Regulation of Human Mesenchymal Stem Cell Functions by an Autocrine Loop Involving NAD⁺Release and P2Y11-Mediated Signaling

Abstract

In several cell types, a regulated efflux of NAD(+) across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD(+) (NAD(+)(e)) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD(+) through Cx43 HC. NAD(+) efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca(2+) (5-6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD(+)(e) activates the purinergic receptor P2Y(11) and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/[Ca(2+)](i) signaling cascade, involving the opening, unique to MSC, of L-type Ca(2+) channels. Extracellular NAD(+) enhanced nuclear translocation of cAMP/Ca(2+)-dependent transcription factors. Moreover, NAD(+), either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E(2) and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD(+)(e). Controls included Cx43-siRNA transfected and/or NAD(+)-glycohydrolase-treated MSC (autocrine effects), and NAD(+)-untreated or P2Y(11)-siRNA-transfected MSC (exogenous NAD(+)). These findings suggest a potential beneficial role of NAD(+)(e) in modulating MSC functions relevant to MSC-based cell therapies.

Purinergic and Store-Operated Ca2+ Signaling Mechanisms in Mesenchymal Stem Cells and Their Roles in ATP-Induced Stimulation of Cell Migration

“…This study examined hDP-MSCs from several donors and obtained consistent evidence to demonstrate that P2X7, P2Y 1 , and P2Y 11 are the purinergic P2 receptors responsible for ATP-induced Ca 21 signaling in hDP-MSCs ( Figs. 1 and 2), confirming expression of these P2 receptors in MSCs reported by some previous studies using BM-MSCs and AT-MSCs [19][20][21][22]28]. Nonetheless, this study observed noticeable variations in the results obtained in cells from different donors, for example, at both mRNA and functional expression levels (Figs.…”

“…Accumulating evidence shows that MSCs release ATP constitutively or in response to mechanical stimulation [17][18][19][20]. Previous studies consistently demonstrated that ATP induced robust Ca 21 responses but reported expression of a bewildering variety of P2X and P2Y receptors in MSCs from different tissues and species [17][18][19][20][21][22][23][24][25][26][27][28] and, as a result, the cognate intrinsic mechanisms remains contentious. There is also evidence for occurrence of storeoperated Ca 21 entry in BM-MSCs [17], but the molecular identity of the Ca 21 channels is still elusive.…”

A critical look at the function of the P2Y11 receptor

“…Nicotinamide adenine dinucleotide (NAD + ) is also capable of activating P2Y 11 and causing an increase in intracellular Ca 2+ , IP 3 , and cAMP in P2Y 11 transfected 1321N1 cells [58,59]. Human mesenchymal stem cells and neutrophils are activated by NAD + presumably through P2Y 11 [60,61]. In these studies, specificity of the response was determined by inhibiting NAD + -signalling with G s and protein kinase A (PKA) inhibitor, NF157 antagonist, and P2RY11 knockdown.…”

“…The effects include a lower rate of CX 3 CL-mediated endothelial killing and migration in natural killer (NK) cells [123], delayed apoptosis in neutrophils [124], and increased chemotaxis in granulocytes [58,59]. In mesenchymal stem cells, NAD + stimulation of P2Y 11 resulted in cytokine release and chemotaxis [60]. P2Y 11 facilitated skin repair by the release of interleukin-6 (IL-6) in keratinocytes following IFNγ-induced ATP stimulation [125] and in LXA 4 -treated bronchial cystic fibrotic epithelium P2Y 11 promoted proliferation, migration, and wound repair [20].…”

Immunoregulation through extracellular nucleotides

“…87,88 P2Y11 activation by extracellular NAD ϩ also enhances the capacity of human bone marrow-derived mesenchymal stem cells to suppress effector T-cell proliferation. 89 Thus, P2Y11 seems to be involved in the immunomodulatory activity of mesenchymal stem cells in the bone marrow as well. Because no rodent ortholog of P2Y11 exists, 23,38 eATP may increase the intracellular cAMP concentration and exert immune suppression in humans, but not in naturally P2Y11-deficient murine cells (Figure 3).…”