Role of ecto-NTPDases on UDP-sensitive P2Y 6 receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women (original) (raw)
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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
Regulation of scu-PA secretion and u-PA Receptor Expression in Osteoblast-like Cells
Cell Structure and Function, 1993
The production of proteolytic emzymes by osteoblasts is considered important for initiating osteoclastic bone resorption. Using the established cell line NY as an example of osteoblast-like cells, the effect of intracellular cyclic AMP (CAMP) and protein kinase C (PKC) on plasminogen activator secretion and its specific binding to the cells were investigated. HT-1080 cells were used as the control. NY cells predominantly secrete single-chain urokinase-type plasminogen activator (scu-PA) and some two-chain u-PA. Both scu-PA and u-PA were present in the cell surface and cell lysate of NY cells, and their distribution in HT-1080 cells was quite similar to that of NY cells. Exposing cells to phorboi myristate acetate (PMA) or dibutyryl cyclic AMP (db CAMP) enhanced the secretion of scu-PA and two-chain u-PA, whereas l-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) decreased scu-PA secretion, indicating that it is enhanced by protein kinase C (PKC) as well as by CAMP in NY cells. On the other hand, in HT-1080 cells, PMA decreased the level of two-chain u-PA secretion into the conditioned medium. The binding assay of 125I-DFP-u-PA to NY cells revealed the presence of a single class of binding sites with a Kd of 2.23 nM and Bmax of 0.82x 106 binding sites/cell. PMA however, altered neither the Kd nor the Bmax. Dibutyryl CAMP increased the Bmax 1.9 fold. Thus, NY cells secrete u-PA and express specific binding sites on the cell surface, which are modulated by CAMP and PKC. The u-PA/u-PA receptor system may contribute to osteoblastic bone resorption.
Differentiation of human osteoblastic cells in culture
Inflammation, 1995
We have followed the synthesis and secretion of urokinase-type plasminogen activator (u-PA) and its inhibitor, PAl-1, and matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP-I) during differentiation of a human osteoblastic cell line, HOS TE85, and the effect of TNF-t~ on this process. Our results show that the ratio of u-PA/PAI-I associated with the cell-matrix components increases during differentiation of these cells over a 14-day period. Although TNF-a suppresses the induced increase in steady-state mRNA levels of u-PA and PAI-1 during maturation of extracellular matrix (ECM), the u-PA/PAI-1 ratio is altered in such a way that PA activity associated with the ECM is higher than control cells. The expression of MMP-1 is low and remains essentially invariant over a culture period of 14 days. TNF-~ enhances MMP-1 transcription nearly 12-fold initially, after which mRNA levels drop off but remain significantly higher than the controls. Activities and steady-state mRNA levels of MMP-2 and MMP-9 increase nearly 15-fold during maturation of the ECM, but the level of TIMP-1 mRNA is not appreciably altered. The presence of TNF-~ suppresses maturation-induced transcription of MMP-2, enhances TIMP-I transcription, but has little effect on MMP-9 mRNA levels. The data show that chronic exposure to TNF-c~ alters the balance between u-PA/PAI-1 and MMPs/TIMP-1, which favors higher activity of proteinases. Accordingly, the presence of TNF-c~ in chronic inflammatory episodes would be expected to alter bone remodeling by inhibiting maturation of ECM and formation of bone.
Stem Cell Research & Therapy
Background Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs. Methods MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluoresc...
Dual Role of P2 Receptors during Osteoblast Differentiation
P2 receptors activated by ATP are expressed in the skeletal system. However, the role of P2 receptors in osteoblast differentiation remains unclear. Methods: Participation of P2 receptors in differentiation was investigated in the preosteoblast MC3T3-M1 cell line. Preosteoblasts were stimulated for 7 or 14 days in the presence of osteogenic medium containing ATP and its analogs, and then alkaline phosphatase (ALP) activity, gene expression analyses, and protein expression were assessed. Results: We observed that ATP and its analogs promoted increased ALP activity after 7 days of treatment.
