Silencing NTPDase3 activity rehabilitates the osteogenic commitment of post-menopausal stem cell bone progenitors (original) (raw)
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Journal of Cellular Physiology, 2012
This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y 2 , P2Y 4 , and P2Y 6 ) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 AE 5 years old, n ¼ 18) undergoing total hip arthroplasty. UTP and UDP (100 mM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca 2þ ] i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y 6 receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPgS was devoid of effect. Selective blockade of P2Y 6 receptors with MRS 2578 prevented [Ca 2þ ] i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y 2 , P2Y 4 , and P2Y 6 receptors. While the expression of P2Y 6 receptors remained fairly constant (7$21 days), P2Y 2 and P2Y 4 became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y 6 receptors coupled to increases in [Ca 2þ ] i . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.
On the role of P2 purinoceptors and ecto-NTPDases in postmenopausal human osteogenesis
2014
PhD thesis-José Bernardo Noronha Matos (2011-2014) 3 ABBREVIATIONS 3'-UTR, Three prime untranslated region 25-OH-Vitamine D, 25-OH-Cholecalciferol a.u., Arbitrary units A438079, 3-[[5-(2,3-Dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride ADO, Adenosine ADPβS, Adenosine 5'-[β-thio]diphosphate ALP, Alkaline phosphatase AMP, Adenosine 5'-monophosphate AP-1, Adapter-related protein complex 1 subunit AR, Androgen receptor ARL 67156, 6-N,N-Diethyl-D-β,γ-dibromomethylene ATP trisodium salt ATP, Adenosine 5'-triphosphate ATPase, Adenosine 5'-triphosphatase BGLAP, Osteocalcin coding gene BMP, Bone morphogenic protein BMPRIA, Bone morphogenic protein receptor type IA BMSCs, Bone marrow stromal cells BRU, Bone remodelling units BTE, Bone tissue engineering BzATP, 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate [Ca 2+ ]i, Intracellular calcium cAMP, 3'-5'-Cyclic adenosine monophosphate CaSR, Calcium sensing receptors CD11b, Integrin alpha M CD14, Monocyte differentiation antigen CD14 CD19, B-lymphocyte antigen CD19 CD34, Haematopoietic progenitor cell antigen CD34 CD39, Apyrase or NTPDase1 CD44, Receptor for hyaluronic acid CD45, Protein tyrosine phosphatase, receptor type C, also known as PTPRC PhD thesis-José Bernardo Noronha Matos (2011-2014) 4 CD49, Integrin alpha subunit or very late antigen CD54, Intercellular adhesion molecule-1 CD73, Ecto-5'-nucleotidase CD79, B-cell antigen receptor complex-associated protein (alpha or beta) chain CD90, Thy-1 membrane glycoprotein CD105, Endoglin CD164, Sialomucin-like 2 protein c-Fos, G0/G1 switch regulatory protein 7 or Proto-oncogene c-Fos CHL, chelerythrine c-Jun, Transcription factor AP-1 c-Kit, Tyrosine-protein kinase Kit COL1A1, Collagen alpha-1(I) chain coding gene COL1A2, Collagen alpha-2(I) chain coding gene CR, Calcitonin receptor CREB, cAMP response element-binding protein CXCR, chemokine receptors CYP27B1, 1α-hydroxylase DAG, Diacylglycerol 1.Introduction
Journal of Cellular Physiology, 2016
Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A 1 , A 2A , A 2B , and A 3) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5 0-di-and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union.
Estrogens Activate Bone Morphogenetic Protein-2 Gene Transcription in Mouse Mesenchymal Stem Cells
Molecular Endocrinology, 2003
Estrogens exert their physiological effects on target tissues by interacting with the estrogen receptors, ER␣ and ER. Estrogen replacement is one the most common and effective strategies used to prevent osteoporosis in postmenopausal women. Whereas it was thought that estrogens work exclusively by inhibiting bone resorption, our previous results show that 17-estradiol (E2) increases mouse bone morphogenetic protein (BMP)-2 mRNA, suggesting that estrogens may also enhance bone formation. In this study, we used quantitative real-time RT-PCR analysis to demonstrate that estrogens increase BMP-2 mRNA in mouse mesenchymal stem cells. The selective ER modulators, tamoxifen, raloxifene, and ICI-182,780 (ICI), failed to enhance BMP-2 mRNA, whereas ICI inhibited E2 stimulation of expression. To investigate if estrogens increase BMP-2 expression by transcriptional mechanisms and if the response is mediated by ER␣ and/or ER, we studied the effects of estrogens on BMP-2 promoter activity in transient transfected C3H10T1/2 cells. E2 produced a dose-dependent induction of the mouse ؊2712 BMP-2 promoter activity in cells cotransfected with ER␣ and ER. At a dose of 10 nM E2, ER␣ induced mouse BMP-2 promoter activity 9-fold,
International Journal of Molecular Sciences, 2015
Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels.
