-ß1. TGF-ß1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-ß1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-ß1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

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.

Hepatocyte fate upon TGF-ß challenge is determined by the matrix environment.

Primary hepatocytes are a versatile tool to investigate all aspects of liver function, and frequently used in drug development and testing. Upon TGF-ß challenge, hepatocytes either undergo an epithelial to mesenchymal transition (EMT) or apoptosis: culture on stiff collagen (monolayer) results in EMT whereas hepatocytes in a soft collagen matrix (sandwich) undergo programmed cell death. In this study, we analyzed the transcriptional programs triggered by TGF-ß under different culture conditions. Our results indicate that TGF-ß initiates an identical transcription profile in hepatocytes irrespective of the cellular environment. The fact that we nevertheless observe two vastly different phenotypes indicates that the matrix environment rather than the TGF-ß induced expression signature is the major determinant of the hepatocellular response. To confirm the impact of the surrounding matrix environment on the hepatocytes׳ phenotype in response to TGF-ß signaling, we studied the effect of Snail overexpression and knockout in both culture conditions. Neither genetic manipulation showed an impact on hepatocytes' fate upon TGF-ß treatment, confirming the crucial role of the surrounding matrix. Our findings provide novel insights into the hepatocellular basis of the fate decision between EMT and apoptotic cell death, and might explain why liver cells can react in very different manners to identical stimuli when tissue remodeling has changed the matrix environment, as occurs in a fibrotic liver.

Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists.

Bone morphogenetic proteins (BMPs) are members of the TGFß family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFß receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose- and time-dependent manner, which can provide an explanation of some properties of the BMP regulatory network.

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.

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.

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.

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.

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.

Intestinal BMP-9 locally upregulates FGF19 and is down-regulated in obese patients with diabetes.

Bone morphogenetic protein (BMP)-9, a member of the TGFß-family of cytokines, is believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients' samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis.

A fast and efficient polymerase chain reaction-based method for the preparation of in situ hybridization probes.

AIMS: In situ hybridization (ISH) is the method of choice for analysis of the local distribution of gene expression in tissue samples at the cellular level. In this study we present a rapid and efficient protocol for the generation of labelled cRNA probes. METHODS AND RESULTS: The protocol is based on the preparation of DNA in vitro transcription templates using polymerase chain reaction (PCR), using primers that include RNA polymerase promoter sequences and size-based purification of PCR fragments containing the target gene-specific cDNA and promoter elements for T7 and SP6 RNA polymerase. The optimized purification protocols ensure high transcription efficiency and target specificity of the labelled cRNA. The cRNA hybridization probes obtained are compatible with established in situ hybridization protocols. CONCLUSIONS: Purified PCR fragment-based in vitro transcription enables preparation of in situ hybridization probes which allow the rapid detection of gene expression distribution in tissue slices from any gene of interest.