p19(ARF) /p14(ARF) controls oncogenic functions of signal transducer and activator of transcription 3 in hepatocellular carcinoma.

UNLABELLED: Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION: These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.

TGF-beta dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells.

BACKGROUND: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-beta. We analyzed the oxidative stress induced by TGF-beta and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs. RESULTS: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-beta administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-beta/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense. CONCLUSION: The data provide evidence that TGF-beta induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model.

It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker-tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.

The plasticity of p19 ARF null hepatic stellate cells and the dynamics of activation.

In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19 ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19 ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, alpha-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-beta1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120(ctn) and beta-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19(ARF) deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.

Orchiectomy upregulates free soluble RANKL in bone marrow of aged rats.

The osteoprotegerin (OPG)/receptor activator of NF-kappaB ligand (RANKL) axis is thought to be involved in the upregulation of bone turnover following sex steroid deficiency. Here, we investigated the effects of orchiectomy (ORX) on bone turnover and free soluble RANKL (sRANKL) in aged rats. Free, bioactive sRANKL is a critical determinant and key mediator for survival and activity of mature osteoclasts. Thirty-three 9-month-old male Fischer-344 rats were either ORX or sham-operated (SHAM). Following in vivo fluorochrome labeling, vehicle (ricinus oil/benzyl benzoate)-treated SHAM and vehicle- or testosterone undecanoate (T, 6 mg/kg s.c. once weekly)-treated ORX rats (n=8-9 each) were killed 2 months after surgery. Vehicle-treated ORX rats showed lower seminal vesicle weight, loss of proximal tibial trabecular bone mineral density, and reduced cortical thickness at the tibial shaft as measured by peripheral quantitative computed tomography relative to SHAM controls. Bone loss in vehicle-treated ORX rats was associated with enhanced bone turnover as evidenced by increases in tibial cancellous bone formation rate, osteoclast numbers, urinary excretion of calcium and deoxypyridinoline, and serum osteocalcin. T treatment of ORX rats restored seminal vesicle weight to SHAM control levels, and completely protected against post-ORX bone loss by suppressing bone turnover. Free sRANKL concentrations in bone marrow supernatants harvested from the proximal femur were about 3-fold higher in vehicle-treated ORX relative to SHAM rats, and returned to SHAM control levels in T-treated ORX rats. mRNA abundance of matrix metalloproteinase-14 (MMP-14) in bone marrow was 4-fold higher in vehicle-treated ORX rats relative to SHAM rats. T treatment of ORX rats suppressed MMP-14 mRNA expression to SHAM control levels. We conclude that orchiectomy increases the concentration of free sRANKL in bone marrow of aged rats. In addition, increased shedding of membrane-bound RANKL by MMP-14 may be a pivotal mechanism resulting in augmented free sRANKL concentrations in the bone marrow environment after androgen withdrawal.

High cortical bone mass phenotype in betacellulin transgenic mice is EGFR dependent.

Signaling through the epidermal growth factor receptor (EGFR) by ligands such as epidermal growth factor (EGF), transforming growth factor alpha (TGFA), and amphiregulin (AREG) has been reported to have effects on skeletal growth. The role of betacellulin (BTC), another EGFR ligand, in skeletal development and bone metabolism is unknown. In previous experiments, transgenic mice overexpressing BTC ubiquitously under the control of the chicken beta-actin promoter (BTC-tg) exhibited stunted growth and disproportionately sized long bones. In this study, we performed a detailed phenotypic analysis of BTC-tg mice at 3, 6, and 9 wk of age. Osteoblastic cells from transgenic mice showed strong expression of BTC as determined by Western blots and by immunohistochemistry on bone sections. In femurs of male and female BTC-tg mice, we found reduced longitudinal bone growth and a pronounced increase in total volumetric BMD. The increased femoral BMD was mainly caused by augmented endocortical bone apposition and subsequent cortical bone thickening. In contrast, vertebral BMD was reduced in BTC-tg mice of both sexes. An overall similar phenotype was found in 6-mo-old BTC-tg mice. The increase in cortical bone mass in the appendicular skeleton of BTC-tg mice was largely blocked when they were crossed into the Egfr (Wa5) background characterized by a dominant negative EGFR. Our study showed that overexpression of BTC results in an EGFR-dependent upregulation of cortical bone mass in the appendicular skeleton of mice, uncovering a potential novel anabolic pathway for cortical bone.

Integration of Ras subeffector signaling in TGF-beta mediated late stage hepatocarcinogenesis.

Immortalized p19(ARF) null hepatocytes (MIM) feature a high degree of functional differentiation and are susceptible to transforming growth factor (TGF)-beta driven growth arrest and apoptosis. In contrast, polarized MIM hepatocytes expressing hyperactive Ha-Ras continue proliferation in cooperation with TGF-beta, and adopt an invasive phenotype by executing an epithelial to mesenchymal transition (EMT). In this study, we analyzed the involvement of Ras subeffectors in TGF-beta mediated hepatocellular EMT by employing MIM hepatocytes, which express Ras mutants allowing selective activation of either mitogen-activated protein kinase (MAPK) signaling (V12-S35) or phosphoinositide 3-OH (PI3)3 kinase (PI3K) signaling (V12-C40). We found that MAPK signaling in MIM-S35 hepatocytes was necessary and sufficient to promote resistance to TGF-beta mediated inhibition of proliferation in vitro and in vivo. MIM-S35 hepatocytes showed also PI3K activation during EMT, however, MAPK signaling on its own protected hepatocytes from apoptosis. Yet, MIM-C40 hepatocytes failed to form tumors and required additional MAPK stimulation to overcome TGF-beta mediated growth arrest. In vivo, the collaboration of MAPK signaling and TGF-beta activity drastically accelerated the cell-cycle progression of the hepatocytes, leading to vast tumor formation. From these data we conclude that MAPK is crucial for the cooperation with TGF-beta to regulate the proliferation as well as the survival of hepatocytes during EMT, and causes the fatal increase in hepatocellular tumor progression.