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All-trans retinoic acid (ATRA) promotes the development and the function of insulin producing cells and induces partial differentiation of pancreatic tumor cells. A number of evidences clearly indicate that the ATRA mediated signaling may have a substantial role in therapeutic approaches based on restoration of functional β-cells. Among the proteins up-regulated by ATRA, Vav1 is involved in maturation and function of haematopoietic cells and is essential for retinoids induced differentiation of tumor promyelocytes. The presence of Vav1 in solid tissues, including pancreas, is considered ectopic and no role in the differentiation of human epithelial cells has so far been described. We demonstrated here that Vav1 sustains the maturation to β-cells of the normal precursors human Biliary Tree Stem/progenitor Cells (hBTSCs) induced by a differentiation medium containing ATRA and that, in the mature normal pancreas, insulin-producing cells express variable levels of Vav1. Using pancreatic ductal adenocarcinoma (PDAC)-derived cells, we also revealed that the ATRA induced up-modulation of Vav1 is essential for the retinoid-induced trans-differentiation of neoplastic cells into insulin producing cells. The results of this study identify Vav1 as crucial molecule in ATRA induced maturation of insulin producing cells and suggest this protein as a marker for new strategies ended to restore functional β-cells. Graphical abstract.

PU.1 is essential for the differentiation of haemopoietic precursors and is strongly implicated in leukaemogenesis, yet the protein interactions that regulate its activity in different myeloid lineages are still largely unknown. In the present study, by combining fluorescent EMSA (electrophoretic mobility-shift assay) with MS, we reveal the presence of hnRNP K (heterogeneous nuclear ribonucleoprotein K) in molecular complexes that PU.1 forms on the CD11b promoter during the agonist-induced maturation of AML (acute myeloid leukaemia)-derived cells along both the granulocytic and the monocytic lineages. Although hnRNP K and PU.1 act synergistically during granulocytic differentiation, hnRNP K seems to have a negative effect on PU.1 activity during monocytic maturation. Since hnRNP K acts as a docking platform, integrating signal transduction pathways to nucleic acid-directed processes, it may assist PU.1 in activating or repressing transcription by recruiting lineage-specific components of the transcription machinery. It is therefore possible that hnRNP K plays a key role in the mechanisms underlying the specific targeting of protein-protein interactions identified as mediators of transcriptional activation or repression and may be responsible for the block of haemopoietic differentiation.

The multidomain protein Vav1, in addition to promote the acquisition of maturation related properties by normal hematopoietic cells, is a key player in the ATRA- and PMA-induced completion of the differentiation program of tumoral myeloid precursors derived from APL. This review is focussed on the role of Vav1 in differentiating promyelocytes, as part of interconnected networks of functionally related proteins ended to regulate different aspects of myeloid maturation. The role of Vav1 in determining actin cytoskeleton reorganization alternative to the best known function as a GEF for small G proteins is discussed, as well as the binding of Vav1 with cytoplasmic and nuclear signaling molecules which provides a new perspective in the modulation of nuclear architecture and activity. In particular, new hints are provided on the ability of Vav1 to determine the nuclear amount of proteins implicated in modulating mRNA production and stability and in regulating the ATRA-dependent protein expression also by direct interaction with transcription factors known to drive the ATRA-induced maturation of myeloid cells. The reviewed findings summarize the major advances in the understanding of additional, non conventional functions connected with the vast interactive potential of Vav1., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Vav1 is a critical signal transducer for both the development and function of normal hematopoietic cells, in which it regulates the acquisition of maturation-related properties, including adhesion, motility, and phagocytosis. Vav1 is also important for the agonist-induced maturation of acute promyelocytic leukemia (APL)-derived promyelocytes, in which it promotes the acquisition of a mature phenotype by playing multiple functions at both cytoplasmic and nuclear levels. We investigated the possible role of Vav1 in the differentiation of leukemic precursors to monocytes/macrophages. Tumoral promyelocytes in which Vav1 was negatively modulated were induced to differentiate into monocytes/macrophages with phorbol-12-myristate-13-acetate (PMA) and monitored for their maturation-related properties. We found that Vav1 was crucial for the phenotypical differentiation of tumoral myeloid precursors to monocytes/macrophages, in terms of CD11b expression, adhesion capability and cell morphology. Confocal analysis revealed that Vav1 may synergize with actin in modulating nuclear morphology of PMA-treated adherent cells. Our data indicate that, in tumoral promyelocytes, Vav1 is a component of lineage-specific transduction machineries that can be recruited by various differentiating agents. Since Vav1 plays a central role in the completion of the differentiation program of leukemic promyelocytes along diverse hematopoietic lineages, it can be considered a common target for developing new therapeutic strategies for the various subtypes of myeloid leukemias.

