*2.8. Integrin-Bound UDCA-LPE Translocated into Lipid Rafts, Which Co-Fractionated with LPE but Not UDCA*

The intracellular transport of a heterobivalent ligand could be determined by one of its receptors [21]. To investigate which receptor determined the localization of UDCA-LPE, we treated CL48 cells with UDCA, LPE or UDCA-LPE for 30 min and cell lysates were subjected to lipid-raft fractionation and the concentrations of UDCA, LPE or UDCA-LPE in 12 fractions were respectively determined by high-performance liquid chromatography-tandem mass spectrometry. UDCA was localized only in non-raft fractions, whereas LPE was present in both raft- and non-raft fractions (Figure 8A), suggesting that UDCA receptors were localized only in non-raft fractions whereas LPE receptors were present in both fractions. UDCA-LPE displayed an integrated localization of both UDCA and LPE and the proportion of UDCA-LPE in raft fractions 1–4 was in parallel to that of LPE (Figure 8A, Inset) suggesting that the initial localization of UDCA-LPE was determined by both UDCA- and LPE-receptors. GRGDSP, which inhibited the binding of UDCA-LPE to integrins, decreased the proportion of UDCA-LPE in non-raft fractions and increased the proportion in lipid-raft fractions, indicating that integrin-bound UDCA-LPE was initially localized in non-raft fractions. After incubation with UDCA-LPE for 2 h

or overnight an increased proportion of UDCA-LPE was detected in lipid rafts in a time-dependent manner (Figure 8B), suggesting a translocation of integrin-bound UDCA-LPE to lipid rafts at a longer incubation time. These data were consistent with the translocation of integrins into lipid rafts by UDCA-LPE treatment (Figure 7), suggesting a co-translocation of integrins with UDCA-LPE. Taken together, the co-translocation of integrins and UDCA-LPE was determined by LPE receptors.

**Figure 8.** Distribution of UDCA, LPE and UDCA-LPE in lipid fractions. (**A**,**B**) Lipid-raft fractionation of CL48 cells after treatment with (**A**) 90 μM UDCA, 90 μM LPE, 90 μM UDCA-LPE for 30 min or 200 μg/mL GRGDSP for 1 h and 90 μM UDCA-LPE for additional 30 min or (**B**) 90 μM UDCA-LPE for 30 min, 2 h or overnight. Separated fractions were subjected to liquid-chromatography mass spectrometry for quantification of UDCA, LPE or UDCA-LPE in UDCA, LPE or UDCA-LPE-treated cells, respectively. In each treated group, the total levels of UDCA, LPE or UDCA-LPE in 12 fractions was normalized as 100%. Data are means ± the standard deviation of three independent experiments. \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05. (**C**) Schematic time-dependent model for anti-fibrogenic effects of UDCA-LPE. The lightning graphic means stimulation: the binding of UDCA-LPE with integrin activates phosphorylation of FAK and SRC. The arrows mean (1) translocation of the UDCA-LPE complex into lipid rafts, which (2) results in the dephosphorylation of FAK and SRC.

#### **3. Discussion**

As effective therapeutic options against liver fibrosis are limited to date, the proposal of novel compounds which target pro-fibrogenic pathways is urgently needed. The bile acid-phospholipid conjugate UDCA-LPE has been proven to exhibit potent anti-fibrogenic functions in vitro and in vivo [5]. In this study, we analysed enforced translocation of integrins by UDCA-LPE as a possible mechanism for its anti-fibrogenic effects. We showed that UDCA-LPE can associate to the RGD-recognition motif in integrins and LPAR1 with its UDCA- and LPE-moiety, respectively. The latter binding acts as a transporter of UDCA-LPE into lipid-rafts occurring simultaneously with an internalization of UDCA-LPE-bound integrins to the ER and the nuclear envelope. The subsequent loss of SRC co-localization with integrins decreased phosphorylation levels of SRC and FAK leading to an inhibition of pro-fibrogenic activity.

Recent studies have reported that TUDCA stimulates integrin-dependent phosphorylation of SRC, FAK, ERK and p38MAPK [22,23]. Similar to TUDCA, UDCA and UDCA-LPE stimulated integrinand FAK-dependent c-Raf and ERK phosphorylation in CL48 cells as well (Figure 4). Interestingly, recent results using a 3D model of integrin α5β1 have shown the importance of the RGD-recognition motif as a sensor of TUDCA. However, TUDCA has an intracellular effect on integrin α5β1 rather than at the plasma membrane [24]. As UDCA and TUDCA are known to be located at the interfacial outer surface of plasma membrane [25,26], this may be the case for the binding of UDCA-LPE to the extracellular domain of integrins as we showed that UDCA-LPE was able to induce integrin internalization at plasma membrane (Figure 1) and that GRGDSP could inhibit the translocation of integrins (Figure 2B–D and Figure 3).

