title: Activation of latent transforming growth factor-beta1 by nitric oxide in macrophages: role of soluble guanylate cyclase and MAP kinases.
abstract: 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.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Mallikarjuna Reddy
authors.initials: MR
authors.lastname: Metukuri
authors.forename: Rajaie
authors.initials: R
authors.lastname: Namas
authors.forename: Chase
authors.initials: C
authors.lastname: Gladstone
authors.forename: Thierry
authors.initials: T
authors.lastname: Clermont
authors.forename: Bahiyya
authors.initials: B
authors.lastname: Jefferson
authors.forename: Derek
authors.initials: D
authors.lastname: Barclay
authors.forename: Linda
authors.initials: L
authors.lastname: Hermus
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
authors.forename: Ruben
authors.initials: R
authors.lastname: Zamora
authors.forename: Yoram
authors.initials: Y
authors.lastname: Vodovotz
mesh_terms: D000818:Animals; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D006162:Guanylate Cyclase / Q000502:physiology*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology*; D015290:Second Messenger Systems; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D014945:Wound Healing / Q000502:physiology
chemical_list: D053773:Transforming Growth Factor beta1; D009569:Nitric Oxide; D048049:Extracellular Signal-Regulated MAP Kinases; D006162:Guanylate Cyclase