Elena Torreggiani

Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO‐PLEX technology

We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast‐specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL‐8, MCP‐1 and VEGF, but did not express IL‐2, IL‐7, IL‐17, eotaxin, G‐CSF and IFN‐γ. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.

Slug gene expression supports human osteoblast maturation

This study aims to define the function of Slug transcription factor in human normal osteoblasts (hOBs). To date, Slug is considered exclusively a marker of malignancy in bone tissue. Here, we identified, for the first time, a role for Slug in hOBs using a knockdown approach. We demonstrated that Slug is positively correlated with osteoblast markers, including Runx2, osteopontin, osteocalcin, Collagen type 1, Wnt/β-catenin signaling mediators, and mineral deposition. At the same time, Slug silencing potentiates the expression of Sox-9, a factor indispensable for chondrogenic development. These data, with the finding that Slug is in vivo recruited by the promoters of Runx2 and Sox-9 genes, suggest that, in hOBs, Slug may act both as positive and negative transcriptional regulator of Runx2 and Sox-9 genes, respectively. In summary, our results support the hypothesis that Slug functions as a novel regulator of osteoblast activity and may be considered a new factor required for osteoblast maturation.

Role of Slug transcription factor in human mesenchymal stem cells

The pathways that control mesenchymal stem cells (MSCs) differentiation are not well understood, and although some of the involved transcription factors (TFs) have been characterized, the role of others remains unclear. We used human MSCs from tibial plateau (TP) trabecular bone, iliac crest (IC) bone marrow and Wharton’s jelly (WJ) umbilical cord demonstrating a variability in their mineral matrix deposition, and in the expression levels of TFs including Runx2, Sox9, Sox5, Sox6, STAT1 and Slug, all involved in the control of osteochondroprogenitors differentiation program. Because we reasoned that the basal expression level of some TFs with crucial role in the control of MSC fate may be correlated with osteogenic potential, we considered the possibility to affect the hMSCs behaviour by using gene silencing approach without exposing cells to induction media. In this study we found that Slug‐silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression. On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type. Interestingly, we demonstrated a direct in vivo regulatory action of Slug by chromatin immunoprecipitation, showing a specific recruitment of this TF in the promoter of Runx2 and Sox9 genes. As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.

Apoptosis of human primary osteoclasts treated with molecules targeting nuclear factor-κB

Osteoclasts (OCs) are involved in several pathologies associated with bone loss, including rheumatoid arthritis, osteoporosis, bone metastasis of myeloma, osteosarcoma, and breast cancer. In this review we determined the effects of natural compounds, including extracts from medicinal plants, on differentiation and survival of human primary OCs obtained from peripheral blood. We found that OCs from umbilical cord blood and peripheral blood behave differently in response to molecules inducing apoptosis in this experimental system. Apoptosis induced by decoy oligonucleotides was reproducibly obtained in OCs from peripheral blood but not in OCs derived from cord blood. With respect to effects of medicinal plants, we found that crude extracts of Emblica officinalis are able to induce specifically programmed cell death of mature OCs without altering the process of osteoclastogenesis. E. officinalis specifically increased the expression levels of Fas, a critical member of the apoptotic pathway. Gel shift experiments BioPharmaNet demonstrate that E. officinalis extracts specifically compete with the binding of a transcription factor involved in osteoclastogenesis NF‐κB to its specific target DNA sequences. This might explain the observed effects of E. officinalis on the expression levels of IL‐6, an NF‐κB‐specific target gene. We suggest the application of natural products as an alternative tool for therapy applied to bone diseases.