Vav1 plays an important role in the all-trans retinoic acid (ATRA)-induced completion of the differentiation program of acute promyelocytic leukemia (APL)-derived cells, in which it strengthens the drug effects and is involved in the regulation of maturation-related proteins, such as the CD11b surface antigen. In both myeloid and lymphoid cells, accumulating data attribute to the multidomain protein Vav1 a functional relevance in the control of gene expression, by direct interaction with chromatin remodeling and/or transcriptional proteins. The present study provides evidence that, in the APL-derived NB4 cell line, Vav1 and the transcription factor PU.1 cooperate in regulating the ATRA-induced CD11b expression. Both chromatin immunoprecipitation (ChIP) experiments and electrophoretic mobility shift assays (EMSA) indicate that Vav1 and PU.1 are recruited to CD11b promoter. Even if the two proteins may participate in diverse protein/DNA complexes, the amounts of complexes including PU.1 seem to be dependent on the interaction of this transcription factor with tyrosine-phosphorylated Vav1. The reported data suggest that the ATRA-induced increase of Vav1 expression and tyrosine phosphorylation may be involved in recruiting PU.1 to its consensus sequence on the CD11b promoter and, ultimately, in regulating CD11b expression during the late stages of neutrophil differentiation of APL-derived promyelocytes.

Unlabelled: Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time., Introduction: The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied., Materials and Methods: Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1beta to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA., Results: Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells., Conclusions: These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.

The differentiation therapy in treatment of acute promyelocytic leukemia (APL), based on the administration of all-trans retinoic acid (ATRA), is currently flanked with the use of As2O3, a safe and effective agent for patients showing a resistance to ATRA treatment. A synergy between ATRA and As3O3 was also reported in inducing granulocytic differentiation of APL-derived cells. We have demonstrated that phospholipase C-beta2 (PLC-beta2), highly expressed in neutrophils and nearly absent in tumoral promyelocytes, largely increases during ATRA treatment of APL-derived cells and strongly correlates with the responsiveness of APL patients to ATRA-based differentiating therapies. Here we report that, in APL-derived cells, low doses of As3O3 induce a slight increase of PLC-beta2 together with a moderate maturation, and cooperate with ATRA to provoke a significant increase of PLC-beta2 expression. Remarkably, the amounts of PLC-beta2 draw a parallel with the differentiation levels reached by both ATRA-responsive and -resistant cells treated with ATRA/As2O3 combinations. PLC-beta2 is not necessary for the progression of tumoral promyelocytes along the granulocytic lineage and is unable to overcome the differentiation block or to potentiate the agonist-induced maturation. On the other hand, since its expression closely correlates with the differentiation level reached by APL-derived cells induced to maturate by drugs presently employed in APL therapies, PLC-beta2 represents indeed a specific marker to test the ability of differentiation agents to induce the release of the maturation blockade of tumoral myeloid precursors.

Vav is one of the genetic markers that correlate with the differentiation of hematopoietic cells. In T and B cells, it appears crucial for both development and functions, while, in non-lymphoid hematopoietic cells, Vav seems not involved in cell maturation, but rather in the response of mature cells to agonist-dependent proliferation and phagocytosis. We have previously demonstrated that the amount and the tyrosine phosphorylation of Vav are up-regulated in both whole cells and nuclei of tumoral promyelocytes induced to granulocytic maturation by ATRA and that tyrosine-phosphorylated Vav does not display any ATRA-induced GEF activity but contributes to the regulation of PI 3-K activity. In this study, we report that Vav accumulates in nuclei of ATRA-treated APL-derived cells and that the down-modulation of Vav prevents differentiation of tumoral promyelocytes, indicating that it is a key molecule in ATRA-dependent myeloid maturation. On the other hand, the overexpression of Vav induces an increased expression of surface markers of granulocytic differentiation without affecting the maturation-related changes of the nuclear morphology. Consistent with an effect of Vav on the transcriptional machinery, array profiling shows that the inhibition of the Syk-dependent tyrosine phosphorylation of Vav reduces the number of ATRA-induced genes. Our data support the unprecedented notion that Vav plays crucial functions in the maturation process of myeloid cells, and suggest that Vav can be regarded as a potential target for the therapeutic treatment of myeloproliferative disorders.

In this study, we have investigated the expression of phospholipase C-beta2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-beta2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-beta2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-beta2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-beta2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-beta2 levels can predict the in vivo responsiveness to ATRA of APL patients.

Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-beta II migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the alpha isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-beta II occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC-alpha to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular significance in light of the proposed use of pharmacological inhibitors of PKC-dependent biochemical pathways for the therapy of cancer disease.

