Soluble endoglin antagonizes Met signaling in spindle carcinoma cells.

Increased levels of soluble endoglin (Sol-Eng) correlate with poor outcome in human cancer. We have previously shown that shedding of membrane endoglin, and concomitant release of Sol-Eng is a late event in chemical mouse skin carcinogenesis associated with the development of undifferentiated spindle cell carcinomas (SpCCs). In this report, we show that mouse skin SpCCs exhibit a high expression of hepatocyte growth factor (HGF) and an elevated ratio of its active tyrosine kinase receptor Met versus total Met levels. We have evaluated the effect of Sol-Eng in spindle carcinoma cells by transfection of a cDNA encoding most of the endoglin ectodomain or by using purified recombinant Sol-Eng. We found that Sol-Eng inhibited both mitogen-activated protein kinase (MAPK) activity and cell growth in vitro and in vivo. Sol-Eng also blocked MAPK activation by transforming growth factor-ß1 (TGF-ß1) and impaired both basal and HGF-induced activation of Met and downstream MAPK. Moreover, Sol-Eng strongly reduced basal and HGF-stimulated spindle cell migration and invasion. Both Sol-Eng and full-length endoglin were shown to interact with Met by coimmunoprecipitation experiments. However, full-length endoglin expressed at the plasma membrane of spindle carcinoma cells had no effect on Met signaling activity, and was unable to inhibit HGF-induced cell migration/invasion. These results point to a paradoxical suppressor role for Sol-Eng in carcinogenesis.

Podoplanin is a substrate of presenilin-1/γ-secretase.

Podoplanin (PDPN) is a mucin-like transmembrane glycoprotein that plays an important role in development and cancer. Here, we provide evidence that the intracellular domain (ICD) of podoplanin is released into the cytosol following a sequential proteolytic processing by a metalloprotease and γ-secretase. Western blotting and cell fractionation studies revealed that HEK293T and MDCK cells transfected with an eGFP-tagged podoplanin construct (PDPNeGFP, 50-63kDa) constitutively express two C-terminal fragments (CTFs): a ∼33kDa membrane-bound PCTF33, and a ∼29kDa cytosolic podoplanin ICD (PICD). While pharmacological inhibition of metalloproteases reduced the expression of PCTF33, treatment of cells with γ-secretase inhibitors resulted in enhanced PCTF33 levels. PCTF33 processing by γ-secretase depends on presenilin-1 (PS1) function: cells expressing a dominant negative form of PS1 (PS1 D385N), and mouse embryonic fibroblasts (MEFs) genetically deficient in PS1, but not in PS2, show higher levels of PCTF33 expression with respect to wild-type MEFs. Furthermore, transfection of PS1 deficient MEFs with wild-type PS1 (PS1 wt) decreased PCTF33 levels. N-terminal amino acid sequencing of the affinity purified PICD revealed that the γ-secretase cleavage site was located between valines 150 and 151, but these residues are not critical for proteolysis. We found that podoplanin CTFs are also generated in cells expressing podoplanin mutants harboring heterologous transmembrane regions. Taken together, these results indicate that podoplanin is a novel substrate for PS1/γ-secretase.

Impaired wound repair in adult endoglin heterozygous mice associated with lower NO bioavailability.

Endoglin (Eng) is a transmembrane glycoprotein that is mainly expressed in endothelial cells, but it is also present in the epidermis and skin appendages. To address the role of Eng in cutaneous wound healing, we compared the kinetics of reepithelialization in Eng heterozygous null (Eng(+/-)) mice and their normal littermates (Eng(+/+)) following skin wounds. The wound area was significantly larger in Eng(+/-) than in Eng(+/+) mice from 2 to 8 days after injury; overall wound closure was delayed by 1 to 2 days. In Eng(+/-) mice, keratinocytes at the wound edges exhibited impaired proliferation but were more migratory, as shown by their elongated morphology and increased keratin 17 expression. Inhibition of nitric oxide (NO) synthesis delayed healing in Eng(+/+) but not in Eng(+/-) mice. Administration of the NO donor LA-803 accelerated wound closure in Eng(+/-) mice, with no effect on normal littermates. The acute stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced Eng expression in mouse epidermal keratinocytes in vivo and in vitro associated with hyperproliferation. Similarly, the skin of Eng(+/-) mice failed to mount a hyperplastic response to acute stimulation with TPA. These results demonstrate an important involvement of Eng in wound healing that is associated with NO bioavailability.

Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-ß-induced oxidative stress and apoptosis.

