Laura Sancillo

Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.

Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse

ABSTRACT The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes.

IGFBP-3 inhibits Wnt signaling in metastatic melanoma cells

In previous works, we have shown that insulin‐like growth factor‐binding protein‐3 (IGFBP‐3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP‐3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti‐tumoral effects of IGFBP‐3. We show that the anti‐tumoral effect of IGFBP‐3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP‐3 binds the Wnt signalosome interacting specifically with its component GSK‐3β. As a consequence, the β‐catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK‐3β is activated through dephosphorylation, becoming free to target cytoplasmic β‐catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP‐3 is a novel and effective inhibitor of Wnt signaling. As IGFBP‐3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling.

P‐Selectin Sustains Extramedullary Hematopoiesis in the Gata1low Model of Myelofibrosis

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P‐selectin (P‐sel) that, by triggering neutrophil emperipolesis, may cause TGF‐β release and disease progression. This hypothesis was tested by deleting the P‐sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1low mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P‐selnullGata1low mice survived splenectomy and lived 3 months longer than P‐selWTGata1low littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P‐sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF‐β content, and corrected the HSC distribution that in Gata1low mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF‐β reduced P‐sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1low mice contained numerous cKITpos activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKITpos hematopoietic cells. These activated fibrocytes were not detected in spleens from P‐selnullGata1low or TGF‐β‐inhibited Gata1low littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1low mice, and possibly in PMF, abnormal P‐sel expression in MK may mediate the pathological cell interactions that increase TGF‐β content in MK and favor establishment of a microenvironment that supports myelofibrosis‐related HSC in spleen. Stem Cells 2016;34:67–82

Involvement of cyclic-nucleotide response element-binding family members in the radiation response of Ramos B lymphoma cells

The aim of the present study was to investigate the role of Cyclic-nucleotide Response Element-Binding (CREB) family members and related nuclear transcription factors in the radiation response of human B lymphoma cell lines (Daudi and Ramos). Unlike the more radiosensitive Daudi cells, Ramos cells demonstrated only a moderate increase in early apoptosis after 3–5 Gy irradiation doses, which was detected with Annexin V/PI staining. Moreover, a significant and dose-dependent G2/M phase accumulation was observed in the same cell line at 24 h after both ionizing radiation (IR) doses. Western blot analysis showed an early increase in CREB protein expression that was still present at 3 h and more evident after 3 Gy IR in Ramos cells, along with the dose-dependent upregulation of p53 and NF-κB. These findings were consistent with real-time RT-PCR analysis that showed an early- and dose-dependent upregulation of NFKB1, IKBKB and XIAP gene expression. Unexpectedly, pre-treatment with SN50 did not increase cell death, but cell viability. Taken together, these findings let us hypothesise that the early induction and activation of NF-κB1 in Ramos cells could mediate necrotic cell death and be linked to other molecules belonging to CREB family and involved in the cell cycle regulation.

Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis

Abstract Patients with primary myelofibrosis have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions. Platelet neutrophil interactions are mediated by P-selectin and even though the megakaryocytes of myelofibrosis patients express normal levels of P-selectin, it remains abnormally localized to the demarcation membrane system rather than being assembled into the α-granules in platelets. Mice carrying the hypomorphic Gata1low mutation express the same megakaryocyte abnormalities presented by primary myelofibrosis patients, including abnormal P-selectin localization to the DMS and develop with age myelofibrosis, a disease that closely resembles human primary myelofibrosis. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1low mice would develop thrombosis with age and, in this case, the role played by P-selectin in the development of the trait. To this aim, Gata1low mice were crossed with P-selnull mice according to standard genetic protocols and Gata1lowP-selwt, Gata1lowP-selnull and Gata1WTP-selnull or Gata1wtP-selwt (as controls) littermates obtained. It was shown that platelet counts, but not hematocrit, are reduced in Gata1low mice. Moreover, platelet microparticles are reduced in Gata1low mice and P-selectin positive platelet microparticles were not found. To determine the phenotypic implications of the different mutations, bleeding time was estimated by a tail cut procedure. Mutant mice were sacrificed and presence of thrombosis was determined by immunohistological staining of organs. Gata1low mice with or without the P-selectin null trait had a prolonged bleeding time compared to wild type mice. However, in Gata1low mice significantly higher frequency of thrombotic events was seen in adult and old Gata1low mice compared to Gata1lowP-selnull mice. Thus, presence of the P-selectin null trait rescued Gata1low mice from the thrombotic phenotype, but did not change the level of platelet microparticles. Taken together these data indicate that abnormal localization of P-selectin, induced by the Gata1low mutation, and thus, increased pathological interactions with leucocytes, is responsible for the increased presence of thrombosis seen in these mice.

