Adult mesenchymal stem cells: is there a role for purine receptors in their osteogenic differentiation?

The role played by mesenchymal stem cells (MSCs) in contributing to adult tissue homeostasis and damage repair thanks to their differentiation capabilities has raised a great interest, mainly in bone regenerative medicine. The growth/function of these undifferentiated cells of mesodermal origin, located in specialized structures (niches) of differentiated organs is influenced by substances present in this microenvironment. Among them, ancestral and ubiquitous molecules such as adenine-based purines, i.e., ATP and adenosine, may be included. Notably, extracellular purine concentrations greatly increase during tissue injury; thus, MSCs are exposed to effects mediated by these agents interacting with their own receptors when they act/migrate in vivo or are transplanted into a damaged tissue. Here, we reported that ATP modulates MSC osteogenic differentiation via different P2Y and P2X receptors, but data are often inconclusive/contradictory so that the ATP receptor importance for MSC physiology/differentiation into osteoblasts is yet undetermined. An exception is represented by P2X7 receptors, whose expression was shown at various differentiation stages of bone cells resulting essential for differentiation/survival of both osteoclasts and osteoblasts. As well, adenosine, usually derived from extracellular ATP metabolism, can promote osteogenesis, likely via A2B receptors, even though findings from human MSCs should be implemented and confirmed in preclinical models. Therefore, although many data have revealed possible effects caused by extracellular purines in bone healing/remodeling, further studies, hopefully performed in in vivo models, are necessary to identify defined roles for these compounds in favoring/increasing the pro-osteogenic properties of MSCs and thereby their usefulness in bone regenerative medicine.

HMGA1 negatively regulates NUMB expression at transcriptional and post transcriptional level in glioblastoma stem cells.

Glioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2-3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth. We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division. Since NUMB expression is fundamental for the fulfillment of asymmetric division in stem cells, and is lost or reduced in many tumors, including GBM, we have investigated the ability of HMGA1 to regulate NUMB expression. Here, we show that HMGA1 negatively regulates NUMB expression at transcriptional level, by binding its promoter and counteracting c/EBP-ß and at posttranscriptional level, by regulating the expression of MSI1 and of miR-146a. Finally, we report that HMGA1 knockdown-induced NUMB upregulation leads to the downregulation of the NOTCH1 pathway. Therefore, the data reported here indicate that HMGA1 negatively regulates NUMB expression in BTSCs, further supporting HMGA1 targeting as innovative and effective anti-cancer therapy.

Involvement of P2X7 Receptors in the Osteogenic Differentiation of Mesenchymal Stromal/Stem Cells Derived from Human Subcutaneous Adipose Tissue.

The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100 µM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca2+ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100 µM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dose-dependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.

Upregulation of Epithelial-To-Mesenchymal Transition Markers and P2X7 Receptors Is Associated to Increased Invasiveness Caused by P2X7 Receptor Stimulation in Human Glioblastoma Stem Cells.

Glioblastoma (GBM) stem cells (GSCs), which contribute to GBM unfavorable prognosis, show high expression levels of ATP/P2X7 receptors (P2X7R). Here, we reported that cells exposure to 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP), a P2X7R agonist, up-regulated the expression of markers associated to epithelial-to-mesenchymal transition (EMT), a process likely contributing to GSC malignancy, and increased GSC migration/invasiveness like the known EMT inducer, Transforming Growth Factor ß1 (TGFß1). These effects were coupled to phosphorylation of SMAD2, a downstream effector in the TGFß pathway, suggesting its involvement in P2X7R-mediated activity in GSCs. All BzATP effects, including a decrease in the caspase 3/7 activity in GSC medium, were mostly counteracted by the P2X7R antagonist A438079. Finally, BzATP increased the subunit expression of two main human P2X7R splice variants, the full-length P2X7A and the truncated P2X7B, lacking the carboxylic tail, which have different functional properties depending on their arrangement. Since up-regulation of A/B subunits might favor their assembly into a heterotrimeric P2X7R with great sensitivity towards agonists and cell energy support, this is in line with increased EMT markers expression, cell migration/invasion and GSC survival observed following P2X7R stimulation. As in GBM microenvironment extracellular ATP levels may activate P2X7R, our data suggest a P2X7R role in GBM recurrence/invasiveness.

A New Investigational Perspective for Purines Against Glioblastoma Invasiveness.

BACKGROUND: Glioblastoma Multiforme (GBM) is the most common and lethal brain malignancy. Recent evidence suggests that the presence of stem-like cells (GSCs) inside the tumor with high self-renewal, resistance to chemotherapy and invasiveness/migration potential is associated with poor GBM prognosis. GSC aggressiveness seems to be linked to an important process involved in tumorigenesis and cancer metastasis called Epithelial-to-Mesenchymal Transition (EMT), which is responsible for several biochemical changes and the acquisition of a more mesenchymal phenotype by GSCs, that enhance their migration, invasiveness and resistance to apoptosis. OBJECTIVE: Since previous reports demonstrated that purines, interacting with their own receptors, exerted anti-tumor effects in GBM and derived cells, we tried to investigate the ability of these compounds to reduce tumor cell migration/invasion acting on EMT-associated genes/activators and/or signal pathways. METHODS: Search in the literature of relevant articles related to the objective. RESULTS: Papers examining the activity of purines on EMT signaling pathways/markers in GSCs are still few whereas literature is more abundant as for other kinds of tumors. CONCLUSION: Considering the significance of EMT in GBM aggressiveness and the promising involvement of purines in this process, we think that further research in this regard may open the way towards a new therapeutic approach for the control of GBM invasiveness/recurrence.

The Role of Wnt Signal in Glioblastoma Development and Progression: A Possible New Pharmacological Target for the Therapy of This Tumor.

Wnt is a complex signaling pathway involved in the regulation of crucial biological functions such as development, proliferation, differentiation and migration of cells, mainly stem cells, which are virtually present in all embryonic and adult tissues. Conversely, dysregulation of Wnt signal is implicated in development/progression/invasiveness of different kinds of tumors, wherein a certain number of multipotent cells, namely "cancer stem cells", are characterized by high self-renewal and aggressiveness. Hence, the pharmacological modulation of Wnt pathway could be of particular interest, especially in tumors for which the current standard therapy results to be unsuccessful. This might be the case of glioblastoma multiforme (GBM), one of the most lethal, aggressive and recurrent brain cancers, probably due to the presence of highly malignant GBM stem cells (GSCs) as well as to a dysregulation of Wnt system. By examining the most recent literature, here we point out several factors in the Wnt pathway that are altered in human GBM and derived GSCs, as well as new molecular strategies or experimental drugs able to modulate/inhibit aberrant Wnt signal. Altogether, these aspects serve to emphasize the existence of alternative pharmacological targets that may be useful to develop novel therapies for GBM.