In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells.

Several recent studies have demonstrated that the direct precursor of vitamin D3, the calcifediol [25(OH)D3], through the binding to the nuclear vitamin D receptor (VDR), is able to regulate the expression of many genes involved in several cellular processes. Considering that itself may function as a VDR ligand, although with a lower affinity, respect than the active form of vitamin D, we have assumed that 25(OH)D3 by binding the VDR could have a vitamin's D3 activity such as activating non-genomic pathways, and in particular we selected mesenchymal stem cells derived from human adipose tissue (hADMSCs) for the in vitro assessment of the intracellular Ca2+ mobilization in response to 25(OH)D3. Our result reveals the ability of 25(OH)D3 to activate rapid, non-genomic pathways, such as an increase of intracellular Ca2+ levels, similar to what observed with the biologically active form of vitamin D3. hADMSCs loaded with Fluo-4 AM exhibited a rapid and sustained increase in intracellular Ca2+ concentration as a result of exposure to 10-5 M of 25(OH)D3. In this work, we show for the first time the in vitro ability of 25(OH)D3 to induce a rapid increase of intracellular Ca2+ levels in hADMSCs. These findings represent an important step to better understand the non-genomic effects of vitamin D3 and its role in endocrine system.

Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line.

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

In Vitro Control of Genes Critical for Parathyroid Embryogenesis by Extracellular Calcium.

BACKGROUND: The expression of the parathyroid transcription factors, encoded by the genes GATA3, GCM2, and MAFB, persists after parathyroid morphogenesis. This suggests a role of these genes in the regulatory program that governs parathyroid function in the adult. Indeed, these 3 genes form a transcriptional cascade able to activate PTH gene expression. MATERIALS AND METHODS: Adult adenoma parathyroid tissues were put in primary cell culture to evaluate the messenger ribonucleic acid (mRNA) expression of the PTH gene, of the genes involved in the calcium regulatory signaling pathway (CaSR, GNA11, and AP2S1), and of the 3 genes (GATA3, GCM2, and MAFB) involved in the parathyroid morphogenesis in the presence of different extracellular calcium concentrations from 0.1 mM to 3.0 mM. AIM: The aim of the study was to investigate whether different extracellular calcium conditions could control the expression of transcription factors critical for parathyroid embryogenesis. RESULTS: The results of the experiments showed that the mRNA expression of GATA3, GCM2, and MAFB genes follows the same response as the PTH gene to extracellular calcium concentrations, with the highest expression at low calcium (0.1 mM) and the lowest at high calcium (3.0 mM). Conversely, the genes involved in the calcium signaling in the parathyroid cells showed a variable response to the extracellular calcium concentrations, with the CaSR and GNA11 genes exhibiting a sensitivity to low calcium concentrations. CONCLUSIONS: These findings indicate that transcription factors recognized for their role in parathyroid embryogenesis show a response to extracellular calcium later in adulthood that parallels the behavior of the PTH gene.

Analysis of differentially expressed microRNAs in MEN1 parathyroid adenomas.

Multiple Endocrine Neoplasia type 1 (MEN1) syndrome is a rare complex tumor-predisposing hereditary disorder, inherited in an autosomal dominant manner (OMIM 131100). MEN1 is characterized by tumors of the parathyroids, the neuroendocrine cells of the gastro-entero-pancreatic tract, and the anterior pituitary. The molecular mechanisms that control parathyroid tumorigenesis are still poorly understood. Here we studied the global microRNAs (miRNAs) expression profile in MEN1 parathyroid adenomas to understand the role of these regulatory factors in MEN1 parathyroid tumorigenesis. miRNA arrays containing 1890 human miRNAs were used to profile seven different MEN1 parathyroid adenomas (four presenting somatic loss of heterozygosity (LOH) at 11q13 and three still retaining one wild type copy of the MEN1 gene). Eight miRNAs in non-LOH MEN1 parathyroid adenomas and two miRNAs in LOH MEN1 parathyroid adenomas resulted to be differentially expressed, with a significant fold change, with respect to the control pool. Six microRNAs also resulted to be differentially expressed between LOH MEN1 parathyroid adenomas and non-LOH MEN1 parathyroid adenomas. Significantly differentially expressed miRNAs were all validated by SYBR green real-time quantitative RT-PCR. Pearson correlation coefficient indicated miR-4258, miR-664 and miR-1301 as the most significant miRNAs. In silico target-prediction and network analysis showed miR-664 and miR-1301 as organized in predicted GRNs with genes interested in parathyroid adenomas and carcinomas. In conclusion, our study identified three new miRNAs involved in the MEN1 parathyroid neoplasia, directly targeting genes associated with the development of different inheritable forms of parathyroid tumors. These identified miRNAs could be revealed as prognostic and diagnostic biomarkers for parathyroid tumors to improve the diagnosis of MEN1 neoplasia and other syndromes.