Despite the fact that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptors (TRAIL-Rs) are expressed in intestinal mucosa, little is known about the biological role of this system in intestinal cell physiology. The expression of surface TRAIL and TRAIL-R1, -R2, -R3, -R4 were examined by flow cytometry in the immortalized human cell line tsFHI under culture conditions promoting growth or growth arrest and expression of differentiated traits. A progressive increase of surface TRAIL expression paralleled tsFHI differentiation, consistently with immunohistochemistry analysis showing an increase of TRAIL immunostaining along the crypt–villus axis in normal jejuneal mucosa. In spite of the presence of TRAIL-R1 and TRAIL-R2 “death receptors,” recombinant TRAIL was not cytotoxic for tsFHI cells. Exposure of tsFHI to recombinant TRAIL rather increased/anticipated the expression levels of the cyclin-dependent kinase inhibitors p21 and p27, which mediate the induction of growth arrest and the stabilization of differentiated traits, respectively, as well as of the canonical differentiation marker DPPIV. The differentiation inducing activity of TRAIL was abolished by pre-incubation with a Fc-TRAIL-R2 chimera. On the other hand, TRAIL did not significantly modulate the levels of osteoprotegerin (OPG), CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP10 spontaneously released or induced by inflammatory cytokines. Taken together, these data suggest that TRAIL might act as a paracrine trophic cytokine on intestinal epithelium, promoting intestinal cell differentiation. J.Cell.Physiol. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Lipid second messengers, particularly those derived from the polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Phosphoinositide 3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in a plethora of cell functions. One of the best-characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt. Recent findings have implicated Akt in cancer progression because it stimulates cell proliferation and suppresses apoptosis. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid molecules are important components of signaling pathways operating within the nucleus. PI3Ks, their lipid products, and Akt have also been identified at the nuclear level. In this review, we shall summarize the most updated findings about these molecules in relationship with the nuclear compartment and provide an overview of the possible mechanisms by which they regulate important cell functions. [Copyright &y& Elsevier]

Abstract: Vav1 is a key molecule in the ATRA-induced acquisition of a mature phenotype by tumoral myeloid precursors. Since ATRA acts throughout events that require extensive changes of nuclear architecture and activity and considering that Vav1 accumulates inside the nuclear compartment of differentiating APL-derived cells, the possible role of this protein in modulating the nuclear proteome was investigated. Membrane-depleted nuclei purified from NB4 cells induced to differentiate with ATRA in the presence of forcedly down-modulated Vav1 were subjected to 2D-DIGE followed by mass spectra analysis. The obtained data demonstrated that, in NB4 cells treated with ATRA, Vav1 is involved in determining the nuclear amount of proteins involved in molecular complexes with DNA and may participate to RNA processing by carrying in the nucleus molecules involved in modulating mRNA production and stability, like hnRNPs and SR proteins. Our results provide the first evidence that, at least in maturation of tumoral myeloid precursors, Vav1 is part of interconnected networks of functionally related proteins ended to regulate different aspects of gene expression. Since defects in mRNA processing are common in tumor development, our data suggest that Vav1 is a potential target molecule for developing new anti-cancer strategies. [Copyright &y& Elsevier]

Abstract: Erythroid differentiation of human erythroleukemia cell line K562 induced by erythropoietin is a complex process that involves modifications at nuclear level, including nuclear translocation of phosphatidyl-inositol 3-kinase. In this work we show that erythropoietin stimulation of K562 cells can induce nuclear translocation of active Akt, a downstream molecule of the phosphatidyl-inositol 3-kinase signaling pathway. Akt shows a peak of activity in whole cell homogenates at earlier stage when compared to the nucleus, which shows a peak delayed of 10min. Akt increases its intranuclear amount and activity rapidly and transiently in response to EPO. Almost all Akt kinase that translocates to the nucleus shows a marked phosphorylation on serine 473. Nuclear enzyme translocation is blocked by the phosphatidyl-inositol 3-kinase inhibitor Ly294002 or Wortmannin. The specific Akt pharmacological inhibitor VI, VII and VIII that act as blocking enzyme activation inhibited translocation as well, whereas Akt inhibitor IX, that inhibits Akt activity, did not block Akt nuclear translocation. When cells were treated by means of siRNA sequences or with the Akt inhibitors the differentiation process was arrested, thus showing the requirement of the nuclear translocation of the active enzyme to differentiate. These findings strongly suggest that the intranuclear translocation of active Akt kinase represents an important step in the signaling pathway that mediates erythropoietin-induced erythroid differentiation. [Copyright &y& Elsevier]

The importance of the Vav family of signal transduction molecules in hematopoietic cells has long been acknowledged, even though its role and its regulatory mechanism are not completely understood. We have previously demonstrated that tyrosine-phosphorylated Vav, also located inside the nucleus of myeloid cells, is up-regulated during maturation of promyelocytic precursors induced by all-trans-retinoic acid (ATRA). Here, we report that the tyrosine phosphorylation of Vav during granulocytic maturation is dependent on the tyrosine kinase Syk and is essential for the morphological changes of the cell nucleus. These ATRA-induced events are independent on the guanine nucleotide exchange activity of Vav. We also found that, in differentiating cells, and in both cytoplasmic and nuclear compartments, tyrosine phosphorylated Vav associates with the regulatory subunit of phosphoinositide 3-kinase (PI 3-K). The Vav/p85 interaction is essential for the ATRA-induced PI 3-K activity and for association of PI 3-K with actin, particularly in the nucleus.Our data indicate an unprecedented crucial function for Vav in modulating the morphological maturation process of myeloid cells in a GDP–GTP exchange factor (GEF)-independent manner and suggest a role of Vav as an adaptor protein responsible of targeting PI 3-K to its intranuclear substrates. [Copyright &y& Elsevier]