Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

Abstract

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.

FIG. 1.

The sphingomyelin/ceramide pathway is required for TNF-α-induced SMC and fibroblast proliferation. (A) TNF-α-induced DNA synthesis was quantified using [³H]thymidine incorporation, as indicated in Materials and Methods. SMC were seeded in RPMI containing 10% FCS and then were starved in RPMI without FCS for 24 h and stimulated by TNF-α (2 ng/ml) after pretreatment with DMAPP (10 μmol/liter, 12 h) or DMS (2 μmol/liter, 1 h) as indicated. (B) Western blot of ERK1/2 kinase activation by TNF-α, showing time course and effect of DMAPP or DMS (used as for panel A). Western blots were revealed by anti-activated (phospho)-ERK1/2 and by anti-ERK2 antibodies. (C and D) Time course of nSMase (C) and SK-1 (D) activation in SMC treated with TNF-α. (E) Effect of exogenous bacterial SMase (100 mU/ml, 1 h) on DNA synthesis in SMC. (F to H) Effect of siRNA targeting human nSMase2 (h-nSM/siRNA) and/or cotransfection of a plasmid coding for the murine nSMase2 (m-nSM-plasmid for rescue experiments, with m-nSM-plasmid being resistant to h-nSM/siRNA) on nSMase activation (F), DNA synthesis (G), and ERK1/2 phosphorylation (H) triggered by TNF-α in human SMC. Results are means ± SEM form three to five separate experiments. *, P < 0.05 (comparison between cells treated with or without TNF-α, as indicated).

FIG. 2.

MMP2 is required for TNF-α-induced proliferation via nSMase activation. (A and B) SMC (A) or MMP2^+/+ or MMP2^−/− fibroblasts (Fbl) (obtained from MMP2^+/+ and MMP2^−/− mice, respectively) (B) were serum starved for 24 h in RPMI and stimulated with TNF-α (T) or 10% FCS (S) in the presence or absence of batimastat (10 nM). DNA synthesis was evaluated by [³H]thymidine incorporation. (C to E) Enzyme activities were measured in culture media (MMP2) and cell lysates (nSMase) from SMC or fibroblasts treated with or without TNF-α (2 ng/ml), batimastat, and MMP2/siRNA. Results are means ± SEM from three separate experiments. *, P < 0.05 (comparison with controls). ns, not significant.

FIG. 3.

Exogenous MMP2 mimics cell signaling induced by TNF-α. (A to C) nSMase and SK-1 activities and DNA synthesis were measured in SMC treated or not with MMP2/siRNA and stimulated for 40 min by exogenous MMP2 (exoMMP2) (1 nM, activated for 2 h by 10 μM APMA) (2). (D) MMP2^−/− and MMP2^+/+ fibroblasts were stimulated by exogenous MMP2 for 40 min as described above, and then nSMase activities were determined. Control experiments for toxicity using activated MMP2 were done simultaneously and exhibited no toxicity for the cells during the time course of the experiment (not shown). Results are means ± SEM from three separate experiments. *, P < 0.01 (comparison between MMP2-treated and untreated cells). ns, not significant.

FIG. 4.

Role of MT1-MMP in TNF-α-induced proliferation via MMP/SMase. (A) Time course of MT1-MMP enzymatic activity in wt SMC, zv-SMC (SMC transfected with an empty pcDNA3-zeo vector), and iMT1-SMC (SMC overexpressing pro-iMT1-MMP, a mutated form of MT1-MMP [MMP-E240A], which is catalytically inactive but cleavable by furin to form iMT1-MMP). Cells were treated with TNF-α (2 ng/ml). MT1-MMP activity in cell lysate was determined using a fluorogenic substrate, as described in Materials and Methods. (B) Western blot of MT1-MMP in SMC and iMT1-SMC treated or not with TNF-α (0 to 120 min) and revealed by anti-MT1-MMP and anti-β-actin (upper and lower panels, respectively). (C to E) Evaluation of MMP2 and nSMase activities and DNA synthesis in wt SMC and iMT1-SMC treated (when indicated) by TNF-α. (F to H) Rescue experiment using mouse fibroblasts transfected with MT1-MMP/siRNA and cotransfected with a plasmid coding for the human form of MT1-MMP (MT1) or with the empty vector (E). After 24 h of transfection, siRNA and plasmid were removed and MT1-MMP (F), MMP2 (G), and nSMase (H) activities were quantified. Inset in panel F, silencing effect of MT1-MMP siRNA on MT1-MMP expression in SMC. (I) Western blot showing ERK1/2 activation in the presence of TNF-α in cells transfected or not with MT1-MMP/siRNA in the presence or absence of MT1-MMP plasmid. Results are means ± SEM from three separate experiments. *, P < 0.05 (comparison between cells treated or not with TNF-α, or as indicated). ns, not significant.