An autoregulatory network between menin and pri-miR-24-1 is required for the processing of its specific modulator miR-24-1 in BON1 cells.

Multiple endocrine neoplasia type 1 (MEN1) is a rare hereditary cancer complex syndrome manifesting a variety of endocrine and non-endocrine neoplasms and lesions. MEN1 is characterized by tumours of the parathyroids, of the neuroendocrine cells of the gastroenteropancreatic tract, and of the anterior pituitary. The MEN1 gene, a tumour suppressor gene, encodes the menin protein. Loss of heterozygosity (LOH) at 11q13 is typical of MEN1 tumours in agreement with Knudson's two-hit hypothesis. We previously showed that the MEN1 parathyroid tumorigenesis is under the control of an "incoherent feedback loop" between miR-24-1 and the menin protein that generates a "Gene Regulatory Network" (GRN) that mimics the second hit of Knudson's hypothesis and that could buffer the effect of the stochastic factors that contribute to the onset and progression of this disease. Here we show, in the BON1 cell line derived from lymphnode metastasis of a human carcinoid tumour of the pancreas, that menin binds specifically to the primary RNA sequence pri-miR-24-1 by promoting the miR-24-1 biogenesis. Network simulation showed a new feed-forward loop between menin, microRNA-24-1 and Musashi-1 proteins. This result shows a novel mechanism whereby menin, a RNA-binding protein, facilitates the processing of its specific miRNA by regulating the dynamics of the menin-miR-24 Gene Regulatory Network at the level of pri-miRNA processing.

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells on nanostructured Ti6Al4V and Ti13Nb13Zr.

Bone tissue engineering and nanotechnology enable the design of suitable substitutes to restore and maintain the function of human bone tissues in complex fractures and other large skeletal defects. Long-term stability and functionality of prostheses depend on integration between bone cells and biocompatible implants. Human adipose tissue-derived mesenchymal stem cells (hAMSCs) have been shown to possess the same ability to differentiate into osteoblasts and to produce bone matrix of classical bone marrow derived stem cells (BMMSCs). Ti6A14V and Ti13Nb13Zr are two different biocompatible titanium alloys suitable for medical bone transplantation. Preliminary results from our Research Group demonstrated that smooth Ti6Al4V surfaces exhibit an osteoconductive action on hAMSCs, granting their differentiation into functional osteoblasts and sustaining bone matrix synthesis and calcification. The purpose of this study is to assay the ability of nanostructured Ti6Al4V and Ti13Nb13Zr alloys to preserve the growth and adhesion of hAMSCs and, mostly, to sustain and maintain their osteogenic differentiation and osteoblast activity. The overall results showed that both nanostructured titanium alloys are capable of sustaining cell adhesion and proliferation, to promote their differentiation into osteoblast lineage, and to support the activity of mature osteoblasts in terms of calcium deposition and bone extracellular matrix protein production.

Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast-like SaOS-2 cells.

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

The negative feedback-loop between the oncomir Mir-24-1 and menin modulates the Men1 tumorigenesis by mimicking the "Knudson's second hit".

