Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts.
CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders induced sustained [Ca(2+)](i) increases in CD38(-) target cells. Co-culture on CD38(+) feeders enhanced the proliferation of CD38(-) target cells over control values and significantly shortened the S phase of cell cycle. These results demonstrate a paracrine process based on Cx43-mediated release of NAD(+), its CD38-catalyzed conversion to extracellular cADPR, and influx of this nucleotide into responsive cells to increase [Ca(2+)](i) and stimulate cell proliferation.
Franco
L
L
G. Gaslini Institute, Largo G. Gaslini 5, 16147 Genova, Italy.
Zocchi
E
E
Usai
C
C
Guida
L
L
Bruzzone
S
S
Costa
A
A
De Flora
A
A
D016475:3T3 Cells; D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D018920:Coculture Techniques; D018031:Connexin 43 / Q000235:genetics / Q000502:physiology; D036563:Cyclic ADP-Ribose; D003600:Cytosol / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins; D051379:Mice; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018031:Connexin 43; D008562:Membrane Glycoproteins; D009097:Multienzyme Complexes; D020319:Oligodeoxyribonucleotides, Antisense; D011994:Recombinant Proteins; D009243:NAD; D036563:Cyclic ADP-Ribose; D000246:Adenosine Diphosphate Ribose; D036541:ADP-ribosyl Cyclase; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1; D002118:Calcium