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Summary Vascular mural cells (vMCs) are essential components of the vertebrate vascular system, controlling blood vessel maturation and homeostasis. Discrete molecular mechanisms have been associated with vMC development and differentiation. The function of hemodynamic forces in controlling vMC recruitment is unclear. Using transgenic lines marking developing vMCs in zebrafish embryos, we find that vMCs are recruited by arterial-fated vessels and that the process is flow dependent. We take advantage of tissue-specific CRISPR gene targeting to demonstrate that hemodynamic-dependent Notch activation and the ensuing arterial genetic program is driven by endothelial primary cilia. We also identify zebrafish foxc1b as a cilia-dependent Notch-specific target that is required within endothelial cells to drive vMC recruitment. In summary, we have identified a hemodynamic-dependent mechanism in the developing vasculature that controls vMC recruitment., Graphical Abstract, Highlights • Blood flow and cilia are required for vascular mural cell (vMC) coverage in zebrafish • Cilia-dependent Notch signaling drives vMC recruitment of arterial-fated vessels • Foxc1b is necessary and sufficient to drive vascular myogenesis in zebrafish, Chen et al. find that primary cilia are responsible for blood-flow-driven Notch activation in arterial vessels of zebrafish embryos. This pathway leads to specific expression of foxc1b, which then drives vMC recruitment and supports vascular myogenesis in zebrafish.

The analysis of lipid molecules in living organism is an important step in deciphering metabolic pathways. Recently, the zebrafish has been adopted as a valuable animal model system to perform in vivo metabolomics studies, however limited methodologies and protocols are currently available to investigate zebrafish lipidome and even fewer to analyze specific classes of lipids. Here we present an HPLC-HRMS based method to rapidly measure multiple prenol lipid molecules from zebrafish tissues. In particular, we have optimized our method for concurrent detection of ubiquinones (Coenzyme Q6, Coenzyme Q9, Coenzyme Q10), cholesterol, vitamin E (α-tocopherol), vitamin K1 and vitamin K2. The purpose of this study was to compare different ionization modes, mobile phases and stationary phases in order to optimize lipid molecules separation. After HPLC-HRMS parameters selection, several extraction conditions from zebrafish embryos were evaluated. We assessed our methodology by quantitation of analytical recovery on zebrafish extracts from wild-type or zebrafish mutants (barolo) affected by impaired biosynthesis of ubiquinones.

Background Long non-coding RNAs (lncRNAs) are a group of non-coding RNAs acting as regulators of gene expression, through interaction with histones or through interaction with complementary DNA sequences, implicated in various human diseases such as cancer, cardiovascular diseases, autoimmune and neurodegenerative disorders and have been reported to be involved in the modulation of neuropathic pain. We recorded Laser Evoked Potentials (LEPs) in order to study: (1) lncRNAs modifications in experimental pain model; (2) correlation between the lncRNA changes and objective measure of pain perception. Material and methods LEPs were recorded in 11 healthy subjects after hand and perioral region stimulation. Three consecutive series were recorded for each stimulation site in order to investigate the habituation. Blood samples were collected immediately before LEP recording (baseline) and after 30-min (post-pain). We screened 84 lncRNAs, involved in autoimmunity and inflammatory response. Results We identified 2 lncRNAs up-regulated at the post-pain time: RP11-819C21.1 (fold change = 8.2; p = 0.038) and ZNRD1 antisense RNA 1 non-protein coding (ZNRD1-AS) (fold change = 6.3; p = 0.037). The up-regulation of both lncRNAs showed a significant positive correlation with the LEP habituation to perioral region stimulation (p = 0.04 and p = 0.01, respectively). Conclusions This is the first study showing lncRNA changes in a pain experimental model. RP11-819C21.1 and ZNRD1-AS shows as direct target miR-19a and miR19b, a class of microRNAs involved in modulation of multiple potassium channel α-subunits. lncRNAs could be involved in the pathophysiology of painful diseases characterized by reduced habituation to pain.

