Regulation of human mesenchymal stem cell functions by an autocrine loop involving NAD+ release and P2Y11-mediated signaling.
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.
Advanced Biotechnology Center, Genova, Italy.
Floriana
F
Fruscione
Sonia
S
Scarfì
Chiara
C
Ferraris
Santina
S
Bruzzone
Federica
F
Benvenuto
Lucrezia
L
Guida
Antonio
A
Uccelli
Annalisa
A
Salis
Cesare
C
Usai
Emanuela
E
Jacchetti
Cristina
C
Ilengo
Silvia
S
Scaglione
Rodolfo
R
Quarto
Elena
E
Zocchi
Antonio
A
De Flora
D000255:Adenosine Triphosphate / Q000378:metabolism; D050156:Adipogenesis; D019898:Autocrine Communication; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D005434:Flow Cytometry; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D010012:Osteogenesis; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015290:Second Messenger Systems
D020746:Calcium Channels, L-Type; D018031:Connexin 43; D016207:Cytokines; C109734:P2RY11 protein, human; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D009243:NAD; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D015232:Dinoprostone; D002118:Calcium