Proteinase-activated receptors (PARs) are a novel family of
G-protein-coupled receptors. PAR2 has been implicated in inflammatory airways disease. Although fibroblasts are pathologically important in the airways, the proinflammatory role of PAR2 in these cells remains unknown. We assessed PAR expression and functionality in human primary bronchial fibroblasts (HPBFs) before assessing PAR2-mediated HPBF proliferation,
cytokine production, and adhesion molecule expression. RT-PCR and flow cytometry demonstrated that HPBFs express hPAR1, hPAR2, and hPAR3, but not hPAR4. Intracellular calcium signaling in HPBFs in response to PAR agonists showed that only hPAR1 and hPAR2 were functional receptors. We used the MTT assay to assess HPBF proliferation. Of the PAR2 agonist
proteinases or selective PAR2-activating
peptides (PAR2-APs) tested, none stimulated HPBF proliferation, whereas
thrombin was a HPBF
growth factor.
mRNA for
IL-8 and
granulocyte colony-stimulating factor (
G-CSF) was upregulated after addition of
SLIGKV-NH2 when assessed by RT-PCR. No significant increase in
G-CSF or
IL-8 protein was detected.
Trypsin stimulated
IL-8 and
G-CSF release from HPBF in a time- and dose-dependent manner.
Leupeptin and soya
trypsin inhibitor abrogated
trypsin-stimulated
cytokine release, indicating a requirement for
trypsin's proteolytic activity.
Trypsin and
SLIGKV-NH2 stimulated an increase in
VCAM-1 expression at 12 h
after treatment, which declined thereafter. PAR2-driven upregulation of
VCAM-1 cell surface expression and the release of
IL-8 and
G-CSF from bronchial fibroblasts may be important in promoting neutrophilic airways
inflammation.