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.

Figure 1

Increased expression of active and latent transforming growth factor-β1 (TGF-β1) protein induced by the nitric oxide donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) in RAW 264.7 macrophage-like cells. Mouse RAW264.7 macrophage-like cells were incubated with medium alone (A and B), with 100 μM SNAP (C and D), or with 1,000 μM SNAP (E and F) for 24 hours. The cells were then immunostained for active TGF-β1 (A, C, and E) or latent TGF-β1 (B, D, and F) as described in “Materials and methods,” and photomicrographs were taken at ×40 magnification. Arrows indicate the region from each respective field digitally magnified and depicted in the insets. Red bar (A) indicates 25 μm.

Figure 2

Nitric oxide induces a dose-dependent increase in active and latent transforming growth factor-β1 (TGF-β1) protein expression in RAW 264.7 macrophage-like cells. (A) RAW 264.7 cells were treated with 10–1,000 μM S-nitroso-N-acetyl-d,l-penicillamine (SNAP) for 24 hours, and the expression of TGF-β1 was analyzed as described in “Materials and methods.” Results shown are the mean ± SEM from six to seven independent experiments (*p < 0.005 vs. control; **p < 0.05 vs. 10 μM SNAP). (B) RAW 264.7 cells were treated with 100 μM SNAP or decomposed SNAP (either by heat treatment [T] or pH change) for 24 hours, and the expression of TGF-β1 was analyzed as described in “Materials and methods.” Results shown are the mean ± SEM. from four independent experiments (*p < 0.05 vs. control and decomposed SNAP (pH), **p < 0.01 vs. decomposed SNAP (T), ^#p < 0.05 vs. control and decomposed SNAP (T), ^##p < 0.01 vs. decomposed SNAP (pH). (C) RAW 264.7 cells were untreated, treated with 100 μM SNAP, or 100 μM decomposed SNAP. Cell lysates or defined amounts (5, 50, or 100 ng) of recombinant human latency-associated peptide (LAP) were subjected to anti-LAP and β-actin Western blot as described in “Materials and methods.” The experiment is representative of three; numbers indicate quantification (mean ± SEM) of LAP expression normalized for β-actin expression.

Figure 3

Increased expression of active and latent transforming growth factor-β1 (TGF-β1) protein induced by S-nitroso-N-acetyl-d,l-penicillamine (SNAP) is mediated partially by soluble guanylate cyclase (sGC). (A) Treatment of RAW 264.7 cells with 100 μM SNAP for 24 hours led to activation of latent TGF-β1 (increased brown stain; quantification shown). Co-treatment with the sGC inhibitor 1,H-[1,2,4]oxadiazole[4,3-a]quinoxalon-1-one (ODQ) reversed the activation of TGF-β1 induced by SNAP. Results shown are the mean ± SEM from four independent experiments (*p < 0.001 vs. control, **p < 0.05 vs. 0.3 μM ODQ, ^#p < 0.05 vs. control; ^$p < 0.05 vs. 0.3 μM ODQ). (B) (active) and (C) (latent): Treatment of RAW264.7 cells with the cell-permeable cGMP analog 8-Br-cGMP (100, 500, or 1,000 μM) shows a concentration- and time-dependent effect on both active (B) and latent (C) TGF-β1. Results shown are the mean ± SEM from six to seven independent experiments (^$p < 0.001, *p < 0.005, **p < 0.01 and ***p < 0.05 vs. control; ^#p < 0.05 vs. 100 μM 8-Br-cGMP). (D) Treatment with 8-Br-cGMP (500 μM) led to an increase in latent TGF-β1 protein levels as detected by Western blotting analysis using anti-latency-associated peptide. A representative blot (insert) and the densitometric analysis of five experiments expressed as the mean ± SEM are shown. (*p=0.002 vs. control; analyzed by Student t-test).