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

Summary Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity., Graphical Abstract Highlights ► UBIAD1 is a Golgi prenyltransferase ► UBIAD1 contributes to the nonmitochondrial pool of CoQ10 ► UBIAD1 protects cardiovascular tissues from NOS-dependent oxidative damage ► UBIAD1 is a target for therapeutic strategies by limiting the side effects of statins, UBIAD1 is identified as a nonmitochondrial CoQ10 biosynthetic enzyme required for oxidative damage protection. Through CoQ10 synthesis, UBIAD1 modulates endothelial nitric oxide synthase activity and nitric oxide signaling necessary for cardiovascular development and homeostasis.

MicroRNAs (e.g. miRNAs), endogenously expressed small non-coding RNAs, control gene expression by regulating target messenger RNA at post-transcriptional level. Recent studies have identified a specific role for microRNAs as key regulators of vascular function and dysfunction. The zebrafish ( Danio rerio ) system is a useful model system to study endothelial cell homeostasis during normal and pathological conditions. In this review, we describe how the zebrafish model is becoming a useful tool to study the role of microRNAs and their target genes in endothelial and smooth muscle cells both in normal and pathological conditions. Finally, we also discuss the many ways to manipulate miRNAs levels in zebrafish for therapeutic approaches.

BRAF gene mutations have been associated with human cancers. Among the naturally occurring mutations, two that involve amino acids of the conserved DFG motif in the activation loop (D594V and G596R), appear to be inactivating. Aim of this study was to analyze the molecular mechanisms involved in the loss of function of B-Raf inactivating mutation G596R. Furthermore, the ability of the B-Raf DFG motif mutants to generate heterodimers with C-Raf and the possible functional consequences of the B-Raf/C-Raf heterodimer formation was examined. Wet molecular experiments in HEK293T cells demonstrate that B-RafG596R is a kinase-impaired mutant. Molecular dynamics simulations show that the loss of function of B-RafG596R depends on a restraining effect of Arg596 on the catalytic residue Asp594, which results in the loss of the appropriate spatial localization and/or conformation of the latter necessary for anchoring ATP to the enzyme. Exploration of B-Raf/C-Raf heterodimer formation indicates the occurrence of functioning heterodimers in the case of all the DFG B-Raf mutants, independently from the expected differences in spatial conformation of the activation loop, although the transforming activity of the mutants appear negligible. In conclusion, this study delivers novel information on the functional properties of the B-Raf DFG motif inactivating mutants and on the mechanisms driving B-Raf/C-Raf heterodimerization and consequent C-Raf transactivation.

Extracellular superoxide dismutase (SOD3) gene therapy has been shown to attenuate tissue damages and to improve the recovery of the tissue injuries, but the cellular events delivering the therapeutic response of the enzyme are not well defined. In the current work, we overexpressed SOD3 in rat hindlimb ischemia model to study the signal transduction and injury healing following the sod3 gene transfer. The data suggest a novel sod3 gene transfer-derived signal transduction cascade through Ras-Mek-Erk mitogenic pathway leading to activation of AP1 and CRE transcription factors, increased vascular endothelial growth factor (VEGF)-A and cyclin D1 expression, increased cell proliferation, and consequently improved metabolic functionality of the injured tissue. Increased cell proliferation could explain the improved metabolic performance and the healing of the tissue damages after the sod3 gene transfer. The present data is a novel description of the molecular mechanism of SOD3-mediated recovery of tissue injury and suggests a new physiological role for SOD3 as a Ras regulatory molecule in signal transduction.

Thyroid cancer is frequently associated with the oncogenic conversion of receptor tyrosine kinases (RTKs) or their downstream signalling molecules. Hence, there is a strong biological rationale for assessing the efficacy of RTK blockade to treat patients who are resistant to or not candidates for treatment with radioactive iodine. The first results of clinical trials based on the use of RTK inhibitors in thyroid cancer patients have recently been published. Here we discuss targeting of specific RTKs as a potential therapeutic strategy for the treatment of thyroid cancer.

