LRP1 regulates remodeling of the extracellular matrix by fibroblasts

Abstract

Low density lipoprotein receptor-related protein (LRP1) is an endocytic receptor for diverse proteases, protease inhibitors, and other plasma membrane proteins, including the urokinase receptor (uPAR). LRP1 also functions in cell-signaling and regulates gene expression. The goal of this study was to determine whether LRP1 regulates remodeling of provisional extracellular matrix (ECM) by fibroblasts. To address this problem, we utilized an in vitro model in which type I collagen was reconstituted and overlaid with fibronectin. Either the collagen or fibronectin was fluorescently-labeled. ECM remodeling by fibroblasts deficient in LRP1, uPAR, or MT1-MMP was studied. MT1-MMP was required for efficient remodeling of the deep collagen layer but not involved in fibronectin remodeling. Instead, fibronectin was remodeled by a system that required urokinase-type plasminogen activator (uPA), uPAR, and exogenously-added plasminogen. LRP1 markedly inhibited fibronectin remodeling by regulating cell-surface uPAR and plasminogen activation. LRP1 also regulated remodeling of the deep collagen layer but not by controlling MT1-MMP. Instead, LRP1 deficiency or inhibition de-repressed a secondary pathway for collagen remodeling, which was active in MT1-MMP-deficient cells but not in uPAR-deficient cells. These results demonstrate that LRP1 regulates ECM remodeling principally by repressing pathways that require plasminogen activation by uPA in association with uPAR.

Fig. 1

ECM remodeling by MT1-MMP-deficient (−/−) and expressing (+/+) fibroblasts. The substratum consisted of fluorescently-labeled fibronectin layered on top of type I collagen. (A) Representative cells are shown. Darkened or black areas represent “areas of clearing” or “AOCs”, which are measured as evidence of ECM remodeling. (B) Quantification of the total AOCs for individual MT1-MMP(−/−) or (+/+) cells. AOCs were measured using image J software (mean ± SEM). (C) MT1-MMP-deficient and wild type skin fibroblasts were plated on fibronectin layered over fluorescein-labeled type 1 collagen and allowed to remodel the ECM for 3 h prior to fixation. Representative cells are shown. Many MT1-MMP-defiicent cells showed no evidence of remodeling. (D) Quantification of the total AOCs for wild type and MT1-MMP-deficient cells (mean ± SEM).

Fig. 2

ECM remodeling by LRP1-deficient MEF-2 cells and LRP1-expressing PEA-10 cells. The substratum consisted of fluorescently-labeled fibronectin layered on top of type I collagen. (A) Representative cells are shown. Darkened or black areas represent AOCs. (B) Quantification of AOCs for individual PEA-10 and MEF-2 cells 2h and 3h after plating. AOCs were measured using image J software (mean ± SEM).

Fig. 3

Effects of protease inhibitors on ECM remodeling. The experimental substratum for these studies consisted of fluorescently-labeled fibronectin layered over type I collagen. (A) PEA-10 and MEF-2 were treated with 25 µM aprotinin or with vehicle. Remodeling was allowed to occur for 3 h. (B) PEA-10 and MEF-2 cells were treated with 0.5 mM amiloride, 10 µM GM6001 or with vehicle (DMSO) and then allowed to remodel ECM for 3 h. (C) PEA-10 and MEF-2 cells were cultured on the ECM surface for 3 h in serum-free medium. Cells were permeabilized and treated with the nuclear stain, DAPI. Note that neither cell type remodeled fibronectin in the absence of serum.

Fig. 4

Remodeling of ECM in which the deep layer of type 1 collagen is fluorescently labeled. The experimental substratum for these studies consisted of fibronectin layered over fluorescently-labeled type I collagen. (A) Representative PEA-10, MEF-2, and B-41 cells are shown. These cells were allowed to remodel the ECM for 3 h prior to fixation. Cells were permeabilized and treated with the nuclear stain, DAPI and Phalloidin conjugated to Alexa 598. AOCs are black or darkened areas devoid of fluorophore. (B) Quantification of the total AOCs for individual PEA-10, MEF-2 and B-41 cells. AOCs were measured using image J software (mean ± SEM). (C) PEA-10 and MEF-2 cells were treated with 25 µM aprotinin, 20 mM εACA, or with vehicle. MEFs were allowed to remodel ECM for 3 h. The bar graph shows that the total AOC associated with individual cells was decreased by aprotinin and εACA (mean ± SEM).

Fig. 5

uPAR is essential for the effects of LRP1 on ECM remodeling. (A) uPAR(+/+) and uPAR(−/−) MEFs were cultured in the presence of GST-RAP (0.2 µM) or GST (0.2 µM), as a control, for 72 h. The cells were then allowed to remodel ECM, in which fluorescently-labeled type 1 collagen was over-coated with fibronectin, for 3 h. Representative cells are shown. (B) AOCs are shown for uPAR(+/+) and uPAR(−/−) cells that were treated with GST-RAP or GST (mean ± SEM).

Fig. 6

LRP1 regulates ECM remodeling in the presence and absence of MT1-MMP. (A) Total RNA was isolated from PEA-10 and MEF-2 cells and analyzed by qPCR using specific primers for MT1-MMP. (B) Equal amounts of cellular protein from detergentsoluble cell extracts of PEA-10 and MEF-2, which were labeled with biotin, were subjected to affinity precipitation. The precipitates were analyzed by immunoblot analysis to determine “surface” MT1-MMP. Whole cell extracts also were subjected to immunoblot analysis to detect “total” MT1-MMP and tubulin, as a loading control. (C) MT1-MMP-deficient and wild-type skin fibroblasts were cultured in the presence of GST-RAP (0.2 µM) or GST for 72 h. Cells were plated on fibronectin layered over fluoresceinlabeled type 1 collagen and allowed to remodel the ECM for 3 h prior to fixation. AOCs were measured using image J software (mean ± SEM). (D) PEA-10 cells were co-transfected to express RFP and with MT1-MMP-specific or non-targeting control siRNA. Total RNA was isolated from these cells and analyzed by qPCR using specific primers for MT1-MMP. (E) RFP-transfected PEA-10 cells in which MT1-MMP was silenced or not were cultured in the presence of GST-RAP (0.2 µM) or GST for 72 h. The cells were plated on fibronectin layered over fluorescein-labeled type 1 collagen. Transfected cells were allowed to remodel the ECM for 3 h prior to fixation. AOCs of RFP-positive cells were measured using image J software (mean ± SEM).