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

Ruiz PA, Jarai G - Fibrogenesis Tissue Repair (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, RH12 5AB, UK. gabor.jarai@novartis.com.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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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).

Mentions: DDRs have been implicated in the expression of several factors involved in cell migration and wound healing such as MMPs [24]. We have previously shown that collagen I induces MMP-10, and MMP-2, but not MMP-9 mRNA expression in NHLFs. Furthermore, collagen I-induced MMP-10 expression was shown to be DDR2 but not DDR1 dependent in these cells [20]. In the present study, collagen I-induced MMP-2 mRNA (Figure 2A) and protein expression (Figure 2B) in NHLFs was significantly inhibited by DDR2-specific, but not DDR1-specific, siRNAs. Taken together, these results suggest an important role of DDR2 in collagen I-induced expression of both MMP-10 and MMP-2.


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

Ruiz PA, Jarai G - Fibrogenesis Tissue Repair (2012)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298810&req=5

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).
Mentions: DDRs have been implicated in the expression of several factors involved in cell migration and wound healing such as MMPs [24]. We have previously shown that collagen I induces MMP-10, and MMP-2, but not MMP-9 mRNA expression in NHLFs. Furthermore, collagen I-induced MMP-10 expression was shown to be DDR2 but not DDR1 dependent in these cells [20]. In the present study, collagen I-induced MMP-2 mRNA (Figure 2A) and protein expression (Figure 2B) in NHLFs was significantly inhibited by DDR2-specific, but not DDR1-specific, siRNAs. Taken together, these results suggest an important role of DDR2 in collagen I-induced expression of both MMP-10 and MMP-2.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, RH12 5AB, UK. gabor.jarai@novartis.com.

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.

No MeSH data available.


Related in: MedlinePlus