SLUG: a new target of lymphoid enhancer factor-1 in human osteoblasts

<p>Abstract</p> <p>Background</p> <p>Lymphoid Enhancer Factor-1 (Lef-1) is a member of a transcription factor family that acts as downstream mediator of the Wnt/β-catenin signalling pathway which plays a critical role in osteoblast proliferation and differentiation. In a search for Lef-1 responsive genes in human osteoblasts, we focused on the transcriptional regulation of the SLUG, a zinc finger transcription factor belonging to the Snail family of developmental proteins. Although the role of SLUG in epithelial-mesenchymal transition and cell motility during embryogenesis is well documented, the functions of this factor in most normal adult human tissues are largely unknown. In this study we investigated SLUG expression in normal human osteoblasts and their mesenchymal precursors, and its possible correlation with Lef-1 and Wnt/β-catenin signalling.</p> <p>Results</p> <p>The experiments were performed on normal human primary osteoblasts obtained from bone fragments, cultured in osteogenic conditions in presence of Lef-1 expression vector or GSK-3β inhibitor, SB216763. We demonstrated that the transcription factor SLUG is present in osteoblasts as well as in their mesenchymal precursors obtained from Wharton's Jelly of human umbilical cord and induced to osteoblastic differentiation. We found that SLUG is positively correlated with RUNX2 expression and deposition of mineralized matrix, and is regulated by Lef-1 and β-catenin. Consistently, Chromatin Immunoprecipitation (ChIP) assay, used to detect the direct Lef/Tcf factors that are responsible for the promoter activity of SLUG gene, demonstrated that Lef-1, TCF-1 and TCF4 are recruited to the SLUG gene promoter "<it>in vivo</it>".</p> <p>Conclusion</p> <p>These studies provide, for the first time, the evidence that SLUG expression is correlated with osteogenic commitment, and is positively regulated by Lef-1 signal in normal human osteoblasts. These findings will help to further understand the regulation of the human SLUG gene and reveal the biological functions of SLUG in the context of bone tissue.</p

Osteoclast-mediated acidic hydrolysis of thermally gelled curdlan component of the bone scaffolds: Is it possible?

Many biomaterials for bone regeneration have recently been produced using thermally gelled curdlan (1,3-ß-d-glucan) as a binder for bioceramics. As the human organism does not produce enzymes having the ability to degrade curdlan, it is not clear what is the fate of curdlan gel after its implantation in the bone. To clarify this point, in this research osteoclasts were cultured on the curdlan gel to show its degradation by acidic hydrolysis. The studies clearly demonstrated microstructural (AFM and SEM imaging) and chemical changes (Raman spectroscopy) on the curdlan surface caused by osteoclast culture. Moreover, degradation test in a cell-free system using HCl solution (pH = 4.5), mimicking environment in the resorption lacuna, showed great weight loss of the sample, release of glucose, and chemical changes typical of curdlan degradation. Thus, the presented research for the first time provides a strong evidence of osteoclast-mediated acidic hydrolysis of thermally obtained curdlan gel.Peer ReviewedPostprint (published version

Hypoxia Preconditioning of Human MSCs: a Direct Evidence of HIF-1α and Collagen Type XV Correlation

Background/Aims: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. Methods: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo – Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. Results: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. Conclusion: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied

Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies

Silencing of anti-chondrogenic microRNA-221 in human mesenchymal stem cells promotes cartilage repair in vivo

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation towards the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the anti-chondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs towards chondrogenic differentiation in the absence of TGF-β. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a pro-chondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. This article is protected by copyright. All rights reserved

Induction of apoptosis of human primary osteoclasts treated with extracts from the medicinal plant Emblica officinalis

