Physiological expression of miR-130a during differentiation of CD34+ human hematopoietic stem cells results in the inhibition of monocyte differentiation.

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

Wnt/CTNNB1 Signal Transduction Pathway Inhibits the Expression of ZFP36 in Squamous Cell Carcinoma, by Inducing Transcriptional Repressors SNAI1, SLUG and TWIST.

The Wnt/CTNNB1 pathway is often deregulated in epithelial tumors. The ZFP36 gene, encoding the mRNA binding protein Tristetraprolin (TTP), is downregulated in several cancers, where it has been described to behave as a tumor suppressor. By this report, we show that Wnt/CTNNB1 pathway is constitutively activated, and ZFP36 expression is downregulated in Squamous Cell Carcinoma (SCC) cell lines compared to normal keratinocytes. Moreover, we suggest that the decrease of ZFP36 expression might depend on the activity of transcriptional repressors SNAI1, SLUG and TWIST, whose expression is induced by Wnt/CTNNB1, highlighting a potential regulatory mechanism underlying ZFP36 downregulation in epithelial cancers.

From Osteoclast Differentiation to Osteonecrosis of the Jaw: Molecular and Clinical Insights.

Bone physiology relies on the delicate balance between resorption and formation of its tissue. Bone resorption depends on a process called osteoclastogenesis in which bone-resorbing cells, i.e., osteoclasts, are produced by the differentiation of more undifferentiated progenitors and precursors. This process is governed by two main factors, monocyte colony-stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). While the former exerts a proliferating effect on progenitors/precursors, the latter triggers a differentiation effect on more mature cells of the same lineage. Bone homeostasis requires a perfect space-time coordination of the involved signals. When osteoclastogenesis is poorly balanced with the differentiation of the bone forming counterparts, i.e., osteoblasts, physiological bone remodelling can turn into a pathological state, causing the systematic disruption of bone tissue which results in osteopenia or osteolysis. Examples of these conditions are represented by osteoporosis, Paget's disease, bone metastasis, and multiple myeloma. Therefore, drugs targeting osteoclastogenesis, such as bisphosphonates and an anti-RANKL monoclonal antibody, have been developed and are currently used in the treatment of such diseases. Despite their demonstrated therapeutic efficacy, these agents are unfortunately not devoid of side effects. In this regard, a condition called osteonecrosis of the jaw (ONJ) has been recently correlated with anti-resorptive therapy. In this review we will address the involvement of osteoclasts and osteoclast-related factors in the pathogenesis of ONJ. It is to be hoped that a better understanding of the biological mechanisms underlying bone remodelling will help in the design a medical therapeutic approach for ONJ as an alternative to surgical procedures.

Magnesium Is a Key Regulator of the Balance between Osteoclast and Osteoblast Differentiation in the Presence of Vitamin D3.

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D3 (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.

MafB is a downstream target of the IL-10/STAT3 signaling pathway, involved in the regulation of macrophage de-activation.

In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-7R genes.

ZFP36 stabilizes RIP1 via degradation of XIAP and cIAP2 thereby promoting ripoptosome assembly.

BACKGROUND: ZFP36 is an mRNA binding protein that exerts anti-tumor activity in glioblastoma by triggering cell death, associated to an increase in the stability of the kinase RIP1. METHODS: We used cell death assays, size exclusion chromatography, Co-Immunoprecipitation, shRNA lentivectors and glioma neural stem cells to determine the effects of ZFP36 on the assembly of a death complex containing RIP1 and on the induction of necroptosis. RESULTS: Here we demonstrate that ZFP36 promotes the assembly of the death complex called Ripoptosome and induces RIP1-dependent death. This involves the depletion of the ubiquitine ligases cIAP2 and XIAP and leads to the association of RIP1 to caspase-8 and FADD. Moreover, we show that ZFP36 controls RIP1 levels in glioma neural stem cell lines. CONCLUSIONS: We provide a molecular mechanism for the tumor suppressor role of ZFP36, and the first evidence for Ripoptosome assembly following ZFP36 expression. These findings suggest that ZFP36 plays an important role in RIP1-dependent cell death in conditions where IAPs are depleted.

The Orosomucoid 1 protein is involved in the vitamin D - mediated macrophage de-activation process.

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

The Capacity of Magnesium to Induce Osteoclast Differentiation Is Greatly Enhanced by the Presence of Zoledronate.

Bisphosphonates (BPs) are successfully used to cure a number of diseases characterized by a metabolic reduction in bone density, such as Osteoporosis, or a neoplastic destruction of bone tissue, such as multiple myeloma and bone metastases. These drugs exert their therapeutic effect by causing a systemic osteoclast depletion that, in turn, is responsible for reduced bone resorption. Unfortunately, in addition to their beneficial activity, BPs can also determine a frightening side effect known as osteonecrosis of the jaw (ONJ). It is generally believed that the inability of osteoclasts to dispose of inflamed/necrotic bone represents the main physiopathological aspect of ONJ. In principle, a therapeutic strategy able to elicit a local re-activation of osteoclast production could counteract ONJ and promote the healing of its lesions. Using an experimental model of Vitamin D3-dependent osteoclastogenesis, we have previously demonstrated that Magnesium is a powerful inducer of osteoclast differentiation. Here we show that, surprisingly, this effect is greatly enhanced by the presence of Zoledronate, chosen for our study because it is the most effective and dangerous of the BPs. This finding allows us to hypothesize that Magnesium might play an important role in the topical therapy of ONJ.

