Iron overload enhances human mesenchymal stromal cell growth and hampers matrix calcification.

BACKGROUND: Iron overload syndromes include a wide range of diseases frequently associated with increased morbidity and mortality. Several organs are affected in patients with iron overload including liver, heart, joints, endocrine glands, and pancreas. Moreover, severe bone and hemopoietic tissue alterations are observed. Because of the role of bone marrow mesenchymal stromal cells (BM-MSCs) in bone turnover and hematopoiesis, iron effects on primary BM-MSCs cultures were evaluated. METHODS: Primary human BM-MSCs cultures were prepared and the effects of iron on their proliferation and differentiation were characterized by biochemical analyses and functional approaches. RESULTS: Addition of iron to the culture medium strongly increased BM-MSCs proliferation and induced their accelerated S phase entry. Iron enters BM-MSCs through both transferrin-dependent and transferrin-independent mechanisms, inducing the accumulation of cyclins E and A, the decrease of p27(Kip1), and the activation of MAPK pathway. Conversely, neither apoptotic signs nor up-regulation of reactive oxygen species were observed. Iron inhibited both differentiation of BM-MSCs into osteoblasts and in vitro matrix calcification. These effects result from the merging of inhibitory activities on BM-MSCs osteoblastic commitment and on the ordered matrix calcification process. CONCLUSIONS: We demonstrated that BM-MSCs are a target of iron overload. Iron accelerates BM-MSCs proliferation and affects BM-MSCs osteoblastic commitment, hampering matrix calcification. GENERAL SIGNIFICANCE: Our study reports, for the first time, that iron, at concentration found in overloaded patient sera, stimulates the growth of BM-MSCs, the BM multipotent stromal cell component. Moreover, iron modulates the physiological differentiation of these cells, affecting bone turnover and remodeling.

Resveratrol: from basic studies to bedside.

Plants produce a remarkable amount of low molecular mass natural products endowed with a large array of pivotal biological activities. Among these molecules, resveratrol (3,5,4'-trihydroxystilbene) has been identified as an important modulator of cell phenotype with a complex and pleiotropic mode of action. Extensive literature regarding its activity, mainly employing cellular models, suggests that this polyphenol controls cell proliferation, induces differentiation, and activates apoptosis and autophagy. The compound also modulates angiogenesis and inflammation. Similarly, studies on implanted cancers and chemical-induced tumors confirm the potential chemotherapeutical interest of the compound. Likewise, several reports clearly demonstrated, in animal models, that the compound might positively affect the development and evolution of chronic diseases including type 2 diabetes, obesity, coronary heart disease, metabolic syndrome, and neurogenerative pathologies. Finally, a number of investigations stated that the toxicity of the molecule is scarce. Despite these promising observations, few clinical trials have yet been performed to evaluate the effectiveness of the molecule both in prevention and treatment of human chronic disease. Preliminary findings therefore suggest the need for more extensive clinical investigations.

A cancer-associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss-of-function.

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27Kip1 , a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer-associated germinal p27Kip1 variant, namely glycine9->arginine-p27Kip1 (G9R-p27Kip1 ) identified in a parathyroid adenoma. We unexpectedly found that G9R-p27Kip1 lacks the major tumor suppressor activities of p27Kip1 including its antiproliferative and pro-apoptotic functions. In addition, G9R-p27Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild-type p27Kip1 -transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R-p27Kip1 on S12, a residue normally never found modified in p27Kip1 . The novel S12 phosphorylation appears responsible for the loss of function of G9R-p27Kip1 since S12AG9R-p27Kip1 recovers most of the p27Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D-p27Kip1 recapitulates G9R-p27Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin-dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post-translational modifications due to generation of novel consensus sequences by cancer-associated missense mutations.

Histone deacetylase inhibitors upregulate p57Kip2 level by enhancing its expression through Sp1 transcription factor.

