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Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-alpha-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is24 h, but it is decreased to approximately 8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-alpha, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-alpha is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.

Elastase/anti-elastase imbalance is a hallmark of emphysema, a chronic obstructive pulmonary disease associated with the rupture and inefficient repair of interstitial elastin. We report that neutrophil elastase (NE) at low physiologic concentrations, ranging from 35 nm to 1 μm, invokes transient, peaking at 15 min, activation of extracellular signal-regulated kinases 1 and 2 (ERK) in elastogenic lung fibroblasts. ERK activation is preceded by the release of soluble 25–26-kDa forms of epidermal growth factor (EGF) and transactivation of EGF receptor (EGFR) in NE-exposed cells. The stimulatory effect of NE on ERK is abrogated in the presence of anti-EGF-neutralizing antibodies, EGFR tyrosine kinase inhibitor (AG1478), and ERK kinase inhibitor (PD98059), as well as abolished in both EGFR-desensitized and endocytosis-arrested fibroblasts. Nuclear accumulation of activated ERK is associated with transient, peaking at 30 min, induction of c-Fos and sustained, observed at 24–48 h, decrease of tropoelastin mRNA levels in NE-challenged cells. Pretreatment of fibroblasts with AG1478 or PD98059 abrogates the NE-initiated tropoelastin mRNA suppression. We conclude that proteolytically released EGF signals directly via EGFR and ERK to down-regulate tropoelastin mRNA in NE-challenged lung fibroblasts.

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

Previously, we have demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in confluent rat lung fibroblasts via the binding of a Fra-1-c-Jun heterodimer to an activator protein-1-cAMP response element in the distal region of the elastin promoter. In the present study, we show that bFGF activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, resulting in the translocation of phosphorylated extracellular signal-regulated kinase 1/2 into the nucleus followed by increased binding of Elk-1 to the serum response element of the c-Fos promoter, transient induction of c-Fos mRNA, and sustained induction of Fra-1 mRNA. The addition of PD-98059, an inhibitor of mitogen-activated protein kinase kinase, abrogates the bFGF-dependent repression of elastin mRNA expression. Comparative analyses of confluent and subconfluent fibroblast cultures reveal significant differences in elastin mRNA levels and activator protein-1 protein factors involved in the regulation of elastin transcription. These findings suggest that bFGF modulates specific cellular events that are dependent on the state of the cell and provide a rationale for the differential responses that can be expected in development and injury or repair situations.