Figure 4

Increased expression of active and latent transforming growth factor-β1 (TGF-β1) protein induced by S-nitroso-N-acetyl-d,l-penicillamine (SNAP) is mediated partially via the mitogen-activated protein kinase pathway. (A—C) Treatment of RAW 264.7 cells with 100 μM SNAP for 24 hours led to activation of latent TGF-β1. (A) Co-treatment with the JNK1/2 inhibitor SP600125 reversed the expression of both latent and active TGF-β1 (reduced brown stain; quantification shown) induced by SNAP. Results shown are the mean ± SEM from five independent experiments (*p < 0.01 and ^#p < 0.005 vs. control). (B) Co-treatment with the ERK1/2 inhibitor PD98059 reversed the expression of both latent and active TGF-β1 (reduced brown stain; quantification shown) induced by SNAP. Results shown are the mean ± SEM from five independent experiments (*p < 0.005 vs. control; **p < 0.05 vs. 0.3 μM PD98059). (C) Co-treatment with the p38 inhibitor SB203580 only slightly reversed the activation of TGF-β1 and had no significant effect on the expression of latent TGF-β1. Results shown are the mean ± SEM from five independent experiments (*p < 0.001 vs. control).

Figure 5

Inhibition of soluble guanylate cyclase (sGC) or mitogen-activated protein kinases reduces S-nitroso-N-acetyl-d,l-penicillamine (SNAP)-induced latent transforming growth factor-β1 (TGF-β1) expression. (A) and (B) Co-treatment with both the ERK1/2 inhibitor PD98059 (3 μM) and the sGC inhibitor 1,H-[1,2,4]oxadiazole[4,3-a]quinoxalon-1-one (ODQ) (3 μM) inhibited the elevation of latent TGF-β1 protein levels induced by SNAP as detected by Western blotting analysis using anti-latency-associated peptide. A representative blot (A) and the densitometric analysis (B) of three independent experiments expressed as the mean ± SEM are shown. (*p < 0.005 vs. control; **p < 0.05 vs. PD98059 and ODQ, analyzed by one-way analysis of variance, followed by the Fisher LSD method).

Figure 6

Neither nitric oxide nor the soluble guanylate cyclase (sGC) and mitogen-activated protein (MAP) kinase signaling pathway affects steady-state transforming growth factor-β1 (TGF-β1) mRNA. RAW264.7 cells were treated with S-nitroso-N-acetyl-d,l-penicillamine (SNAP) (100 μM) in the absence or presence of the MAPK inhibitors (3 μM) or soluble guanylate cyclase inhibitor (3 μM) for 24 hours. Total RNA was isolated, followed by Northern blotting and analysis for TGF-β1 mRNA (20 μg total RNA/lane) as described in “Materials and methods.” A representative blot (A) and the densitometric quantification (B) of three independent experiments are shown.

Figure 7

Effect of nitric oxide and cGMP on ERK1/2 kinase phosphorylation. RAW264.7 macrophage-like cells were incubated with medium alone (control), S-nitroso-N-acetyl-d,l-penicillamine (SNAP) (100 μM), or 8-Br-cGMP (500 μM) for 30 minutes, 1, and 3 hours as indicated. Cell lysates were prepared and protein was isolated, followed by Western blotting and analysis for phospho-ERK1/2 and total ERK1/2 (A) or phospho-JNK1/2 and total JNK1/2 (B) as described in “Materials and methods.” A representative blot of six independent experiments for each panel, followed by the densitometric analysis of those experiments, expressed as the mean ± SEM, are shown. (*p=0.010 vs. control; analyzed by the Mann–Whitney rank sum test).

Figure 8

Effect of mitogen-activated protein kinases on soluble guanylate cyclase -induced expression of active and latent transforming growth factor-β1 (TGF-β1). RAW264.7 macro-phage-like cells were treated with 8-Br-cGMP (500 μM) in the absence or presence of the p38 inhibitor SB203580 (SB, 3 μM), the ERK1/2 inhibitor PD98059 (PD, 3 μM), or the JNK inhibitor SP600125 (SP, 3 μM), for 1 hour and 3 hours. The cells were then immunostained for active (A) or latent TGF-β1 (B) and analyzed as described in “Materials and methods.” Results shown are the mean ± SEM from eight independent experiments and represent fold change vs. cells treated with 8-Br-cGMP alone.