We have analysed the expression of the HMGA2 gene in a panel of normal and neoplastic thyroid tissues by immunohistochemistry and quantitative RT-PCR. HMGA2 protein was detectable in four out of 21 follicular carcinomas, 30 out of 45 papillary carcinomas, and 11 out of 12 undifferentiated carcinomas. As far as follicular thyroid adenomas are concerned, only three cases of the 31 analysed showed HMGA2 protein expression, whereas it was absent in seven normal thyroid tissues and in 12 hyperplastic nodules. Quantitative RT-PCR showed that almost all the papillary thyroid carcinomas and 13 out of 16 follicular thyroid carcinomas express much higher HMGA2 specific mRNA levels in comparison to normal thyroids and adenomas. Therefore, our data support the quantitative RT-PCR analysis of HMGA2 expression, rather than immunohistochemistry, as a powerful tool for the diagnosis of thyroid neoplasias. (c) 2008 Elsevier Ltd. All rights reserved.

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demonstrate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we characterized Tyr(981), a known binding site for c-Src, as a major determinant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr(981)-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions.

Cutaneous wound healing is a highly coordinated physiological process that rapidly and efficiently restores skin integrity. Reepithelization is a crucial step during wound healing, which involves migration and proliferation of keratinocytes to cover the denuded dermal surface. Recent advances in wound biology clarified the molecular pathways governing keratinocyte reepithelization at wound sites. These new findings point towards novel therapeutic targets and provide suitable methods to promote faster tissue regeneration in vivo.

Noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer.

RINGWe have isolated a new human RING-finger gene finger family show a variety of functions and are mostly (RNF4) that encodes a 190-amino-acid protein. RNF4, involved in the regulation of development and cell difin addition to the carboxyl-terminally located RING- ferentiation (Saurin et al., 1996). Some viral RINGfinger motif, contains two putative nuclear localiza- finger proteins are involved in transactivation of viral tion signals and stretches of acidic amino acids that or cellular genes and are essential for viral pathogenicare similar to the activation domains of some tran- ity (Saurin et al., 1996). Most of the human RINGscription factors. RNF4 was expressed at low levels in finger proteins have been localized in the nucleus or in all human tissues examined, with the notable excep- tion of very high expression in the testis. The mouse both the cytoplasm and the nucleus and are involved homolog of RNF4 was abundantly expressed in embry- in signal transduction and in oncogenesis (Saurin et onic tissues from the earliest days postgestation and al., 1996). The RING-finger motif of TRAF2 is required exhibited a ubiquitous pattern of expression as as- for the formation of the TRAF2/TANK complex, which sessed by in situ hybridization.We havemapped RNF4 in turn mediates NF-kB activation upon ligand binding to 4p16.3, a chromosome region associated with sev- to CD40 or type II tumor necrosis factor receptor eral genetic and neoplastic diseases. RNF4 spans 47 (Cheng and Baltimore, 1996). Several proteins conkb, is composed of eight exons, and maps immediately taining RING-finger motifs participate in oncogenesis proximal to the anonymous locus D4S183, between the through different mechanisms. Chromosomal translohuntingtin (HD) and the fibroblast growth factor re- cations in different neoplasias generate chimeric proceptor 3 (FGFR3) genes.

We have isolated a rat thyroid cDNA encoding a novel rat receptor-type tyrosine phosphatase protein. This gene, on the basis of its homology to another tyrosine phosphatase, the recently isolated human DEP-1/HPTP eta, has been named r-PTP eta. In rat thyroid cells the r-PTP eta gene acts as a differentiation marker, Indeed, the block of thyroid cell differentiation induced by viral and cellular oncogenes is associated with the inhibition or marked reduction of the expression of this gene and its expression is positively regulated by thyrotropin, the physiological stimulator of thyroid cell growth. (C) 1997 Academic Press.

The effect of thyrotropin and norepinephrine on cytosolic calcium levels were evaluated in normal (PC C13) and transformed (PC E1A, PC Py and PC E1APy) rat thyroid epithelial cell lines. A different pattern of response to both norepinephrine and thyrotropin was observed among the distinct cell lines. In PC C13 the cytosolic calcium rise induced by norepinephrine, characterized by an early transient spike followed by a second phase of sustained calcium levels, was greatly enhanced by thyrotropin. The effect of norepinephrine on calcium concentrations was less affected by thyrotropin in PC C13 transformed by the adenovirus E1A oncogene. Conversely, in Polyoma middle-T transformed PC C13 the increase in cytoplasmic calcium was still sensitive to thyrotropin. The most malignant PC E1APy were totally independent of thyrotropin. © 1993.