P2 receptor expression, signaling and function in osteoclasts
Frontiers in bioscience (Scholar edition), 2011
Skeletal development and bone remodeling depend on the coordinated activity of osteoblasts and osteoclasts, which are responsible for bone formation and resorption, respectively. Mature osteoclasts result from the fusion of precursor cells, and they are large, multinucleated, highly specialized cells. Cellular release of ATP and UTP occurs in response to a variety of stimuli including mechanical stimulation, which occurs in the bone environment. ATP and UTP or their metabolites can then act on P2 receptors in the plasma membrane to induce various responses in bone cells. The influence of these receptors on osteoclast physiology and bone physiology in general is beginning to be understood, but much work is still required. This review focuses on P2 receptors in osteoclasts, their expression, signaling and function in the regulation of osteoclast formation, resorptive activity and survival.
TNAP, TrAP, ecto-purinergic signaling, and bone remodeling
Journal of Cellular Biochemistry, 2008
Bone remodeling is a process of continuous resorption and formation/mineralization carried out by osteoclasts and osteoblasts, which, along with osteocytes, comprise the bone multicellular unit (BMU). A key component of the BMU is the bone remodeling compartment (BRC), isolated from the marrow by a canopy of osteoblast-like lining cells. Although much progress has been made regarding the cytokinedependent and hormonal regulation of bone remodeling, less attention has been placed on the role of extracellular pH (pH e). Osteoclastic bone resorption occurs at acidic pH e. Furthermore, osteoclasts can be regarded as epithelial-like cells, due to their polarized structure and ability to form a seal against bone, isolating the lacunar space. The major ecto-phosphatases of osteoclasts and osteoblasts, acid and alkaline phosphatases, both have ATPase activity with pH optima several units different from neutrality. Furthermore, osteoclasts and osteoblasts express plasma membrane purinergic P2 receptors that, upon activation by ATP, accelerate bone osteoclast resorption and impair osteoblast mineralization. We hypothesize that these ecto-phosphatases help regulate [ATP] e and localized pH e at the sites of bone resorption and mineralization by pH-dependent ATP hydrolysis coupled with P2Y-dependent regulation of osteoclast and osteoblast function. Furthermore, osteoclast cellular HCO À 3 , formed as a product of lacunar V-ATPase H þ secretion, is secreted into the BRC, which could elevate BRC pH e , in turn affecting osteoblast function. We will review the existing data addressing regulation of BRC pH e , present a hypothesis regarding its regulation, and discuss the hypothesis in the context of the function of proteins that regulate pH e .
Gene, 2015
Bone development and homeostasis require the interplay between several cell types, including mesenchymal osteoblasts and osteocytes, as well as hematopoietic osteoclasts. Recent evidence suggests that cell proliferation, differentiation and apoptosis of both mesenchymal and hematopoietic stem cells, which are fundamental for tissue regeneration and treatment of degenerative diseases, is controlled by P2 receptors (i.e., P2X and P2Y receptors). Both types of P2 receptors are versatile transducers of diverse signals activated by extracellular nucleotides like ATP that are released in response to tissue injury, infection or shear stress. The P2X family of receptors has been shown to mediate multiple signaling events including the influx of calcium, activation of mitogen activated protein kinases (MAPKs) and induction of AP-1 family members known to regulate bone development. Support for the significance of P2X 7 in regulating bone development and homeostasis has been provided by several studies focusing on animal models and single nucleotide polymorphisms. P2 receptors are functionally expressed in both bone forming osteoblasts and bone resorbing osteoclasts, while recent findings also suggest that these receptors translate mechanical stimuli in osteocytes. Their ability to respond to external nucleotide analogs renders these cell surface proteins excellent targets for skeletal regenerative therapies. This overview summarizes mechanisms by which nucleotide receptors control skeletal cells and contribute to bone tissue development remodeling and repair.