Pulsed Electro-Magnetic Field (PEMF) Effect on Bone Healing in Animal Models: A Review of Its Efficacy Related to Different Type of Damage.

Biophysical energies are a versatile tool to stimulate tissues by generating biopotentials. In particular, pulsed electromagnetic field (PEMF) stimulation has intrigued researchers since the 1970s. To date, many investigations have been carried out in vivo, but a gold standard treatment protocol has not yet been defined. The main obstacles are represented by the complex setting of PEMF characteristics, the variety of animal models (including direct and indirect bone damage) and the lack of a complete understanding of the molecular pathways involved. In the present review the main studies about PEMF stimulation in animal models with bone impairment were reviewed. PEMF signal characteristics were investigated, as well as their effect on molecular pathways and osseous morphological features. We believe that this review might be a useful starting point for a prospective study in a clinical setting. Consistent evidence from the literature suggests a potential beneficial role of PEMF in clinical practice. Nevertheless, the wide variability of selected parameters (frequency, duration, and amplitude) and the heterogeneity of applied protocols make it difficult to draw certain conclusions about PEMF effectiveness in clinical implementation to promote bone healing. Deepening the knowledge regarding the most consistent results reported in literature to date, we believe that this review may be a useful starting point to propose standardized experimental guidelines. This might provide a solid base for further controlled trials, to investigate PEMF efficacy in bone damage conditions during routine clinical practice.

Targeting Chemotherapy to Decondensed H3K27me3-Marked Chromatin of AML Cells Enhances Leukemia Suppression.

Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death-promoting and growth-inhibitory genes.In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities. SIGNIFICANCE: Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy.See related commentary by Adema and Colla, p. 359.

Magnesium favors the capacity of vitamin D3 to induce the monocyte differentiation of U937 cells.

The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized.

Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis.

Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients' cohort, it could be used for further studies to design an updated classification model for MF patients.

The vitamin D3/Hox-A10 pathway supports MafB function during the monocyte differentiation of human CD34+ hemopoietic progenitors.

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34(+) hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D(3) monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D(3) receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D(3)/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

TFE3 transcription factor regulates the expression of MAFB during macrophage differentiation.

Transcription Factor for Immunoglobulin Heavy-Chain Enhancer 3 (Tfe3) is a transactivator of metabolic genes that are regulated through an EBox located in their promoters. It is involved in physiological processes such as osteoclast and macrophage differentiation, as well as in pathological processes such as translocations underlying different cancer diseases. MAFB is a basic region/leucine zipper transcription factor that affects transcription by binding specific DNA regions known as MARE. It plays a pivotal role in regulating lineage-specific hematopoiesis by repressing transcription of erythroid specific genes in myeloid cells and enhancing expression of macrophage and megakaryocytic genes. Here we have shown MAFB to be highly induced in human hematopoietic cells undergoing macrophage differentiation following Tfe3 ectopic expression, and to be down regulated, compared to the controls, in the same cell population following Phorbol Esters (PMA) dependent differentiation coupled to Tfe3 gene silencing. Electrophoretic mobility shift assays identified a Tfe3-binding site (EBox) in the MAFB promoter region that is conserved in different mammalian species. MAFB promoter was transactivated by co-expression of Tfe3 in reporter gene assays while deletion or mutation of the MAFB EBox prevented transactivation by Tfe3. Both of these genes were previously included in the group of transcription factors able to drive macrophage differentiation. The observation that MAFB belongs to the Tfe3 regulon suggests the existence of a pathway where these two gene families act synergistically to determine differentiation.

Cadherins down-regulation: towards a better understanding of their relevance in colorectal cancer.

The down-regulation of cadherin expression in colorectal cancer (CRC) has been widely studied. However, existing data on cadherin expression are highly variable and its relevance to CRC development has not been completely established. This review examines published studies on cadherins whose down-regulation has been already demonstrated in CRC, trying to establish a relationship with promoter methylation, the capacity to influence the Wnt / CTNNB1 (catenin beta 1, beta-catenin) signalling pathway and the clinical implications for disease outcome. Moreover, it also analyses factors that may explain data variability and highlights the importance of considering the altered subcellular localization of the examined cadherins. The results of this survey reveal that thirty of one hundred existing cadherins appear to be down-regulated in CRC. Among these, ten are cadherins, sixteen are protocadherins, equally divided between clustered and non clustered, and four are cadherin - related. These findings suggest that, to better define the role played by cadherin down-regulation in CRC pathogenesis, the expression of multiple rather than individual cadherins should be taken into account and further functional studies are necessary to clarify the relative ability of individual cadherins to inhibit CTNNB1 therefore acting as tumor suppressors.

Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts.

The mRNA-destabilizing protein tristetraprolin (TTP), encoded by the ZFP36 gene, is known to be able to end inflammatory responses by directly targeting and destabilizing mRNAs encoding pro-inflammatory cytokines. We analyzed its role in psoriasis, a disease characterized by chronic inflammation. We observed that TTP is downregulated in fibroblasts deriving from psoriasis patients compared to those deriving from healthy individuals and that psoriatic fibroblasts exhibit abnormal inflammasome activity compared to their physiological counterpart. This phenomenon depends on TTP downregulation. In fact, following restoration, TTP is capable of directly targeting for degradation NLRP3 mRNA, thereby drastically decreasing inflammasome activation. Moreover, we provide evidence that ZFP36 undergoes methylation in psoriasis, by virtue of the presence of long stretches of CpG dinucleotides both in the promoter and the coding region. Besides confirming that a perturbation of TTP expression might underlie the pathogenesis of psoriasis, we suggest that deregulated inflammasome activity might play a role in the disease alongside deregulated cytokine expression.

PPARdelta is a ligand-dependent negative regulator of vitamin D3-induced monocyte differentiation.

A number of reports indicate that peroxisome proliferator-activated receptor (PPAR) delta is involved in the molecular control of monocyte-macrophage differentiation. In this regard, the recent demonstration that PPARdelta is a primary response gene of 1alpha,25-dihydroxyvitamin D3 (VD), i.e. a powerful inducer of such process, allowed us to hypothesize the existence of a cross talk between PPARdelta and VD receptor pathways. To address this issue, we analyzed the effects promoted by stimulation with PPARdelta ligands and by overexpression of this nuclear receptor in monoblastic cell lines undergoing exposure to VD. The results obtained evidenced that, although promoting a weak differentiation effect by themselves, PPARdelta ligands efficiently co-operated with VD treatment. In spite of this, PPARdelta overexpression exerted a remarkable inhibitory effect on monocyte-macrophage differentiation induced by VD that was, at least partly, reverted by stimulation with a highly specific PPARdelta ligand. These data indicate that, although acting through a ligand-dependent modality, PPARdelta is a negative regulator of VD-mediated monocyte differentiation, allowing us to hypothesize a role of the investigated nuclear receptor in the differentiation block of M5 type (monoblastic) acute myeloid leukemias (AMLs). Bioinformatic analysis of a microarray database, containing the expression profiles of 285 AML cases, further supported this hypothesis demonstrating the existence of a subset of M5 type (monoblastic) AMLs that overexpress PPARdelta gene.

Loss of expression of µ-protocadherin and protocadherin-24 in sporadic and hereditary nonpolyposis colorectal cancers.

Colorectal cancer (CRC) is a neoplastic disease in which normal mucosa undergoes a process of malignant transformation due to the progressive accumulation of molecular alterations affecting proto-oncogenes and oncosuppressor genes. Some of these modifications exert their carcinogenic potential by promoting a constitutive activation of the ß-catenin signaling proliferation pathway, and when present, loss of cadherin expression also significantly contributes to the same effect. Using a combined approach of molecular and immunohistochemical analysis, we have previously demonstrated that most sporadic CRCs exhibit a down-regulated expression of a cadherin, named µ-protocadherin, that is generally observed in association with a higher proliferation rate and a worse prognosis. The aim of this report was to perform a comparative immunohistochemical assessment of µ-protocadherin and a similar cadherin, named protocadherin-24, in sporadic CRC and hereditary nonpolyposis colorectal cancer. The data obtained put in evidence that double-negative CRCs, lacking both the analyzed protocadherins, are more represented among sporadic tumors, whereas double-positive CRCs, maintaining their expression, exhibit an opposite trend. As expected, loss of protocadherin expression was accompanied by nuclear localization of ß-catenin and increased positivity of the Ki-67 proliferation marker. This finding is consistent with the different clinical evolution of the 2 considered CRC sets according to which patients with hereditary nonpolyposis colorectal cancer experience a better prognosis as compared with those affected by a sporadic CRC.

KLF4 Mediates the Effect of 5-ASA on the ß-Catenin Pathway in Colon Cancer Cells.

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing µ-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores µ-protocadherin expression and promotes the sequestration of ß-catenin to the plasma membrane. Here, we show that 5-ASA-induced µ-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA-mediated ß-catenin inhibition is caused by µ-protocadherin-dependent sequestration of ß-catenin to the plasma membrane and by the direct binding of KLF4 to ß-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA. Cancer Prev Res; 11(8); 503-10. ©2018 AACR.

Loss of ZFP36 expression in colorectal cancer correlates to wnt/ ß-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of ZEB1, SOX9 and MACC1.

The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/ß-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.