Identification of a novel pathway of transforming growth factor-ß1 regulation by extracellular NAD+ in mouse macrophages: in vitro and in silico studies.

Extracellular ß-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-ß1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-ß1 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-ß1, 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-ß1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-ß1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-ß1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-ß1.

Activation of latent transforming growth factor-beta1 by nitric oxide in macrophages: role of soluble guanylate cyclase and MAP kinases.

The inducible nitric oxide (NO) synthase and the cytokine transforming growth factor-beta1 (TGF-beta1), both central modulators of wound healing, interact reciprocally: TGF-beta1 generally suppresses iNOS expression, while NO can induce and activate latent TGF-beta1. We have shown that chemical NO activates recombinant human latent TGF-beta1 by S-nitrosation of the latency-associated peptide (LAP), a cleaved portion of pro-TGF-beta1 that maintains TGF-beta1 in a biologically-inactive state. We hypothesized that cell-associated TGF-beta1 could be activated by NO via known NO-inducible signaling pathways (soluble guanylate cyclase [sGC] and mitogen-activated protein [MAP] kinases). Treatment of mouse RAW 264.7 macrophage-like cells with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) led to a dose- and time-dependent increase in cell-associated active and latent TGF-beta1, as assessed by quantitative immunocytochemistry for active TGF-beta1 vs. LAP and partially validated by western blot analysis. Treatment with the sGC inhibitor 1,H-[1,2,4]oxadiazole[4,3-a]quinoxalon-1-one (ODQ) reduced both active and latent TGF-beta1 dose-dependently. SNAP, in the presence or absence of ODQ or the MAP kinase inhibitors, did not affect steady-state TGF-beta1 mRNA levels. Treatment with inhibitors specific for JNK1/2, ERK1/2, and p38 MAP kinases suppressed SNAP-induced active and latent TGF-beta1. Treatment with the cell-permeable cGMP analog 8-Br-cGMP increased both active and latent TGF-beta1. However, TGF-beta1 activation induced by 8-Br-cGMP was not blocked by MAP kinase inhibitors. Our findings suggest that NO activates latent TGF-beta1 via activation of sGC and generation of cGMP and separately via MAP kinase activation, and may shed insight into the mechanisms by which both cGMP production and MAP kinase activation enhance wound healing.