Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis

Abstract

The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis.

Figure 1

Coagulation proteases increase uPA and PAI-1 expression in the culture supernatant of 4T1 and 67NR breast cancer cell lines. (A-B) Real-time PCR analysis of TF, PAR-1, PAR-2, uPA, and PAI-1 mRNA expression in 4T1 cells (A) and 67NR cells (B). Cells were grown to confluence and starved overnight. Results are shown as mean ± SEM of at least 3 independent experiments. (C-E) Serum starved confluent cell monolayers were incubated for 24 hours with the following coagulation factors: mFVIIa (10nM), FX (130nM), mFVIIa (10nM) and FX (130nM), FXa (125nM), or thrombin (FIIa; 20nM). Levels of uPA in treated 4T1 cells (C) and 67NR cells (D) were determined by ELISA. The amount of PAI-1 released from the treated 4T1 (E) and 67NR (F) cell lines was measured by a PAI-1 ELISA. Results are shown as mean ± SEM of at least 5 independent experiments. *P ≤ .05 and **P ≤ .001 (control versus protease treated).

Figure 2

Silencing PAR-1 and PAR-2 in 4T1 cells. (A-B) Real-time PCR analysis of PAR-1 (A) and PAR-2 (B) mRNA expression in 4T1^ΔPAR-1 and 4T1^ΔPAR-2 cells expressed as a percentage of the 4T1^GFP control. PAR-1 and PAR-2 levels were normalized to HPRT mRNA. uPA protein (C) and PAI-1 protein (D) were measured by ELISA after 24 hour incubation with FXa (125nM) or thrombin (20nM). Results are shown as mean ± SEM of 3 independent experiments. *P ≤ .05 and **P ≤ .001 (4T1^GFP versus 4T1^ΔPAR-1), ^¶P ≤ .05 and ^¶¶P ≤ .001 (4T1^GFP versus 4T1^ΔPAR-2), ^§P ≤ .05 and ^§§P ≤ .001 (4T1^ΔPAR-1 versus 4T1^ΔPAR-2).

Figure 3

Timecourse of uPA and PAI-1 mRNA and protein expression in 4T1 cells stimulated with FXa or thrombin. Levels of uPA mRNA (A) and protein (B) were determined by real-time PCR and ELISA, respectively. PAI-1 mRNA (C) and protein (D) induction were also measured using real-time PCR and ELISA. uPA and PAI-1 mRNA levels were normalized to HPRT mRNA. Results are shown as mean ± SEM of at least 3 independent experiments. *P ≤ .05 and **P ≤ .001 (control versus FXa treated). ^§P ≤ .05 and ^§§P ≤ .001 (control versus thrombin treated).

Figure 4

Stimulation of 4T1 cells with FXa or thrombin induces uPA secretion. (A) Intracellular uPA staining. Subconfluent 4T1 cells were starved overnight then incubated with SFM, FXa, or thrombin for 30 minutes. The cells were then fixed and permeablized on glass chamber slides. Cells were incubated with both uPA-fluorescein isothiocyanate (green) and GM130-Alexa Fluor555 (red) antibodies, then counterstained with DAPI (blue). Slides were viewed on an Olympus BX51WI fluorescence microscope fitted with an Olympus DP70 cooled digital color camera. Total magnification is 400 × (10 × ocular; 40 × objective). DP Controller version 2.2.1.227 software was used for image acquisition. GraphicConverter X V5.4 was used to compile images. (B-C) Serum starved 4T1 cells were incubated with FXa (125nM) or thrombin (20nM) for 1 hour. Cellular (B) and culture supernatant (C) uPA expressed as percentage of total uPA in 4T1 cells. Results are shown as mean ± SEM of 3 independent experiments. *P ≤ .05 (control versus FXa treated) and ^§P ≤ .05 (control versus thrombin treated). (D) Cells were treated with BFA (10 μg/mL) or vehicle control then stimulated with FXa (125nM) or thrombin (20nM) for 1 hour. uPA was quantified in cell culture supernatant. **P ≤ .05 (control versus FXa treated) and ^§P ≤ .05 (control versus thrombin treated). (E) uPA levels in the culture supernatant of 4T1 cells treated with 100nM or 200nM PMA for 1 hour. *P ≤ .05 and **P ≤ .001 (control versus PMA treated). Results are shown as mean ± SEM of 3 independent experiments.

Figure 5

Coagulation proteases activate the secretory pathway in the 4T1 cell line. (A) 4T1 cells were treated for the indicated amount of time with SFM, FXa, thrombin, or 200nM PMA. Blots were probed with antibodies for p-PKCμ and GAPDH. Data shown is representative of 3 independent experiments. (B) 4T1^GFP, 4T1^ΔPAR-1, and 4T1^ΔPAR-2 cells were treated with SFM, FXa, thrombin, or PMA for 5 minutes. Blots were probed with antibodies for p-PKCμ and GAPDH. Phosphorylated PKCμ was quantified by dividing the background-corrected p-PKCμ signal intensity by the background corrected GAPDH signal intensity. The blot shown is representative of at least 3 independent experiments. Quantification results are shown as mean ± SEM of at least 3 independent experiments. *P ≤ .05 (4T1^GFP versus 4T1^ΔPAR-1) and ^#P ≤ .05 (4T1^ΔPAR-1 versus 4T1^ΔPAR-2).

Figure 6

Proposed model of how coagulation protease activation of PAR-1 and PAR-2 regulates uPA and PAI-1 expression in mouse breast cancer cell lines. PAR-1 and PAR-2 can exist as individual receptors or in a complex. Cells, such as the 67NR cell line (left panel), express PAR-1 only whereas 4T1 cells express PAR-1 and a PAR-1/PAR-2 complex (right panel). The configuration of PAR-1 and PAR-2 determines the expression of uPA and PAI-1 in response to different coagulation proteases.