<p>Abstract</p> <p>Background</p> <p>Osteoclasts (OCs) are involved in rheumatoid arthritis and in several pathologies associated with bone loss. Recent results support the concept that some medicinal plants and derived natural products are of great interest for developing therapeutic strategies against bone disorders, including rheumatoid arthritis and osteoporosis. In this study we determined whether extracts of <it>Emblica officinalis </it>fruits display activity of possible interest for the treatment of rheumatoid arthritis and osteoporosis by activating programmed cell death of human primary osteoclasts.</p> <p>Methods</p> <p>The effects of extracts from <it>Emblica officinalis </it>on differentiation and survival of human primary OCs cultures obtained from peripheral blood were determined by tartrate-acid resistant acid phosphatase (TRAP)-positivity and colorimetric MTT assay. The effects of <it>Emblica officinalis </it>extracts on induction of OCs apoptosis were studied using TUNEL and immunocytochemical analysis of FAS receptor expression. Finally, <it>in vitro </it>effects of <it>Emblica officinalis </it>extracts on NF-kB transcription factor activity were determined by gel shift experiments.</p> <p>Results</p> <p>Extracts of <it>Emblica officinalis </it>were able to induce programmed cell death of mature OCs, without altering, at the concentrations employed in our study, the process of osteoclastogenesis. <it>Emblica officinalis </it>increased the expression levels of Fas, a critical member of the apoptotic pathway. Gel shift experiments demonstrated that <it>Emblica officinalis </it>extracts act by interfering with NF-kB activity, a transcription factor involved in osteoclast biology. The data obtained demonstrate that <it>Emblica officinalis </it>extracts selectively compete with the binding of transcription factor NF-kB to its specific target DNA sequences. This effect might explain the observed effects of <it>Emblica officinalis </it>on the expression levels of interleukin-6, a NF-kB specific target gene.</p> <p>Conclusion</p> <p>Induction of apoptosis of osteoclasts could be an important strategy both in interfering with rheumatoid arthritis complications of the bone skeleton leading to joint destruction, and preventing and reducing osteoporosis. Accordingly, we suggest the application of <it>Emblica officinalis </it>extracts as an alternative tool for therapy applied to bone diseases.</p

The Adequacy of Experimental Models and Understanding the Role of Non-coding RNA in Joint Homeostasis and Disease

RNA-mediated processes by non-coding RNA (ncRNAs) namely microRNAs, long ncRNAs and circular RNAs, as all epigenetic mechanisms are particularly sensitive to the effects of tissue microenvironment and environmental factors. In recent years research has focused on the development of smart cell culture in vitro systems one step closer to natural conditions, paying particular attention to the cellular microenvironment and cell culture conditions. 3D cell culture and co-culture systems based on cultivating a single cell population or different cell populations combined together, have found growing interest as useful tools to better understand cell biology and to offer more physiological relevant results by tightly controlling experimental parameters. The development of these in vitro models is a promising approach even if the limited availability of human tissue from which to obtain the cells have to take into account. Such an approach by using tissue specimens of human origin can allow the realization of suitable 3D in vitro models overcoming the limits of traditional 2D monolayer cell cultures, or expensive animal models that often cannot accurately recapitulate human etiopathogenesis and are not suited to develop novel drugs. It is widely recognized that in a 3D environment cells tend to be more subjected to morphological and functional changes differently to those grown in simplistic cellular monolayer. Another important issue is the methods matter regarding the employment of cell culture conditions that have to take into account the physiological parameters such as oxygen concentration, chemical and biophysical components. The applicability of results from in vitro studies to in vivo situations, especially as regards the molecules involved in regulatory mechanisms, is directly dependent on the degree of similarity between the in vitro experimental condition and the in vivo environment. We believe a major effort and investment of time in this direction by the scientific community is necessary. The effort should be toward the improvement and the use of technology which allows cells from a specific donor to grow and behave in vitro in a manner that more closely represents that experienced by their native counterparts. This approach will likely have a significant impact on the understanding the real role of critical regulators of tissue homeostasis such as ncRNAs, and on improving drug discovery. This objective can be achieved through different types of initiatives that connect the scientists who deal with joint homeostasis and disease, such as: (1) the creation of an international research Consortium dedicated to support the development and optimization of 3D cell culture models, (2) specific workshops for promoting the development of guidelines in order to minimize controversies on mechanisms of disease and potential therapeutic targets, (3) the creation of a blog managed by a joint scientific organization that promotes debate and where it is possible to meet the experts. Certainly the biggest challenge is to convince those scientists to move from their already well-established 2D, and often successfully funded, cellular models. Therefore, we think that a critical point is represented by adequate funding policy that takes these issues into account and makes ad hoc funds available for studying and developing more relevant experimental models

