Extracellular signal-Regulated Kinase 5 (ERK5) is required for the Yes-associated protein (YAP) co-transcriptional activity.

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several physio-pathological cellular processes, by integrating multiple cell autonomous and microenvironmental cues. YAP is the main downstream effector of the Hippo pathway, a tumor-suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signal-regulated kinase 5 (ERK5)/mitogen-activated protein kinase in the regulation of YAP activity. By means of ERK5 inhibition/silencing and overexpression experiments, and by using as model liver stem cells, hepatocytes, and hepatocellular carcinoma (HCC) cell lines, we provided evidence that ERK5 is required for YAP-dependent gene expression. Mechanistically, ERK5 controls the recruitment of YAP on promoters of target genes and its physical interaction with the transcriptional partner TEAD; moreover, it mediates the YAP activation occurring in cell adhesion, migration, and TGFß-induced EMT of liver cells. Furthermore, we demonstrated that ERK5 signaling modulates YAP activity in a LATS1/2-independent manner. Therefore, our observations identify ERK5 as a novel upstream Hippo-independent regulator of YAP activity, thus unveiling a new target for therapeutic approaches aimed at interfering with its function.

YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation.

Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified. The aim of this study was to evaluate the role of YAP in the regulation of epithelial/hepatocyte differentiation and to clarify how a transducer of general stimuli can integrate tissue-specific molecular mechanisms determining specific cell outcomes. By means of YAP silencing and overexpression we demonstrated that YAP has a functional role in the repression of epithelial/hepatocyte differentiation by inversely modulating the expression of Snail (master regulator of the epithelial-to-mesenchymal transition and liver stemness) and HNF4α (master regulator of hepatocyte differentiation) at transcriptional level, through the direct occupancy of their promoters. Furthermore, we found that Snail, in turn, is able to positively control YAP expression influencing protein level and subcellular localization and that HNF4α stably represses YAP transcription in differentiated hepatocytes both in cell culture and in adult liver. Overall, our data indicate YAP as a new member of the HNF4/Snail epistatic molecular circuitry previously demonstrated to control liver cell state. In this model, the dynamic balance between three main transcriptional regulators, that are able to control reciprocally their expression/activity, is responsible for the induction/maintenance of different liver cell differentiation states and its modulation could be the aim of therapeutic protocols for several chronic liver diseases.

TGFß Impairs HNF1α Functional Activity in Epithelial-to-Mesenchymal Transition Interfering With the Recruitment of CBP/p300 Acetyltransferases.

The cytokine transforming growth factor ß (TGFß) plays a crucial role in the induction of both epithelial-to-mesenchymal transition (EMT) program and fibro-cirrhotic process in the liver, where it contributes also to organ inflammation following several chronic injuries. All these pathological situations greatly increase the risk of hepatocellular carcinoma (HCC) and contribute to tumor progression. In particular, late-stage HCCs are characterized by constitutive activation of TGFß pathway and by an EMT molecular signature leading to the acquisition of invasive and metastatic properties. In these pathological conditions, the cytokine has been shown to induce the transcriptional downregulation of HNF1α, a master regulator of the epithelial/hepatocyte differentiation and of the EMT reverse process, the mesenchymal-to-epithelial transition (MET). Therefore, the restoration of HNF1α expression/activity has been proposed as targeted therapeutic strategy for liver fibro-cirrhosis and late-stage HCCs. In this study, TGFß is found to trigger an early functional inactivation of HNF1α during EMT process that anticipates the effects of the transcriptional downregulation of its own gene. Mechanistically, the cytokine, while not affecting the HNF1α DNA-binding capacity, impaired its ability to recruit CBP/p300 acetyltransferases on target gene promoters and, consequently, its transactivating function. The loss of HNF1α capacity to bind to CBP/p300 and HNF1α functional inactivation have been found to correlate with a change of its posttranslational modification profile. Collectively, the results obtained in this work unveil a new level of HNF1α functional inactivation by TGFß and contribute to shed light on the early events triggering EMT in hepatocytes. Moreover, these data suggest that the use of HNF1α as anti-EMT tool in a TGFß-containing microenvironment may require the design of new therapeutic strategies overcoming the TGFß-induced HNF1α inactivation.

HDAC1 inhibition by MS-275 in mesothelial cells limits cellular invasion and promotes MMT reversal.

Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in a variety of conditions including peritoneal dialysis (PD), post-surgery adhesions and peritoneal metastases. The acquisition of invasive and pro-fibrotic abilities by mesothelial cells (MCs) through induction of MMT, a cell-specific form of EMT, plays a main role in this process. Aim of this study was to evaluate possible effects of histone deacetylase (HDAC) inhibitors, key components of the epigenetic machinery, in counteracting MMT observed in MCs isolated from effluent of PD patients. HDAC inhibitors with different class/isoform selectivity have been used for pharmacological inhibition. While the effect of other inhibitors was limited to a partial E-cadherin re-expression, MS-275, a HDAC1-3 inhibitor, promoted: (i) downregulation of mesenchymal markers (MMP2, Col1A1, PAI-1, TGFß1, TGFßRI) (ii) upregulation of epithelial markers (E-cadherin, Occludin), (iii) reacquisition of an epithelial-like morphology and (iv) marked reduction of cellular invasiveness. Results were confirmed by HDAC1 genetic silencing. Mechanistically, MS-275 causes: (i) increase of nuclear histone H3 acetylation (ii) rescue of the acetylation profile on E-cadherin promoter, (iii) Snail functional impairment. Overall, our study, pinpointing a role for HDAC1, revealed a new player in the regulation of peritoneal fibrosis, providing the rationale for future therapeutic opportunities.