ERα and AP-1 interact in vivo with a specific sequence of the F promoter of the human ERα gene in osteoblasts†

Estrogen‐responsive genes often have an estrogen response element (ERE) positioned next to activator protein‐1 (AP‐1) binding sites. Considering that the interaction between ERE and AP‐1 elements has been described for the modulation of bone‐specific genes, we investigated the 17‐β‐estradiol responsiveness and the role of these cis‐elements present in the F promoter of the human estrogen receptor alpha (ERα) gene. The F promoter, containing the sequence analyzed here, is one of the multiple promoters of the human ERα gene and is the only active promoter in bone tissue. Through electrophoretic mobility shift (EMSA), chromatin immunoprecipitation (ChIP), and re‐ChIP assays, we investigated the binding of ERα and four members of the AP‐1 family (c‐Jun, c‐fos, Fra‐2, and ATF2) to a region located approximately 800 bp upstream of the transcriptional start site of exon F of the human ERα gene in SaOS‐2 osteoblast‐like cells. Reporter gene assay experiments in combination with DNA binding assays demonstrated that F promoter activity is under the control of upstream cis‐acting elements which are recognized by specific combinations of ERα, c‐Jun, c‐fos, and ATF2 homo‐ and heterodimers. Moreover, ChIP and re‐ChIP experiments showed that these nuclear factors bind the F promoter in vivo with a simultaneous occupancy stimulated by 17‐β‐estradiol. Taken together, our findings support a model in which ERα/AP‐1 complexes modulate F promoter activity under conditions of 17‐β‐estradiol stimulation. J. Cell. Physiol. 216: 101–110, 2008.

Human mesenchymal stem cells seeded on extracellular matrix-scaffold: Viability and osteogenic potential

The development and the optimization of novel culture systems of mesenchymal osteoprogenitors are some of the most important challenges in the field of bone tissue engineering (TE). A new combination between cells and extracellular matrix (ECM)‐scaffold, containing ECM has here been analyzed. As source for osteoprogenitors, mesenchymal stem cells obtained from human umbilical cord Whartons Jelly (hWJMSCs), were used. As ECM‐scaffold, a powder form of isolated and purified porcine urinary bladder matrix (pUBM), was employed. The goals of the current work were: (1) the characterization of the in vitro hWJMSCs behavior, in terms of viability, proliferation, and adhesion to ECM‐scaffold; (2) the effectiveness of ECM‐scaffold to induce/modulate the osteoblastic differentiation; and (3) the proposal for a possible application of cells/ECM‐scaffold construct to the field of cell/TE. In this respect, the properties of the pUBM‐scaffold in promoting and guiding the in vitro adhesion, proliferation, and three‐dimensional colonization of hWJMSCs, without altering viability and morphological characteristics of the cells, are here described. Finally, we have also demonstrated that pUBM‐scaffolds positively affect the expression of typical osteoblastic markers in hWJMSCs. J. Cell. Physiol. 227: 857–866, 2012.

Influence of obstetric factors on osteogenic potential of umbilical cord-derived mesenchymal stem cells

Whartons jelly from the umbilical cord is a noncontroversial source of mesenchymal stem cells (WJMSCs) with high plasticity, proliferation rate and ability to differentiate towards multiple lineages. WJMSCs from different donors have been characterized for their osteogenic potential. Although there is large evidence of WJMSCs plasticity, recently scientific debate has focused on MSCs selection, establishing predictable elements to discriminate the cells with most promising osteoprogenitor cell potential.In the present study a comparative study between the presence of osteoblastic markers and different parameters that pertain to both the newborn and the mother was performed. Umbilical cords were collected after all patients signed the informed consent and local ethical commettee approved the study. Obstetric parameters, including babys gender and birth weight, mothers age at delivery, gestational stage at parturition and mode of delivery were examined. After characterization and expansion, WJMSCs were analyzed for two osteoblastic markers, alkaline phosphatase (ALP) activity, and the expression level of RUNX-2 transcription factor, and for their ability to deposit mineralized matrix after osteogenic induction.We found that osteoblastic potential was not influenced by babys gender and mode of delivery. On the contrary, the highest degree of osteoblastic potential has been shown by WJMSCs with RUNX-2 high basal levels, selected from umbilical cords of the heaviest term babies.Even if further evaluation is required, our hypothesis is that our findings may help in selecting the optimal umbilical cord donors and in collecting high potential Whartons jelly-derived osteoprogenitors efficiently.