Journal of Cellular Physiology, 2012
Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH‐induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4‐knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH‐responsiveness permitted further studies with single WT and KO clo...
Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality
American Journal of Physiology-Endocrinology and Metabolism
A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 ( Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4−/− mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4−/− MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, ...
Journal of Bone and Mineral Research, 1999
in culture was demonstrated by reverse transcriptase-polymerase chain reaction. Following the withdrawal of 17-estradiol, MOB cultures showed a significant increase in the number of cells synthesizing stromelysin-1; this effect was enhanced by stimulation with either interleukin-1 or interleukin-6. Northern blot analysis showed only a slight increase in stromelysin-1 mRNA message following the withdrawal of 17-estradiol. Our data show an unexpected up-regulation of stromelysin-1 synthesis by osteoblasts both in vivo and in vitro following estrogen withdrawal. Although this effect was not reflected in a significant change in stromelysin-1 mRNA expression in vitro, there is evidence to suggest a role for this enzyme in the early stages of bone loss during the pathogenesis of osteoporosisSeven-week-old CD-1 mice, either ovariectomized (OVX) or sham-operated (SHAM), were kept under standard laboratory conditions for 4 weeks. Animals were killed and the lumbar vertebrae (L1-L4) and distal femoral head bone samples removed. Samples for histology and histomorphometry were processed and embedded as below. Other samples were allocated as follows: left-sided explants, L1 and L4, were mounted in Tissue-Tek, snap frozen in liquid nitrogen, and stored at -70°C until sectioned (T 0 ); equivalent right-sided explants, L2 and L3, were cultured for 18 h in 5 M monensin in Dulbecco's modified Eagle's medium supplemented with 10% charcoal-stripped FCS and antibiotics to increase the level of intracellular MMPs as previously described, and subsequently snap frozen.
Molecular Medicine Reports, 2017
To identify novel candidate genes associated with osteoporosis, RNA-sequence analysis of human mesenchymal stem cells (hMSCs) from patients with osteoporosis (G3) and osteopenia (G2), and healthy controls (G1) was performed. Differentially expressed genes (DEGs) from among the three groups were identified. DEGs were separated into nine groups according to their gene expression patterns: UU (up and up), UF (up and flat), UD (up and down), FU (flat and up), FF (flat and flat), FD (flat and down), DU (down and up), DF (down and flat), and DD (down and down). Among the 42 DEGs between G3 and G1, eight candidate genes, namely stimulated by retinoic acid 6 (STRA6), melanophilin, neurotrophic receptor tyrosine kinase 2, cartilage oligomeric matrix protein, collagen type XI α 1 chain, integrin subunit β 2, monooxygenase DBH-like 1 and selenoprotein P, were selected, as they demonstrated consistent gene expression patterns of UU, FU, FD, and DD. Among these eight genes, STRA6 was highly expressed in the osteoporosis group and based on additional data from quantitative polymerase chain reaction analysis, it was selected for further study. In order to investigate whether STRA6 served a functional role in osteoblast or adipocyte differentiation, the effects of STRA6 expression changes in pluripotent stem cell C3H10T1/2, preosteoblast MC3T3-E1 and stromal ST2 cell lines were examined. Bone morphogenetic protein 2 enhanced STRA6 expression only at the early stage of osteoblast differe ntiation, and overexpression of STRA6 temporally inhibited the expression of osteoblastogenesis markers, including runt related transcription factor 2, bone sialoprotein and osteocalcin. Furthermore, the knockdown of STRA6 slightly enhanced nodule formation at the late stage of osteoblast differentiation, and overexpression of STRA6 in ST2 cells enhanced adipocyte differentiation. Taken together, STRA6 expression could be associated with the pathogenesis of osteoporosis by promoting adipocyte differentiation over osteoblast differentiation in the hMSC population.
Journal of Cellular Biochemistry, 2001
In the mouse, ovariectomy (OVX) leads to signi®cant reductions in cancellous bone volume while estrogen (17b-estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) a and b expression of primary culture MSCs isolated from OVX and sham-operated mice. MSCs, treated in vitro with 10 À7 M E2, displayed a signi®cant increase in ERa mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERb mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up-regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF-b1, BMP-2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERa was the predominant form expressed in MSCs obtained from both OVX and sham-operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERa expression, osteogenic gene expression and, inhibits apoptosis and ERb expression in MSCs obtained from OVX and sham-operated mice. Coexpression of ERa, but not ERb, and osteogenic differentiation markers might indicate that ERa function as an activator and ERb function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERa activation.