Regulation of Rac1 activation by the low density lipoprotein receptor-related protein

Abstract

The low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1-expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR-/- MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK- cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR-/- MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal-regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.

Figure 1.

Characterization of uPAR-deficient MEFs. (A) Genomic DNA PCR was performed to detect the intact uPAR allele in A1, B1, and C1 cells, using primers that hybridize with exon 3 of the uPAR gene. The PAI-1 allele was also detected as an internal control for total DNA. The lane labeled “control” shows DNA from the tail of a normal mouse. (B) Cell-surface uPAR in uPAR−/− (A1 and A2), uPAR+/− (B1 and B2), and uPAR+/+ (C1 and C2) cells was detected by plasminogen activation assay. 0.5 mM amiloride was added to some cultures to inhibit uPA-specific plasminogen activation. Substrate hydrolysis was standardized based on cell protein. (C) Expression of LRP-1 in uPAR−/− (A1), uPAR+/− (B1), and uPAR+/+ (C1) cells and in MEF-1 (LRP-1-positive) and MEF-2 (LRP-1-negative) cells was detected by immunoblot analysis using an antibody 11H4.

Figure 2.

Migration of uPAR-deficient MEFs. (A) uPAR−/− (A1 and A2), uPAR+/− (B1 and B2), and uPAR+/+ (C1 and C2) MEFs were allowed to migrate through Transwell membranes that were precoated with purified vitronectin or type I collagen. Migrating cells were detected by crystal violet staining. (B) uPAR−/− (A1), uPAR+/− (B1), and uPAR+/+ (C1) MEFs were treated with DMSO (−) or 10 μM PD098059 (+) for 15 min at RT and then allowed to migrate through Transwell membranes that were precoated with 20% FBS. Migrating cells were detected by crystal violet staining. (C) A1, B1, and C1 MEFs were cotransfected to express dominant-negative MEK1 (DN-MEK1) and pEGFP-N1. Control cells were cotransfected with equal amounts of empty vector (pBK-CMV) and pEGFP-N1. After culturing for 24 h, the cells were allowed to migrate for 4 h through serum-coated membranes. Migration was determined by fluorescence microscopy. Results are expressed relative to the migration rate of A1 cells that were cotransfected with pBK-CMV and pEGFP-N1 (mean ± SEM).

Figure 3.

uPAR regulates Rac1 activation in MEFs. (A) uPAR−/− (A1 and A2) and uPAR+/+ (C1 and C2) MEFs were cultured in serum-supplemented medium for 18 h and then extracted to detect GTP-loaded Rac1. (B) uPAR−/− (A1 and A2) and uPAR+/+ (C1 and C2) MEFs were cotransfected with constructs encoding N17Rac1 and pEGFP-N1 or with pBK-CMV (empty vector) and pEGFP-N1. Cell migration was studied using serum-coated membranes. Migration was determined by fluorescence microscopy. Results are expressed relative to the migration rate of A1 cells that were cotransfected with pBK-CMV and pEGFP-N1 (mean ± SEM).

Figure 4.

uPAR expression alters the morphology of MEFs. uPAR+/+ C1 cells and uPAR−/− A1 cells were plated in serum-coated chamber slides for the indicated times and stained with rhodamine-conjugated phalloidin. Images were collected by fluorescence microscopy. Bar, 10 μm.

Figure 5.

LRP-1 expression suppresses ERK activation in serum-deprived MEFs. (A) MEF-1 (LRP+/+) and MEF-2 (LRP−/−) cells were extracted without serum deprivation or after serum deprivation for 18 h. Phosphorylated and total ERK were detected by immunoblot analysis. (B) ERK phosphorylation in MEF-2 cells was determined as a function of time after serum deprivation.

Figure 6.

LRP-1 suppresses Rac1 activation. (A) MEF-1 and MEF-2 cells were cultured in DME and 10% FBS for 18 h and then extracted to detect activated and total Rac1. The level of activated Rac1 (standardized based on total Rac1) was determined by densitometry scanning using ImageQuant software to generate the values shown in the bar graph (mean ± SEM, n = 3). The asterisk indicates statistical significance (unpaired t test, P < 0.05). (B) MEF-2 cells were transfected to express VLDLr or with empty vector (pRc/CMV). Expression of the VLDLr and Rac1 activation were determined 4 d later. The level of activated Rac1 in VLDLr-expressing MEF-2 cells, compared with vector-transfected control cells, is shown in the bar graph (mean ± SEM, n = 3). The asterisk indicates statistical significance (unpaired t test, P < 0.05). (C) uPAR-deficient A1 cells and uPAR-expressing C1 cells were cultured in the absence (−) or presence (+) of 200 nM GST-RAP for 3 d. Rac1 activation was then determined. The values shown in the bar graph are standardized against uPAR−/− cells that were not treated with RAP (mean ± SEM, n = 3). The asterisk indicates a statistical significance between C1 cells that were cultured in the presence and absence of RAP (unpaired t test, P < 0.05). (D) LM-TK⁻ and L929 cells were cultured in the presence or absence of 200 nM GST-RAP for 3 d. Rac1 activation was then determined. The bar graph shows values that were standardized against activated Rac1 in LM-TK⁻ cells or L929 cells that were not RAP-treated (mean ± SEM, n = 3). The asterisk indicates that Rac1 activation in L929 cells treated with RAP was significantly different from Rac1 activation in the same cells that were not treated with RAP (unpaired t test, P < 0.05). In control experiments, we demonstrated that GST does not affect Rac1 activation (not depicted).

Figure 7.

The morphology of LRP-1-expressing and -deficient MEFs. MEF-1 and MEF-2 cells were plated in serum-coated chamber slides for the indicated times and stained with rhodamine-conjugated phalloidin. Images were collected using the fluorescence microscope. Bar, 10 μm.

Figure 8.

ERK and Rac1 in migration of LRP-1-deficient MEFs. (A) MEF-1 and MEF-2 cells were treated with 10 μM of the MEK inhibitor, PD098059 or with vehicle (DMSO) for 15 min at RT, and were then allowed to migrate through serum-coated Transwell membranes. The number of migrating cells was determined by crystal violet staining. (B) MEF-1 and MEF-2 cells were cotransfected to express dominant-negative MEK1 (DN-M) or dominant-negative Rac1 (DN-R) and GFP. Control cells were transfected with empty vector (pBK-CMV) and pEGFP-N1. Cell migration experiments were performed 24 h after transfection. Migration was determined by fluorescence microscopy. Results are expressed relative to the migration rate in MEF-1 cells transfected with pBK-CMV and pEGFP-N1 (n = 4). The asterisk indicates statistical significance at the P < 0.05 level.

Figure 9.

Effects of LRP-1 neutralization on MEF migration in the absence of uPAR. (A) uPAR−/− (A1 and A2), uPAR+/− (B1 and B2), and uPAR+/+ (C1 and C2) MEFs were cultured in the presence or absence of 200 nM GST-RAP for 3 d. Cells were allowed to migrate through Transwell membranes that were precoated with FBS. Migration was determined by crystal violet staining. The asterisk indicates that the bar is significantly different from the control (no RAP treatment; unpaired t test, P < 0.01, n = 3). (B) Migration of LM-TK⁻ and L929 cells was studied after culturing these cells in the presence or absence of GST-RAP for 3 d. Cells were allowed to migrate through Transwell membranes that were precoated with vitronectin or fibronectin. Cell migration was determined by crystal violet staining. The asterisk indicates statistical significance (unpaired t test, P < 0.01; n = 3).