Discoidin domain receptors regulate the migration of primary human lung fibroblasts through collagen matrices

Abstract

Background: The two discoidin domain receptors (DDRs), DDR1 and DDR2 are receptor tyrosine kinases (RTKs) with the unique ability among RTKs to respond to collagen. We have previously shown that collagen I induces DDR1 and matrix metalloproteinase (MMP)-10 expression through DDR2 activation and a Janus kinase (JAK)2 and extracellular signal-regulated kinase (ERK)1/2-mediated mechanism in primary human lung fibroblasts suggesting that these signaling pathways play a role in fibroblast function. Fibroblasts can traverse basement membrane barriers during development, wound healing and pathological conditions such as cancer and fibrosis by activating tissue-invasive programs, the identity of which remain largely undefined. In the present work, we investigated the role of DDRs and DDR-associated signal transduction in these processes.

Results: Transwell migration experiments showed that normal human lung fibroblast (NHLF) transmigration through collagen I-coated inserts is mediated by DDR2 and the DDR2-associated signaling kinases JAK2 and ERK1/2, but not DDR1. Additionally, experiments with specific small interfering (si)RNAs revealed that collagen I-induced expression of MMP-10 and MMP-2 is DDR2 but not DDR1 dependent in NHLFs. Our data showed that collagen I increases NHLF migration through collagen IV, the main component of basement membranes. Furthermore, basal and collagen I-induced NHLF migration through collagen IV-coated inserts was both DDR2 and DDR1 dependent. Finally, DDR2, but not DDR1 was shown to be involved in fibroblast proliferation.

Conclusions: Our results suggest a mechanism by which the presence of collagen I in situations of excessive matrix deposition could induce fibroblast migration through basement membranes through DDR2 activation and subsequent DDR1 and MMP-2 gene expression. This work provides new insights into the role of DDRs in fibroblast function.

Figure 1

Collagen I induces normal human lung fibroblast (NHLF) migration through collagen IV. NHLFs were grown on non-coated 8.0 μm polycarbonate inserts, or inserts coated with collagen IV (10 μg/cm²) or fibronectin (5 μg/cm²). Cells were serum-starved for 24 h and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for 24 h. The bottom chamber was treated with cell dissociation buffer and acetomethoxycalcein (calcein AM) for 1 h at 37°C. Fluorescence of solution with detached cells was measured at 485 nm excitation and 520 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, **P < 0.01).

Figure 2

Collagen I induces matrix metalloproteinase (MMP)-2 expression through discoidin domain receptor (DDR)2, but not DDR1 in normal human lung fibroblasts (NHLFs). NHLFs were reverse transfected with negative control small interfering (si)RNA, and DDR2-specific and DDR1-specific siRNA using lipofectamine RNAiMAX. At 48 h after transfection, NHLFs were serum-starved for 24 h and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for 16 h. Total RNA was isolated, reverse transcribed and real-time quantitative PCR was performed using the TaqMan system with specific primers and TaqMan probes for human, MMP-2 (A), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The expression changes (fold increase) were calculated relative to unstimulated control cells after normalizing with GAPDH. Results are representative of mean fold increase ± SD of three independent experiments performed in triplicate (n = 9, *P < 0.05). NHLFs were transfected with DDR2-specific or DDR1-specific siRNAs or negative control siRNA prior to starvation and 16 h of collagen I stimulation. MMP-2 (B) protein was measured in the culture supernatant by ELISA. Results are representative of mean fold increase ± SD of three independent experiments performed in triplicate (n = 9, *P < 0.05).

Figure 3

Normal human lung fibroblast (NHLF) migration through collagen I and collagen IV is discoidin domain receptor (DDR)2-dependent. NHLFs were reverse transfected with negative control small interfering (si)RNA, or specific siRNA for DDR2, DDR1 (A, C), Janus kinase (JAK)2, or extracellular signal-regulated kinase (ERK)1/2 (B, D) using lipofectamine RNAiMAX. At 48 h after transfection, NHLFs were serum-starved for 24 h and transferred to 8.0 μm polycarbonate inserts coated with collagen I (10 μg/cm²) (A, B) or collagen IV (10 μg/cm²) (C, D). Where indicated, cells were incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for an additional 16 h (E). The bottom chamber was treated with cell dissociation buffer and acetomethoxycalcein (calcein AM) for 1 h at 37°C. The fluorescence of solution with detached cells was measured at 485 nm excitation and 520 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, *P < 0.05, **P < 0.01).

Figure 4

Normal human lung fibroblast (NHLF) migration through collagen IV is matrix metalloproteinase (MMP)-2-dependent. NHLFs were reverse transfected with negative control small interfering (si)RNA, or specific siRNA for MMP-10, or MMP-2 using lipofectamine RNAiMAX. At 48 h after transfection, NHLFs were serum-starved for 24 h and transferred to 8.0 μm polycarbonate inserts coated collagen IV (10 μg/cm²). Cells were incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for an additional 16 h. The bottom chamber was treated with cell dissociation buffer and acetomethoxycalcein (calcein AM) for 1 h at 37°C. The fluorescence of solution with detached cells was measured at 485 nm excitation and 520 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, *P < 0.05).

Figure 5

Normal human lung fibroblast (NHLF) proliferation is discoidin domain receptor (DDR)2, but not DDR1 dependent. NHLFs were reverse transfected with DDR2-, DDR1-specific small interfering (si)RNA or negative control siRNA using lipofectamine RNAiMAX. At 48 h after transfection, NHLFs where transferred to a 96-well black walled plate at 3,000 cells/well. Cells were serum-starved for 24 h and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for an additional 16 h. Cell proliferation was determined using the DELFIA proliferation assay kit and measuring time-resolved fluorescence at 340 nm excitation and 615 nm emission. Results are representative of mean fold increase ± SD of two independent experiments performed in triplicate (n = 6, **P < 0.01).