Mechanisms of tissue inhibitor of metalloproteinase 1 augmentation by IL-13 on TGF-β1–stimulated primary human fibroblasts

Abstract

TGF-beta induces expression of tissue inhibitor of metalloproteinase 1 (TIMP-1), a potent inhibitor of matrix metalloproteinases that controls extracellular matrix metabolism and deposition. IL-13 alone does not induce TIMP-1, but in combination with TGF-beta it augments TIMP-1 expression. Although these interactions have implications for remodeling in asthma, little is understood regarding the mechanisms controlling TIMP-1 product. To explore the role of Smads and mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in the TIMP-1 augmentation by IL-13+TGF-beta1 in primary human airway fibroblasts. Real-time PCR, Western blot, ELISA, and transient transfection were used to evaluate the mechanisms of TIMP-1 augmentation. IL-13 enhanced TGF-beta1-induced Smad-2 and Smad-3 phosphorylation, transient transfection with dominant-negative Smad-2 or Smad-3 decreased TIMP-1 mRNA expression in the presence of TGF-beta1 and IL-13+TGF-beta1 through inhibition of Smad-2 or Smad-3 phosphorylation. ERK phosphorylation was increased by IL-13 and IL-13+TGF-beta1. MEK-ERK inhibition decreased TIMP-1 mRNA/protein to a greater degree after IL-13+TGF-beta1 stimulation versus TGF-beta1 alone. MEK-ERK inhibition also significantly increased Akt phosphorylation under all conditions and decreased Smad-3 phosphorylation in the presence of IL-13+TGF-beta1. In contrast, phosphoinositide-3 kinase-Akt inhibition increased phosphorylation of ERK and Smads, leading to increased TIMP-1. These results indicate that IL-13 augments TGF-beta1-induced TIMP-1 expression through increased Smad phosphorylation. These increases occur as TGF-beta1 downregulates IL-13-induced phosphoinositide-3 kinase activation while leaving the positive effect of IL-13-induced ERK on Smad signaling. This augmentation of TGF-beta1-induced TIMP-1 by IL-13 could contribute to the fibrosis and airway remodeling seen in the presence of T(H)2 inflammation in asthma.