Building Bridges between Clinical and Forensic Toxicology Laboratories.

BACKGROUND: Clinical and forensic toxicology can be defined as two disciplines involving the detection, identification and measurement of xenobiotics in biological and non-biological samples to assist in the diagnosis, treatment, prognosis and prevention of poisonings and to disclose causes and contributory causes of fatal intoxications, respectively. OBJECTIVE: This article explores the close connections between clinical and forensic toxicology in overlapping areas of interest. METHODS: An update has been carried out of the following seven areas of interest in analytical toxicology: doping control, Sudden Cardiac Death (SCD), brain death, Sudden Infant Death Syndrome (SIDS) and Munchausen Syndrome by Proxy (MSBP), prenatal exposure to drugs and Fetal Alcohol Syndrome (FAS), Drug-Facilitated Crimes (DFC) and intoxications by new psychoactive substances (NPS). RESULTS: While issues such as SCD, SIDS or doping control are investigated mainly in forensic laboratories, others such as prenatal exposure to drugs or FAS are mainly treated in clinical laboratories. On the other hand, areas such MSBP, DFC or the intoxications by NPS are of interest in both laboratories. Some of these topics are initially treated in hospital emergency departments, involving clinical laboratories and sometimes lately derived to forensic laboratories. Conversely, cases with initial medicallegal implications and fatalities are directly handled by forensic toxicology, but may trigger further studies in the clinical setting. CONCLUSION: Many areas of common interest between clinical and forensic laboratories are building bridges between them. The increasing relationships are improving the growth, the reliability and the robustness of both kinds of laboratories.

Quantification of Methamphetamine «Shabu¼ in Biological Matrices to Detect Prenatal Exposure: A Case Report and a Literature Review.

BACKGROUND: Methamphetamine misuse represents an increasing global public health problem. Its consumption during pregnancy becomes a relevant issue, since it has clinical consequences for the child's health and the pregnant woman. Despite this, there are only few data in the literature that include analytical results in the matrices used to detect prenatal exposure. OBJECTIVES: 1) Present a case report of prenatal methamphetamine exposure with toxicological analytical confirmation in biological matrices; and 2) Perform a compilation of prenatal methamphetamine exposure studies and case reports which include toxicological analytical results. METHODS: Prenatal methamphetamine exposure was confirmed using a traditional "screen with reflex" approach. Methamphetamine and amphetamine were quantified in urine, meconium and hair samples of the neonate and mother by gas chromatography-mass spectrometry. Also, a detailed revision of the existent literature that provides information on the analytical toxicology results has been included. RESULTS: In the neonatal biological matrices test results of methamphetamine/amphetamine were: urine 2,966.43/1,638.71 ng/mL, meconium 1,450/<0.1 ng/g and hair 36.54/9.66 ng/mg. In the maternal biological matrices, test results were: urine 13,393.89/3,074.95 ng/mL and hair 11.29/3.37 ng/mg (0-3 cm), 4.68/2.58 (3-6 cm), 6.43/3.13 ng/mg (6-9 cm) and 4.72/2.49 ng/mg (9-12 cm). These results confirm a recent and continued regular substance use throughout pregnancy including delivery. CONCLUSION: The data provided will be useful for clinical purposes to improve the diagnostic and follow- up of acute and chronic intoxications. Additionally, results will be used to support interpretations in the field of forensic and legal medicine.

Overexpression of the Vitronectin V10 Subunit in Patients with Nonalcoholic Steatohepatitis: Implications for Noninvasive Diagnosis of NASH.

Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL). Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment of the ECM protein vitronectin (VTN) was then selected as a promising circulating biomarker in discriminating NASH. The analysis of sera of patients provided these major findings: the circulating VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits. In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive discrimination of NASH from simple steatosis.

Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes.

In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that, in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines as in vitro models of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

Molecular Mechanisms Underlying Peritoneal EMT and Fibrosis.

Peritoneal dialysis is a form of renal replacement alternative to the hemodialysis. During this treatment, the peritoneal membrane acts as a permeable barrier for exchange of solutes and water. Continual exposure to dialysis solutions, as well as episodes of peritonitis and hemoperitoneum, can cause acute/chronic inflammation and injury to the peritoneal membrane, which undergoes progressive fibrosis, angiogenesis, and vasculopathy, eventually leading to discontinuation of the peritoneal dialysis. Among the different events controlling this pathological process, epithelial to mesenchymal transition of mesothelial cells plays a main role in the induction of fibrosis and in subsequent functional deterioration of the peritoneal membrane. Here, the main extracellular inducers and cellular players are described. Moreover, signaling pathways acting during this process are elucidated, with emphasis on signals delivered by TGF-ß family members and by Toll-like/IL-1ß receptors. The understanding of molecular mechanisms underlying fibrosis of the peritoneal membrane has both a basic and a translational relevance, since it may be useful for setup of therapies aimed at counteracting the deterioration as well as restoring the homeostasis of the peritoneal membrane.