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met(-/-) oval cells) are more sensitive to TGF-ß-induced apoptosis than cells expressing a functional Met (Met(flx/flx)), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-ß induced a mitochondria-dependent apoptotic cell death in Met(flx/flx) and Met(-/-) oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-ß-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met(-/-) oval cells. TGF-ß also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-ß-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-ß. Notably, oxidative stress-related events were strongly amplified in Met(-/-) oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-ß-induced apoptosis and increased sensitivity of Met(flx/flx) oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met(-/-) oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Met(flx/flx) oval cells, whereas no effect was observed in Met(-/-) oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-ß-induced oxidative stress and apoptosis.

The role of the TGF-ß coreceptor endoglin in cancer.

Endoglin (CD105) is an auxiliary membrane receptor of transforming growth factor beta (TGF-ß) that interacts with type I and type II TGF-ß receptors and modulates TGF-ß signaling. Endoglin is overexpressed in the tumor-associated vascular endothelium, where it modulates angiogenesis. This feature makes endoglin a promising target for antiangiogenic cancer therapy. In addition, recent studies on human and experimental models of carcinogenesis point to an important tumor cell-autonomous role of endoglin by regulating proliferation, migration, invasion, and metastasis. These studies suggest that endoglin behaves as a suppressor of malignancy in experimental and human epithelial carcinogenesis, although it can also promote metastasis in other types of cancer. In this review, we evaluate the implication of endoglin in tumor development underlying studies developed in our laboratories in recent years.

Snail1 suppresses TGF-beta-induced apoptosis and is sufficient to trigger EMT in hepatocytes.

Although TGF-ß suppresses early stages of tumour development, it later contributes to tumour progression when cells become resistant to its suppressive effects. In addition to circumventing TGF-ß-induced growth arrest and apoptosis, malignant tumour cells become capable of undergoing epithelial-to-mesenchymal transition (EMT), favouring invasion and metastasis. Therefore, defining the mechanisms that allow cancer cells to escape from the suppressive effects of TGF-ß is fundamental to understand tumour progression and to design specific therapies. Here, we have examined the role of Snail1 as a suppressor of TGF-ß-induced apoptosis in murine non-transformed hepatocytes, rat and human hepatocarcinoma cell lines and transgenic mice. We show that Snail1 confers resistance to TGF-ß-induced cell death and that it is sufficient to induce EMT in adult hepatocytes, cells otherwise refractory to this transition upon exposure to TGF-ß. Furthermore, we show that Snail1 silencing prevents EMT and restores the cell death response induced by TGF-ß. As Snail1 is a known target of TGF-ß signalling, our data indicate that Snail1 might transduce the tumour-promoting effects of TGF-ß, namely the EMT concomitant with the resistance to cell death.

Deletion of the Met tyrosine kinase in liver progenitor oval cells increases sensitivity to apoptosis in vitro.

The hepatocyte growth factor (HGF)/Met signaling system is essential for liver development, homeostasis, and function. In this study, we took advantage of a liver-specific, Met-conditional knockout mouse generated in our laboratory to address the molecular mechanisms of HGF/Met signaling in adult liver progenitor cell (oval cell) biology. For this purpose, we isolated oval cells from 3,5-diethoxycarbonyl-1,4-dihydro-collidine-treated Met(flx/flx) mice and established oval cell-derived cell lines that carried either functional (Met(flx/flx)) or a nonfunctional (Met(-/-)) met gene using virus-mediated Cre-loxP recombination. Oval cells lacking Met tyrosine kinase activity displayed neither Met phosphorylation nor activation of downstream targets and were refractory to HGF stimulation. Although Met(-/-) and Met(flx/flx) cells proliferated at similar rates under 10% serum, Met-deficient cells demonstrated decreased cell viability and were more prone to apoptosis when challenged with either serum starvation or the pro-apoptotic cytokine transforming growth factor-beta. Treatment with HGF reduced transforming growth factor-beta-mediated cell death in Met(flx/flx) but not Met(-/-) cells. Importantly, Met(flx/flx) and Met(-/-) cells both constitutively expressed hgf, and conditioned medium from serum-starved oval cells exhibited anti-apoptotic activity in Met(flx/flx) cells. Furthermore, serum-starved Met(flx/flx) cells showed persistent activation of the Met tyrosine kinase, suggesting HGF/Met autocrine regulation. In conclusion, these data reveal a critical, functional role for Met in oval cell survival through an autocrine mechanism.

Isolation and characterization of a putative liver progenitor population after treatment of fetal rat hepatocytes with TGF-beta.