Histone deacetylase inhibitors (HDACIs) represent a new class of targeted anticancer agents. Here, we evaluate the effects of butyrate (BuA) and other HDACIs on p57(Kip2), a cyclin-dependent kinase inhibitor (cki). We observed that inhibitors of class I/II histone deacetylases (HDACs), but not of class III HDACs, induce a remarkable accumulation of p57(Kip2) in several cells. The cki upregulation is associated with an increased gene expression that was not prevented by cycloheximide, indicating that HDACIs affect directly p57(Kip2) transcription. The characterization of p57(Kip2) promoter indicates that the first 165 bp are mostly involved in the BuA effects. Chromatin immunoprecipitation studies demonstrated that the BuA treatment causes the recruitment of Sp1 transcription factor. The Sp1 importance was confirmed by the reduction of BuA effects by mithramycin A (an Sp1 antagonist) and, most stringently, by Sp1 downregulation due to Sp1 siRNA. Moreover, both the treatments reduce the p57(Kip2) transcription in untreated cells, suggesting that Sp1 is required for the constitutive cki expression. Studies employing plasmids containing parts of the 165 bp of p57(Kip2) promoter indicate that the promoter region between -87 and -113 bp, which includes two putative Sp1 consensus sequences, plays a critical role in the response to HDACIs. Since this p57(Kip2) promoter region also embraces the consensus sequence for the transcriptional repressor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2), we evaluated whether this factor is involved into the BuA effect. When CTIP2 was downregulated by a specific siRNA, we observed the enhancement of BuA activity on p57(Kip2) expression suggesting that CTIP2 might also be involved in HDACIs effects.

Histone Deacetylase Inhibitors Increase p27(Kip1) by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation.

Histone deacetylase inhibitors (HDACIs) represent an intriguing class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important clinical activities at well tolerated doses. HDACIs also interfere with the aging process and are involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation, and apoptosis. Here, we evaluated the effects of HDACIs on p27(Kip1), a key cyclin-dependent kinase inhibitor (CKI). We observed that HDACI-dependent antiproliferative activity is associated with p27(Kip1) accumulation due to a reduced protein degradation. p27(Kip1) removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27(Kip1) stability through downregulation of Skp2 protein levels. Skp2 decline is only partially due to a reduced Skp2 gene expression. Conversely, the protein decrease is more profound and enduring compared to the changes of Skp2 transcript. This argues for HDACIs effects on Skp2 protein posttranslational modifications and/or on its removal. In summary, we demonstrate that HDACIs increase p27(Kip1) by hampering its nuclear ubiquitination/degradation. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities.

Resveratrol mimics insulin activity in the adipogenic commitment of human bone marrow mesenchymal stromal cells.

Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent cells capable of differentiating toward osteoblatic and adipocytic phenotypes. BM-MSCs play several key roles including bone remodeling, establishment of hematopoietic niche and immune tolerance induction. Here, we investigated the effect of resveratrol (RSV), a therapeutically promising natural polyphenol, on the commitment of human BM-MSCs primary cultures. Cell differentiation was evaluated by means of morphological analysis, specific staining and expression of osteogenic and adipocytic master genes (Runx-2, PPARγ). To maintain BM-MSC multipotency, all experiments were performed on cells at very early passages. At any concentration RSV, added to standard medium, did not affect the phenotype of confluent BM-MSCs, while, when added to osteogenic or adipogenic medium, 1 µM RSV enhances the differentiation toward osteoblasts or adipocytes, respectively. Conversely, the addition of higher RSV concentration (25 µM) to both differentiation media resulted exclusively in BM-MSCs adipogenesis. Surprisingly, the analysis of RSV molecular effects demonstrated that the compound completely substitutes insulin, a key component of adipogenic medium. We also observed that RSV treatment is associated to enhanced phosphorylation of CREB, a critical effector of insulin adipogenic activity. Finally, our observations contribute to the mechanistic elucidation of the well-known RSV positive effect on insulin sensitivity and type 2 diabetes mellitus.

P27Kip1 serine 10 phosphorylation determines its metabolism and interaction with cyclin-dependent kinases.

p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its "adaptability" to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.

Resveratrol and cancer treatment: is hormesis a yet unsolved matter?

