As nonmicrobicidal effects of LL-37 have been reported to be receptor mediated, we investigated whether HCECs express FPRL1, a known receptor for LL-37.
17 RT-PCR was performed to study FPRL1 expression in HCECs
(Fig. 5A) . Both SV40-HCECs and P-HCECs expressed FPRL1 mRNA (
n = 3, each). FPRL1 is a receptor linked to a Gi protein. To determine whether LL-37-induced migration is mediated via a G-protein-coupled receptor (GPCR) such as FPRL1, we studied the effect of pertussis toxin (PTX, a Gi inhibitor). PTX alone did not affect cell migration (data not shown). As shown in
Figure 5B , pretreatment with PTX either partially (100 ng/mL PTX) or almost completely (250 ng/mL PTX) eliminated LL-37-stimulated SV40-HCEC migration (
n = 3,
P < 0.05, Student’s
t-test), suggesting involvement of a GPCR. To determine whether FPRL1 was the GPCR involved, we then studied the effect of WRW
4 (a FPRL1-specific peptide antagonist) on LL-37-induced SV40-HCECs migration. WRW
4 partially reduced LL-37-mediated migration by up to 57% (
Fig. 5C ,
n = 5). Similar findings with PTX and WRW
4 were observed with P-HCECs (
n = 2–3). When WRW
4 was tested at 50 μM, 75% and 79% inhibition of LL-37-stimulated migration were seen in SV40-HCECs (
n = 1) and P-HCECs (
n = 2), respectively. To study the involvement of mitogen activated protein kinase (MAPK; p38MAPK; c-Jun-N-terminal-kinase, c-JNK; extracellular signal-regulated kinase, ERK1/2), tyrosine kinase (TK), protein kinase C (PKC), phosphatidyl inositol-3 kinase (PI
3K), and epidermal growth factor receptor (EGFR) signaling in LL-37-induced HCEC migration, assays were performed using SV40-HCECs preincubated with inhibitors of these cellular signaling pathways. When tested alone, each inhibitor did not have a significant effect on cell migration (data not shown). As shown in
Figure 6A , the p38 MAPK inhibitor SB600125 attenuated LL-37-stimulated migration by 25% (
n = 3), but the decrease did not reach statistical significance (
P = 0.08, Student’s
t-test). PD98059 (an ERK1/2 inhibitor) and SP203580 (a c-JNK inhibitor) inhibited the stimulatory effect of LL-37 on cell migration by 60% and 78%, respectively. As shown in
Figure 6B , the TK inhibitor genistein partially (by 49%) blocked LL-37 induced cell migration, whereas H-7 (a PKC inhibitor) had no inhibitory effect (
n = 3–4). Of the inhibitors tested, the PI
3K inhibitor LY294002 was the most effective and inhibited LL-37-mediated migration by 92%. AG1478 (an EGFR-TK inhibitor) was found to inhibit migration in a concentration-dependent manner and effectively reduced LL-37-mediated migration by up to 85% (
Fig. 6C ,
n = 2–3). All these observations were confirmed in experiments with P-HCECs (
n = 1–2).