Human osteoclasts/osteoblasts 3D dynamic co-culture system to study the beneficial effects of glucosamine on bone microenvironment

Glucosamine (GlcN) functions as a building block of the cartilage matrix and its multifaceted roles in promoting joint health has been extensively investigated. On the contrary, the role of GlcN in osteogenesis and bone tissue is poorly understood, mainly due to the lack of adequate experimental models. Consequently, the benefit of GlcN in bone disorders remains controversial. In order to broaden the pharmacological relevance and potential therapeutic/nutraceutic efficacy of GlcN, we investigated the effect of GlcN treatment on human primary osteoclasts (hOCs) and osteoblasts (hOBs) that were grown with 2D traditional methods or co-cultured in a more complex culture system one step closer to the in vivo bone microenvironment, consisting in a three-dimensional (3D) dynamic system (RCCS-4TM bioreactor, Synthecon™). In this condition osteoclastogenesis was supported by hOBs and sizeable self assembling aggregates were obtained. The differentiated osteoclasts were evaluated by the tartrate-resistant acid phosphatase assay (TRAP), osteogenic differentiation was monitored by analyzing both mineral matrix deposition through Alizarin Red staining, and expression of specific osteogenic markers through RT-qPCR. We found that DONA® crystalline glucosamine sulfate (i.e. the original GlcN sulfate product) was effective in decreasing the osteoclastic cell differentiation and function. Osteoclasts from OA donors were more sensitive than those from healthy donors. At the same time, DONA® showed anabolic effects on osteoblasts both in 2D conventional cell culture and in osteoclasts/osteoblasts 3D dynamic co-culture system. Here we demonstrated for the first time, as far as we know, the effectiveness of a 3D dynamic co-culture system to provide useful information on the spectrum of action of GlcN on bone microenvironment. This can pave the way to better define the potential applications of a compound such as GlcN which is positioned between pharmaceuticals and nutraceuticals. Therefore, based on our observations, we hypothesize that GlcN could have potential benefits either in the treatment of osteopenic diseases such as osteoporosis, or in the bone health maintenance

Emerging potential of gene silencing approaches targeting anti-chondrogenic factors for cell-based cartilage repair

The field of cartilage repair has exponentially been growing over the past decade. Here, we discuss the possibility to achieve satisfactory regeneration of articular cartilage by means of human mesenchymal stem cells (hMSCs) depleted of anti-chondrogenic factors and implanted in the site of injury. Different types of molecules including transcription factors, transcriptional co-regulators, secreted proteins, and microRNAs have recently been identified as negative modulators of chondroprogenitor differentiation and chondrocyte function. We review the current knowledge about these molecules as potential targets for gene knockdown strategies using RNA interference (RNAi) tools that allow the specific suppression of gene function. The critical issues regarding the optimization of the gene silencing approach as well as the delivery strategies are discussed. We anticipate that further development of these techniques will lead to the generation of implantable hMSCs with enhanced potential to regenerate articular cartilage damaged by injury, disease, or aging

Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells.

(O)estrogen receptor-alpha (ERalpha), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERalpha gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at -117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of gamma-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PF a and PF b, that do not present binding sites for known transcription factors and that are involved in a strong DNA-protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERalpha gene expression in bone. These data provide evidence for different promoter usage of the ERalpha gene through the recruitment of tissue-specific transcription activators and co-regulators