Identification of a novel pathway of transforming growth factor-β1 regulation by extracellular NAD+ in mouse macrophages: in vitro and in silico studies

• Published in: The Journal of biological chemistry 2012-09, Vol.287 (37), p.31003-31014
• Main Authors: Zamora, Ruben, Azhar, Nabil, Namas, Rajaie, Metukuri, Mallikarjuna R, Clermont, Thierry, Gladstone, Chase, Namas, Rami A, Hermus, Linda, Megas, Cristina, Constantine, Gregory, Billiar, Timothy R, Fink, Mitchell P, Vodovotz, Yoram
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calcium Ionophores - pharmacology; Cell Biology; Cell Line; Cyclic ADP-Ribose - analogs & derivatives; Cyclic ADP-Ribose - genetics; Cyclic ADP-Ribose - metabolism; Cyclic ADP-Ribose - pharmacology; Macrophages - cytology; Macrophages - metabolism; Mice; Mice, Mutant Strains; Models, Biological; NAD - genetics; NAD - metabolism; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; Transforming Growth Factor beta1 - genetics; Transforming Growth Factor beta1 - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.344309

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.