FIG. 5.

Exogenous MT1-MMP triggers cell signaling (activation of MMP2, nSMase, and SK-1) and DNA synthesis. (A to D) Effect of exogenous MT1-MMP (Exo MT1, 1 nM) activated by 10 μM APMA. The MMP2, nSMase, and SK-1 activities and DNA synthesis were evaluated in SMC treated or not with activated MT1-MMP (Exo MT1) for 40 min, as indicated in Materials and Methods. (E and F) MMP2^−/− and MMP2^+/+ fibroblasts were stimulated by exogenous active MT1-MMP for 40 min as described above, and then MMP2 and nSMase activities were determined. Control experiments for toxicity using activated MT1-MMP were done simultaneously and exhibited no toxicity for the cells during the time course of the experiment (not shown). Results are means ± SEM from three separate experiments. *, P < 0.01 (comparison between cells treated or not with exo MT1-MMP).

FIG. 6.

Furin is required for TNF-α-induced MMP/nSMase/SK-1 activation. (A) Time course of furin activation in wt SMC, furin-silenced SMC (pretreated with specific siRNA for 24 h), SMC/PDX (SMC transfected with pCR/CMV-PDX plasmid), and SMC treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl-ketone (FI) at 25 μM for 16 h and then treated with TNF-α (2 ng/ml) and time course of furin in MMP2^+/+ or MMP2^−/− fibroblasts treated with TNF-α (2 ng/ml). (B) Western blot of MT1-MMP activation induced by TNF-α in wt SMC or SMC/PDX. Membranes were blotted with anti-MT1-MMP and anti-β-actin antibodies (the arrow indicates the active form of MT1-MMP). (C to F) Determination of MT1-MMP, MMP2, nSMase, and SK-1 activities in wt SMC, SMC/PDX, FI-treated SMC, and furin-silenced SMC stimulated with TNF-α (2 ng/ml). (G) TNF-α-induced ERK1/2 phosphorylation in wt SMC and SMC/PDX. Western blots were labeled with anti-activated-ERK1/2 and ERK2 antibodies. Results are representative of three separate experiments. (H) DNA synthesis induced by TNF-α in wt SMC, furin-silenced SMC, SMC/PDX, and FI-treated SMC. Results are means ± SEM from three or four separate experiments. *, P < 0.05 (comparison between cells treated or not with TNF-α).

FIG. 7.

Role of TGN and vesicular transport in furin and nSMase activation by TNF-α. Brefeldin A (10 μM) or monensin (10 μM) was incubated with SMC for 6 h before stimulation with TNF-α and furin (A) or nSMase (B) activity determination. Brefeldin and monensin controls were done simultaneously and exhibited no toxicity for the cells during the time course of the experiment (not shown). Results are means ± SEM from three separate experiments. *, P < 0.05 (comparison between treated and untreated cells at time zero).

FIG. 8.

Schematic diagram of the signaling pathways activated by mitogenic concentrations of TNF-α and roles of furin, MT1-MMP, and MMP2 in the activation of the nSMase in mesenchymal cells. nSMase is the first step of the sphingomyelin/ceramide pathway, which leads to S1P generation (when the intermediate steps, including ceramidase and SK, are working). S1P can in turn interact with Edg/S1P receptors, which trigger a signaling mitogenic cascade involving ERK1/2 activation. This effect is mimicked by adding exogenous activated MT1-MMP and MMP2 (light blue). The sites of action of the molecular and pharmacological inhibitors used in the paper are indicated by green labels. Sm, sphingomyelinase.