Role of ecto‐NTPDases on UDP‐sensitive P2Y₆ receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women

Abstract

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 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca(2+)](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 UTPγS was devoid of effect. Selective blockade of P2Y(6) receptors with MRS 2578 prevented [Ca(2+)](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(2+)](i) . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

Purinergic signalling in the musculoskeletal system

“…Recently, extracellular nucleotides have been reported to modulate the differentiation of human MSCs, with the P2X6, P2Y 4 and P2Y 14 receptors identified as playing pivotal roles in this process [292,293]. UDP activation of P2Y 6 receptors has also been shown to regulate the osteogenic differentiation of the primary MSCs from postmenopausal women [291]. The same study also demonstrated that NTPDases influence which osteoblast progenitors are driven into proliferation or differentiation [291].…”

“…UDP activation of P2Y 6 receptors has also been shown to regulate the osteogenic differentiation of the primary MSCs from postmenopausal women [291]. The same study also demonstrated that NTPDases influence which osteoblast progenitors are driven into proliferation or differentiation [291]. Furthermore, a detailed investigation using knockout mice identified that the P2Y 13 receptor regulates the terminal differentiation of MSCs into osteoblasts or adipocytes [207].…”

“…UTP and, to a lesser extent, ATP were also shown to stimulate human MSC migration [290]. Human MSCs express all P2Y receptors and the P2X3-7 receptors [291,292]. Recently, extracellular nucleotides have been reported to modulate the differentiation of human MSCs, with the P2X6, P2Y 4 and P2Y 14 receptors identified as playing pivotal roles in this process [292,293].…”

P2X7 nucleotide receptor signaling potentiates the Wnt/β-catenin pathway in cells of the osteoblast lineage

“…Data are means ± SEM (n = 3 independent preparations) compared to Wnt3a alone for up to 24 h post-treatment. As nucleotides are rapidly degraded once released into the extracellular milieu [32,33], it is likely that the prolonged potentiation of canonical Wnt signaling is due at least in part to changes in expression of secondary factors or pathway components. One possibility is that activation of P2X7 receptors leads to alterations in the expression of Wnt pathway components.…”

Purinergic signalling-evoked intracellular Ca2+ concentration changes in the regulation of chondrogenesis and skeletal muscle formation

“…Adipose tissue-derived stem cells (ATSCs) and dental follicle cells (DFCs) derived from the ectomesenchyme expressed some P2X (P2X 3,4,5,6,7 ) and all 8 P2Y receptor subtypes; interestingly, no mRNA transcripts of P2X 1 and P2X 2 receptors were identified in either cell type [90,91]. Extracellular nucleotides acting through P2 receptors (primarily P2X 6 , P2Y 4 , P2Y 6 , P2Y 13 and P2Y 14 ) are important modulators of MSC differentiation towards the osteogenic and adipogenic lineages [90][91][92]. The purinergic concept of MSC differentiation is further supported by a recent study showing substantial changes in ecto-nucleotide metabolism and ecto-nucleotidase expression profiles following chondrogenic differentiation of human umbilical cord-derived MSCs [93].…”