Plants produce many low molecular mass natural compounds endowed with biological activity. Among them, resveratrol (3,5,4'-trihydroxystilbene) has been demonstrated to be able to affect a plethora of pivotal cellular molecular processes, including transduction pathways and gene expression. These activities result, in turn, in several different cell phenotypes. Particularly, frequent effects of resveratrol treatment appear to be the reduction of growth and the activation of programmed cell death. Accordingly, a number of trials are currently under development to evaluate the possibility of using resveratrol in cancer therapy, both as single agent or in association with other anticancer compounds. However, some reports suggest that, at low concentrations, not only resveratrol does not inhibit the proliferation and/or the survival of cells but, conversely, it induces proliferation and/or protects cells against toxic agents. On the basis of these biphasic effects, it has been proposed that resveratrol belongs to the so-called hormetic compounds. Hormesis is an expression employed by toxicologists to describe a U-shaped (or J-shaped) dose response characterized by a beneficial effect at low doses and a toxic (or inhibitory) activity at high dose. In this review, we will reappraise data that might suggest or disprove that resveratrol is endowed with clear hormetic properties.

p57(Kip2) and cancer: time for a critical appraisal.

p57(Kip2) is a cyclin-dependent kinase inhibitor belonging to the Cip/Kip family, which also includes p21(Cip1) and p27(Kip1). So far, p57(Kip2) is the least-studied Cip/Kip protein, and for a long time its relevance has been related mainly to its unique role in embryogenesis. Moreover, genetic and molecular studies on animal models and patients with Beckwith-Wiedemann syndrome have shown that alterations in CDKN1C (the p57(Kip2) encoding gene) have functional relevance in the pathogenesis of this disease. Recently, a number of investigations have identified and characterized heretofore unexpected roles for p57(Kip2). The protein appears to be critically involved in initial steps of cell and tissue differentiation, and particularly in neuronal development and erythropoiesis. Intriguingly, p27(Kip1), the Cip/Kip member that is most homologous to p57(Kip2), is primarily involved in the process of cell cycle exit. p57(Kip2) also plays a critical role in controlling cytoskeletal organization and cell migration through its interaction with LIMK-1. Furthermore, p57(Kip2) appears to modulate genome expression. Finally, accumulating evidence indicates that p57(Kip2) protein is frequently downregulated in different types of human epithelial and nonepithelial cancers as a consequence of genetic and epigenetic events. In summary, the emerging picture is that several aspects of p57(Kip2)'s functions are only poorly clarified. This review represents an appraisal of the data available on the p57(Kip2) gene and protein structure, and its role in human physiology and pathology. We particularly focus our attention on p57(Kip2) changes in cancers and pharmacological approaches for modulating p57(Kip2) levels.

The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells.

BACKGROUND: The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs), the knowledge of their effects on normal cells is of pivotal importance. DESIGN AND METHODS: We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs). RESULTS: Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase. CONCLUSIONS: Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.

Targeting p27Kip1 protein: its relevance in the therapy of human cancer.

INTRODUCTION: Cell division cycle progression is achieved by a sequential and stringently concerted activation of a family of serine-threonine kinases, namely the cyclin-dependent kinases (CDKs). p27(Kip1) is a pivotal CDK inhibitor and a tight modulator of CDK-dependent phenotypes. Thus, p27(Kip1) plays a fundamental role in key cellular processes such as proliferation, differentiation, apoptosis, substrate adhesion and motility. Intriguingly, when p27(Kip1) is localized in the nucleus, it acts as an antiproliferative protein, while, in the cytosol, p27(Kip1) promotes cytoskeleton remodeling and might positively influence metastatization. Downregulation of p27(Kip1) nuclear level or its cytosolic mislocalization are consistently correlated with poor prognosis of numerous types of human epithelial and non-epithelial cancers. AREAS COVERED: This review illustrates the basic structural features of p27(Kip1) protein, its metabolism and alterations in human malignancies, along with describing anticancer strategies based on targeting p27(Kip1). EXPERT OPINION: Given the role of p27(Kip1) in the control of cell proliferation and its decreased level observed in malignancies with poor outcome, drugs able to handle the protein levels and localization might represent an important goal for novel specific and effective anticancer strategies. Although no convincing proofs have been reported, putative negative consequences of p27(Kip1) targeting might be also conceivable.

EPO receptor gain-of-function causes hereditary polycythemia, alters CD34 cell differentiation and increases circulating endothelial precursors.

BACKGROUND: Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. METHODOLOGY/PRINCIPAL FINDINGS: We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G-->T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34(+) cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. CONCLUSIONS/SIGNIFICANCE: Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.