The design of heterobivalent ligands to target two different receptors has previously been used for pharmacological purposes [27]. As a novel heterobivalent ligand (Figure 8C), UDCA-LPE was not only able to bind to integrins with its UDCA-moiety but also triggered LPE/LPAR1 signalling through its LPE-moiety (Figure 5A–C). The stimulation of UDCA and LPE signalling occurred in the first 5 min of UDCA-LPE treatment by association of UDCA-LPE with integrins and LPAR1 (Figure 8, left). This process may be equivalent to UDCA + LPE treatment. However, the character of UDCA-LPE to bridge integrins and LPE receptors, which was confirmed by co-immunoprecipitation of integrins and LPAR1 (Figure 5D), rendered UDCA-LPE to have a specific function in pulling integrins into the intracellular transport pathway of LPE. We hypothesize that this results in the translocation of integrins from the plasma membrane to the ER and the nuclear envelop observed at a longer incubation time (Figure 8, right). Additional experiments have to further evaluate the localization and intracellular trafficking of endocytosed integrins. Our results showed that LPE alone had no effect on the localization of integrins and that UDCA-LPE-induced translocation of integrins and the inhibition of integrin signalling were dependent on the LPE-moiety of UDCA-LPE.

It has been reported that LPAR1 is localized partially in lipid rafts [28] and that disruption of lipid rafts impairs the function of LPAR1 [29,30]. As LPAR1 is a receptor of LPE [16], we also found that ~18% of LPE was localized in lipid rafts upon LPE treatment for 30 min (Figure 8A). Although UDCA has been reported to antagonize the deoxycholate-induced cholesterol depletion [31], it has been shown that UDCA owns a much higher affinity to non-raft than to lipid-raft fractions [25]. Consistent with this, almost no UDCA was detectable in raft fractions of UDCA-treated cells (Figure 8A). The localization of integrins in non-rafts and LPE endocytic transport pathway destined in lipid-rafts indeed allowed an opportunity for UDCA-LPE to be the mediator for integrin translocation (Figure 8C). UDCA-LPE was translocated into lipid rafts via LPE/LPAR1 axis (Figure 8) concomitant with its internalization (Figure 7) via UDCA/integrin axis.

The general mechanism for endocytic transport of LPE has not been well understood. LPARs are normally localized in both clathrin and caveolar endocytic microdomains and the latter is thought to respond to LPAR internalization because of LPAR co-localization with caveolin-1 in the nucleus [28]. It has been shown that LPAR-induced gene expression is insensitive to caveolea-disrupting agents filipin and methyl-β-cyclodextrin [28], suggesting that LPAR internalization does not necessarily rely on the

structure of caveolea. Our data also supported this notion as filipin or methyl-β-cyclodextrin treatment did not inhibit integrin translocation induced by UDCA-LPE (data not shown). The independency from caveolea was one of the features of UDCA-LPE-induced internalization of integrins which may be different from the previously reported integrin endocytosis/recycling pathway [32].

Integrins cross-talk with crucial pro-fibrogenic pathways such as TGFβ1 and PDGF signalling [33] and are therefore regarded as attractive therapeutic targets for the treatment of fibrotic disease. Most inhibitors of integrins including antibodies and cyclic RGD-containing peptides [34,35] have focused on the inhibition of integrin-induced cell-to-ECM and cell-to-cell interactions. However, the use of RGD peptides for fibrosis treatment is quite limited [36,37] because of their lack of persistent effects [38]. Due to multiple binding sites of integrins for ECM [39], an exclusive blockade of RGD-recognition motif may not completely disrupt the binding of integrins to ECM. Here, we could demonstrate that UDCA-LPE not only occupied the RGD-binding sites in integrins but also induced integrin internalization which completely disrupted the ECM-binding to integrins at the plasma membrane (Figure 8C). Thus, UDCA-LPE emerged as an effective inhibitor of RGD-binding integrins more potent than the typical RGD-containing peptide.

It is well-recognized that integrin-induced signalling plays a crucial role in fibrogenesis and that the downstream proteins FAK and SRC play an essential during pro-fibrotic signalling [40,41]. RGD peptide has been reported to activate integrins [42,43], which may also promote fibrogenic signalling. Our data supported this notion as we found that RGD peptide was able to induce integrin signalling (Figure 4D). Unlike RGD peptide, by removing the activator of FAK and SRC UDCA-LPE treatment led to persistent inhibition of integrin signalling after long incubation of CL48 cells and HHStec cells (Figures 2 and 8C, right) thus displaying a very potent anti-fibrogenic effect.

In present study, we demonstrated a possible novel pharmacological tool for integrin inhibition, where UDCA-LPE did not function as a direct inhibitor of integrins per se but as a heterobivalent ligand bridging between integrins and LPAR1. By the action of LPE/LPAR1 transporters in cells, UDCA-LPE was able to induce the translocation of integrins leading to a loss of co-localization with SRC, which resulted in dephosphorylation of FAK and SRC and inhibition of downstream fibrogenic targets. This elucidated mechanism of action renders UDCA-LPE as a drug candidate for the treatment of liver fibrosis.