The "in vitro" establishment of a physiological model of bipotential liver progenitors would be useful for analyzing the molecular mechanisms involved in regulating growth and differentiation, as well as studying their potential role/s in liver physiology and pathology. The transforming growth factor-beta (TGF-beta) induces de-differentiation of fetal rat hepatocytes (FH), concomitant with changes in morphology. The aim of this work was to isolate and characterize this population of TGF-beta-treated fetal hepatocytes (TbetaT-FH) and test whether they can behave as liver progenitors. The TbetaT-FH isolated cell lines show high expression of Thy-1 and low expression of c-Kit. They express liver-specific proteins, such as albumin and alpha-fetoprotein, and mesenchymal markers, such as vimentin. TbetaT-FH maintain expression of the hnf3beta gene, but lose expression of hnf1beta, hnf4, and hnf6. They express c-met and show an increase in proliferation in response to HGF. Interestingly, the transdifferentiation process is coincident with changes in the expression of genes related to the oxidative metabolism. TbetaT-FH cultured in the presence of EGF + DMSO change morphology, towards epithelial cells, gaining expression of CK19 and c-Kit, markers found in hepatoblasts and bile duct cells. Furthermore, TbetaT-FH form duct-like structures when cultured on Matrigel. TbetaT-FH show also potential to revert to an hepatocyte phenotype when submitted to a long-term "in vitro" differentiation protocol towards hepatocytic lineage. In summary, our results support the hypothesis that hepatocytes can function as facultative liver stem cells and demonstrate that TGF-beta might play an essential role in the transdifferentiation process.

Activation of NADPH oxidase by transforming growth factor-beta in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-kappaB-dependent mechanism.

The TGF-beta (transforming growth factor-beta) induces survival signals in foetal rat hepatocytes through transactivation of EGFR (epidermal growth factor receptor). The molecular mechanism is not completely understood, but both activation of the TACE (tumour necrosis factor alpha-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-alpha and HB-EGF (heparin-binding epidermal growth factor-like growth factor) appear to be involved. In the present study, we have analysed the molecular mechanisms that mediate up-regulation of the EGFR ligands by TGF-beta in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-beta, and the existence of an amplification loop triggered by initial activation of the EGFR, have been studied. Results indicate that DPI (diphenyleneiodonium) and apocynin, two NOX (NADPH oxidase) inhibitors, and SB431542, an inhibitor of the TbetaR-I (TGF-beta receptor I), block up-regulation of EGFR ligands and Akt activation. Different members of the NOX family of genes are expressed in hepatocytes, included nox1, nox2 and nox4. TGF-beta up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TbetaR-I-dependent manner. TGF-beta mediates activation of the nuclear factor-kappaB pathway, which is inhibited by DPI and is required for up-regulation of TGF-alpha and HB-EGF. In contrast, EGFR activation is not required for TGF-beta-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-beta in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-beta-treated cells.

Autocrine production of TGF-beta confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands.

Transforming growth factor-beta (TGF-beta) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdés et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-beta-treated fetal hepatocytes: TbetaT-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes TbetaT-FH to die after serum withdrawal. TbetaT-FH expresses high levels of transforming growth factor-alpha (TGF-alpha) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-alpha antibody restores the capacity of cells to die. TGF-beta, which is expressed by TbetaT-FH, mediates up-regulation of TGF-alpha and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-beta confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands.

Involvement of EGF receptor and c-Src in the survival signals induced by TGF-beta1 in hepatocytes.

Transforming growth factor beta1 (TGF-beta1) belongs to a family of polypeptide factors, whose cytostatic and apoptotic functions help restrain the growth of mammalian cells. Although solid data established the role of TGF-beta's as suppressor factors in tumorigenic processes, in the context of an advanced stage of disease, TGF-beta's could also play a pro-oncogenic role. We have previously shown that TGF-beta1 induces both pro- and anti-apoptotic signals in foetal rat hepatocytes. In this work, we have focused on its anti-apoptotic mechanism. We show that TGF-beta1 activates the epidermal growth factor receptor (EGFR) and phosphorylates c-Src. EGFR is required for Akt activation. Blocking EGFR signalling amplifies the apoptotic response to TGF-beta1. TGF-beta1 induced a rapid activation of the tumour necrosis factor-alpha-converting enzyme (TACE/ADAM (a disintegrin and metalloprotease) 17). Inhibitors of TACE considerably attenuated Akt activation, which suggests that TGF-beta1 activates EGF signalling in hepatocytes by promoting shedding of EGF-like ligands. The activation of c-Src by TGF-beta1 is EGFR dependent and is required for full Akt phosphorylation and cell survival. Inhibition of EGFR does not block the epithelial-mesenchymal transition (EMT) induced by TGF-beta1 in hepatocytes, which indicates that activation of EGFR plays an essential role in impairing apoptosis, but it is dispensable for the EMT process.