IL-1beta signals through the EGF receptor and activates Egr-1 through MMP-ADAM

Abstract

The immediate-early gene Egr-1 controls the inducible expression of many genes implicated in the pathogenesis of a range of vascular disorders, yet our understanding of the mechanisms controlling the rapid expression of this prototypic zinc finger transcription factor is poor. Here we show that Egr-1 expression induced by IL-1beta is dependent on metalloproteinases (MMP) and a disintegrin and a metalloproteinase (ADAM). Pharmacologic MMP/ADAM inhibitors and siRNA knockdown prevent IL-1beta induction of Egr-1. Further, IL-1beta activates Egr-1 via the epidermal growth factor receptor (EGFR). This is blocked by EGFR tyrosine kinase inhibition and EGFR knockdown. IL-1beta induction of Egr-1 expression is reduced in murine embryonic fibroblasts (mEFs) deficient in ADAM17 despite unbiased expression of EGFR and IL-1RI in ADAM17-deficient and wild-type mEFs. Finally, we show that IL-1beta-inducible wound repair after mechanical injury requires both EGFR and MMP/ADAM. This study reports for the first time that Egr-1 induction by IL-1beta involves EGFR and MMP/ADAM-dependent EGFR phosphorylation.

Figure 1. IL-1beta-inducible Egr-1 expression is MMP/ADAM-dependent.

Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). (A) Egr-1 mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. (B) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. (C) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. (D) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. (E) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.

Figure 2. Induction of Egr-1 by IL-1beta is EGFR-dependent.

Growth-quiescent SMCs were incubated with EGFR inhibitors AG1478 (5 µM) and PD153035 (5 µM) for 30 min, and exposed to IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). (A) Egr-1 mRNA levels in AG1478- and PD153035-treated SMCs incubated with IL-1beta by qPCR. Data were normalized to beta-actin. (B) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with AG1478 or PD153035. (C) Western blotting for EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with EGFR siRNA (0.1 µM) and treated with IL-1beta for 30 min. (D) Western blotting for ErbB4, EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ErbB4 siRNA (0.1 µM) and treated with IL-1beta for 30 min. (E) Egr-1 mRNA levels in AG825 (10 µM for 30 min)-treated SMCs incubated with IL-1beta for 30 min by qPCR. Data were normalized to beta-actin. (F) Western blotting for Egr-1 or beta-actin in total extracts of SMCs pretreated with AG825 (10 µM for 30 min) then stimulated with IL-1beta for another 30 min.

Figure 3. IL-1beta trans-activates EGFR in a time-dependent manner and it is dependent on MMP activity.

(A) Growth-quiescent SMCs were stimulated with IL-1beta (10 ng/ml) for the indicated times. Total protein extracts were resolved by SDS-PAGE and subjected to Western blot analysis using antibodies for Egr-1, EGFR phospho-Tyr⁸⁴⁵, total EGFR and beta-actin. Alternatively, Western blotting for EGFR phospho-Tyr⁸⁴⁵ or EGFR of total extracts of SMCs treated with (B) AG1478 (5 µM), (C) GM6001+ or GM6001- (25 µM) (D) TAPI-1 (10 µM), PD153035 (5 µM) for 30 min followed by IL-1beta stimulation for 5 min.

Figure 4. ADAM17 is required for IL-1beta–inducible Egr-1 expression.

(A) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. (B) Interaction of ¹²⁵I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10⁴ cells/well) were incubated with increasing amounts of ¹²⁵I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.

Figure 5. MMP and EGFR inhibitors block SMC wound repair.

Growth-quiescent SMCs were incubated with GM6001+ or GM6001- (25 µM), TAPI-1 (10 µM), AG1478 (5 µM), PD153035 (5 µM) or vehicle for 30 min followed by in vitro scraping injury and addition of IL-1beta (10 ng/ml). Photographs of the denuded zone were taken at 24 h.