UDCA-LPE modulates different signaling pathways involved in hepatic fibrogenesis

Background: Liver fibrosis with deposition and remodeling of extracellular matrix (ECM) is regulated.by different signaling pathways. Ursodeoxycholyl Lysophosphatidylethanolamide (UDCA-LPE) is a synthetic bile acid-phospholipid conjugate with hepatoprotective and anti-fibrotic functions in vitro and in vivo. In this study we aim to elucidate signaling pathways, which mediate anti-fibrogenic action of UDCA-LPE.

Results: At membrane level ECM interacts with integrin receptors, which promotes pro-fibrogenic signaling. Focal Adhesion Kinase (FAK) and SRC play a central role in integrin signaling. After phosphorylation in response to integrin engagement, FAK and SRC kinase mediate activation of myofibroblasts. After UDCA-LPE treatment, p-FAK (Tyr 925 and Tyr 576/577) and p-SRC (Tyr416) are reduced in CL48 liver cells. The reduction of p-FAK and p-SRC is time dependent. The second messenger cAMP and cAMP dependent Protein Kinase A (PKA) can also suppress ECM production by inhibiting hepatic stellate cells differentiation. We determined PKA activity by using the antibody for PKA substrates (RRXS*/T*). PKA is activated 1 minute after UDCA-LPE treatment with a peak after 15 min. Phosphorylation of ERK, B-RAF and C-RAF by UDCA-LPE was also investigated by western blot. UDCA-LPE induced pERK (Thr 202/Tyr 204) after 1 minute, the activation maximized at 15 min and lasted more than 2 hours. ERK is phosphorylated by MEK, which is mediated by B-RAF and C-RAF in CL48 cells. UDCA-LPE induced pB-RAF (Ser 445) and pC-RAF (Ser 338) after 1 minute. We next investigated the crosstalk between FAK, ERK and PKA signaling pathways. FAK autophosphorylation inhibitor Y15 significantly increased the phosphorylation of ERK. After Y15 treatment phosphorylation of ERK became less sensitive to UDCA-LPE and activation of C-RAF by UDCA-LPE was inhibited. These results suggest that activation of C-RAF and ERK by UDCA-LPE is dependent on FAK inhibition. EGFR inhibitor AG1478 also reduced UDCA-LPE mediated accumulation of pERK and inhibited C-RAF phosphorylation, suggesting activation of C-RAF and ERK by UDCA-LPE is EGFR dependent. PKA inhibitor Rp-cAMP reduced pERK and inhibited B-RAF phosphorylation, suggesting activation of B-RAF and ERK is PKA dependent.

Conclusions: UDCA-LPE disturbs pro-fibrogenic integrin signaling by inhibiting FAK and SRC activity. FAK inhibition was identified as an upstream event of activation of the EGFR/C-RAF/ERK pathway. UDCA-LPE further activates PKA, which subsequently induces B-RAF/ERK signaling. By inhibiting pro-fibrogenic pathways with concomitant activation of pro-proliferative ERK signaling, UDCA-LPE may improve hepatofibrogenesis and accelerate liver regeneration.

Corresponding author: Pathil, Anita

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