The role of angiogenesis, inflammation and estrogen receptors in breast implant capsules development and remodeling

BACKGROUND Capsular contracture is the most common complication following breast implant placement. The multiple factors unbalancing the physiological response to the foreign body have not been fully elucidated. The aim of this study was to investigate the role of neo-angiogenesis, inflammation and estrogen receptors in peri-prosthetic tissue development and remodeling. METHODS The study enrolled 31 women who underwent expander substitution with definitive implant. Specimens were stained with hematoxylin/eosin, Masson trichrome, immunohistochemistry and immunofluorescence for alpha-smooth muscle actin, estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), Collagen type I and III, CD31 (as a marker of neo-angiogenesis) and vascular endothelial growth factor (VEGF). Inflammatory infiltration was quantified and analyzed. Transmission electron microscopy was performed for ultrastructural evaluation. RESULTS Myofibroblasts, mainly localized in the middle layer of capsular tissue, expressed VEGF, ER-α and ER-β. ER-β expression positively correlated with Collagen type I deposition (p= 0.025). Neo-angiogenesis was predominant in the middle layer. CD31 expression positively correlated with Collagen type I expression (p=0.009) and inflammatory infiltration grade (p= 0.004). The degree of inflammatory infiltration negatively correlated with the time from implantation (p = 0.022). DISCUSSION The middle layer is key in the development and remodeling of capsular tissue. Myofibroblasts produce VEGF, that induces neo-angiogenesis. New vessels formation is also correlated to the inflammatory response. Collagen deposition is associated with ER-β expression and neo-angiogenesis. These findings may prelude to targeted pharmacologic therapies able to control such interactions, thus hampering the self-sustaining loop promoting the progression of physiologic fibrosis toward pathologic contracture.

The fragile X mental retardation protein regulates tumor invasiveness-related pathways in melanoma cells

The fragile X mental retardation protein (FMRP) is lacking or mutated in patients with the fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. FMRP affects metastasis formation in a mouse model for breast cancer. Here we show that FMRP is overexpressed in human melanoma with high Breslow thickness and high Clark level. Furthermore, meta-analysis of the TCGA melanoma data revealed that high levels of FMRP expression correlate significantly with metastatic tumor tissues, risk of relapsing and disease-free survival. Reduction of FMRP in metastatic melanoma cell lines impinges on cell migration, invasion and adhesion. Next-generation sequencing in human melanoma cells revealed that FMRP regulates a large number of mRNAs involved in relevant processes of melanoma progression. Our findings suggest an association between FMRP levels and the invasive phenotype in melanoma and might open new avenues towards the discovery of novel therapeutic targets.

Modifications in Human Oral Fibroblast Ultrastructure, Collagen Production, and Lysosomal Compartment in Response to Electronic Cigarette Fluids

BACKGROUND Many smokers have recently turned to electronic cigarettes (e-cigarettes) because they have been marketed as a cheaper, safer smokeless alternative to traditional cigarettes and a possible smoking cessation tool. Although the safety of these electronic devices is still not fully known, there is evidence of their cytotoxicity on cells belonging to the oral cavity. In a previous study by the authors, the increase of reactive oxygen production and Bax expression, followed by the occurrence of apoptosis, was demonstrated in human gingival fibroblasts (HGFs). The aim of this paper is to further investigate the effects of the e-cigarette liquids (with and without nicotine) on the same experimental model. METHODS HGFs were treated with e-cigarette fluids containing nicotine (final concentration 1 mg/mL) and the equivalent volume of a fluid without nicotine, for periods ≤48 hours. Lactate dehydrogenase assay (LDH), electronic microscopy analysis, collagen I production, flow cytometry lysosome compartment evaluation, and western blotting light chain 3 (microtubule-associated protein 1A/1B-LC3) expression were performed. RESULTS Fluids containing nicotine exerted cytotoxicity as demonstrated by increased levels of LDH, in parallel to the presence of numerous vacuoles in the cytoplasm, a decrease in collagen I production, and augmented LC3 II expression. Autophagic vesicles and more procollagen I molecules were present in the cytoplasm of fibroblasts exposed to nicotine-free fluids. In the same samples, time-dependent activation of the lysosomal compartment with no changes in LC3 expression was detected. CONCLUSION E-cigarette fluids (with and without nicotine) trigger molecular and morphologic responses in oral fibroblasts, raising concerns about their role in the pathogenesis of oral diseases.

The thrombopoietin/MPL axis is activated in the Gata1low mouse model of myelofibrosis and is associated with a defective RPS14 signature

Myelofibrosis is characterized by hyperactivation of thrombopoietin signaling which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. Since mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop myelofibrosis1 (Zingariello M. et al. 2015), we investigated whether the thrombopoietin axis is hyperactive in this model. Gata1low mice contained 2-times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (5-times greater than normal) and protein (2-times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the thrombopoietin/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona-fide model of myelofibrosis which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.