Sex hormone receptor levels in laryngeal carcinoma: a comparison between protein and RNA evaluations

The larynx is a secondary sex organ, and the hormone dependence of laryngeal carcinomas is considered an interesting matter of speculation. However, while tumors of other secondary sex organs, including the prostate, breast, and endometrium, have been recognized as hormone-dependent cancers, the laryngeal carcinomas are still subject to controversy. In this study, samples from 15 laryngeal carcinomas obtained at the time of surgery were assayed for specific estrogen alpha, progesterone, and androgen receptor expression, both at mRNA and protein levels. Detectable levels of specific estrogen and progesterone receptors, 53.3 and 73.3%, respectively, were found in the tumors. This positive detection by immunohistochemical analysis was higher in tumors than in normal mucosa adjacent to the tumor areas and was correlated with the absence of metastatic lymph nodes. No androgen receptor protein was detected in any sample analyzed, even if quantitative RT-PCR revealed high mRNA levels specific for this receptor. A strict correspondence between protein and mRNA hormone receptor levels was not found. This is in agreement with the transcriptional and protein synthesis mechanisms, and it is also compatible with the complex larynx tumorigenesis.

Induction of estrogen receptor α expression with decoy oligonucleotide targeted to NFATc1 binding sites in osteoblasts

The nuclear factor of activated T cell cytoplasmic 1 (NFATc1) is a member of the NFAT family and is strictly implicated in the growth and development of bone. Most studies have focused on the effects of NFATc1 activation on osteoclastogenesis. On the contrary, the specific roles of NFAT in osteoblast differentiation are not well understood and, in some instances, reports of its role are contradictory. In the present study, we demonstrated that NFATc1 was involved in the transcriptional regulation of human estrogen receptor α (ERα) gene in SaOS-2 osteoblastic like cells. NFATc1 was specifically recruited “in vivo” at C and F distal promoters of ERα gene. In addition, it is here identified as the negative transcription factor removed by the RA4-3′decoy oligonucleotide able to induce ERα expression in osteoblasts. Ca2+/calcineurin-NFAT-mediated signaling pathways and ERα-dependent signals are involved in diverse cellular reactions by regulating gene expression under both physiological and pathological conditions. Therefore, our data might be useful for proper manipulation of NFATc1- and ERα-mediated cellular reactions in different bone disorders, such as osteoporosis.

Transcription factor decoy against NFATc1 in human primary osteoblasts

The present study describes, for the first time, the removal of the nuclear factor of activated T cells cytoplasmic 1 (NFATc1) by a decoy approach in human primary osteoblasts (hOBs). hOBs with different NFATc1 expression levels were used. The functionality of endogenous NFAT proteins in our experimental model was analyzed by monitoring the transcriptional activity on a luciferase reporter construct driven by three copies of an NFAT response element (pNFAT-TA-luc). Cell treatment with the decoy against NFATc1 resulted in a significant increase in the expression of osteoblastic markers, including ERα and ColXV. On the contrary, the expression of Runx2, which is known to not be transcriptionally regulated by NFATc1, was not altered, indicating the specificity of the decoy effect. To our knowledge, this is the first time that transcription factor decoy has been successful in hOBs to allow the investigation of the role of NFATc1 in an experimental model that, compared to the use of cell lines, more closely resembles an in vivo model. In addition, by using chromatin immunoprecipitation we found that in vivo NFATc1 is recruited on the ColXV gene promoter. The specific role of NFATc1 in osteoblast differentiation is not well understood, however, our findings reinforce the action of NFATc1 in the transcriptional program of osteoblasts, also supporting the therapeutic potential for the proper manipulation of NFATc1-mediated events in different bone disorders. At the same time, our data add important information on the regulation of the expression of ColXV, which only recently has been proposed as an osteoblastic marker.