Multiple endocrine neoplasia type 1 (MEN1) syndrome is a rare hereditary cancer disorder characterized by tumors of the parathyroids, of the neuroendocrine cells, of the gastro-entero-pancreatic tract, of the anterior pituitary, and by non-endocrine neoplasms and lesions. MEN1 gene, a tumor suppressor gene, encodes menin protein. Loss of heterozygosity at 11q13 is typical of MEN1 tumors, in agreement with the Knudson's two-hit hypothesis. In silico analysis with Target Scan, Miranda and Pictar-Vert softwares for the prediction of miRNA targets indicated miR-24-1 as capable to bind to the 3'UTR of MEN1 mRNA. We investigated this possibility by analysis of miR-24-1 expression profiles in parathyroid adenomatous tissues from MEN1 gene mutation carriers, in their sporadic non-MEN1 counterparts, and in normal parathyroid tissue. Interestingly, the MEN1 tumorigenesis seems to be under the control of a "negative feedback loop" between miR-24-1 and menin protein, that mimics the second hit of Knudson's hypothesis and that could buffer the effect of the stochastic factors that contribute to the onset and progression of this disease. Our data show an alternative way to MEN1 tumorigenesis and, probably, to the "two-hit dogma". The functional significance of this regulatory mechanism in MEN1 tumorigenesis is also the basis for opening future developments of RNA antagomir(s)-based strategies in the in vivo control of tumorigenesis in MEN1 carriers.

The regulatory network menin-microRNA 26a as a possible target for RNA-based therapy of bone diseases.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, interplaying with transcription factors in complex regulatory networks. Menin is the product of the MEN1 oncosuppressor gene, responsible for multiple endocrine neoplasia type 1 syndrome. Recent data suggest that menin functions as a general regulator of transcription. Menin expression modulates mesenchymal cell commitment to the myogenic or osteogenic lineages. The microRNA 26a (miR-26a) modulates the expression of SMAD1 protein during the osteoblastic differentiation of human adipose tissue-derived stem cells (hADSCs). We used siRNA silencing against MEN1 mRNA and pre-miR-26 mimics to study the interplay between them and to investigate the interplay between menin and miR-26a as regulators of osteogenic differentiation in the hADSCs. We found that in hADSCs the siRNA-induced silencing of MEN1 mRNA resulted in a down regulation of miR-26a, with a consequent up-regulation of SMAD1 protein. Chromatin immunoprecipitation (ChIP) showed that menin occupies the miR-26-a gene promoter, thus inducing its expression and confirming that menin is a positive regulator of miR-26a. In conclusion, results from this study evidenced, for the first time, a direct interaction between menin transcription factor and miRNA, interaction that seems to play a pivotal role during the hADSCs osteogenesis, thus suggesting a novel target for bone disease RNA-based therapy.

In vitro effects of oestrogens, antioestrogens and SERMs on pancreatic solid pseudopapillary neoplasm-derived primary cell culture.

BACKGROUND: Solid-pseudopapillary neoplasms of the pancreas (SPNs) are uncommon tumours usually frequent in young women. Although the pathogenesis of SPNs is uncertain a potential influence of the sex hormone milieu on the biology of these tumours has been suggested. The controversial expression of oestrogen receptors (ERs) in SPNs, provide a rationale for studying the effects of oestrogenic molecules on SPN development. METHODS: The expression of a large series of hormonal ligands and receptors was evaluated in tissue specimens and in a primary cell culture (SPNC), obtained from a SPN in young female patient. The effects of 17beta-oestradiol (17betaE2), ICI 182,780 and tamoxifen (Tam) on cell replication and growth were examined. RESULTS: We have established SPNC primary line. Immunocytochemical analysis was positive for vimentin, cyclin D1 and beta-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERalpha, ERbeta and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17betaE2 and Tam respectively were proved in SPNC. CONCLUSION: In conclusion, we provide the first direct evidence that oestrogenic molecules can influence proliferation of SPNC, offering future strategies in the control of this neoplasia via selective ER modulators.

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces.

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.