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

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 Ca²⁺. Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca²⁺ ionophores recapitulated the effects of NAD⁺ on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca²⁺ chelation, and antagonism of L-type Ca²⁺ 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.