PD-MSCsPRL-1 decreased ER stress in a rat model of BDL and in hepatocytes
In the UPR pathway, PERK-eIF2α-ATF4-CHOP signaling is implicated in liver diseases [23]. To analyze UPR pathway activation by PD-MSCPRL−1 transplantation, we assessed the expression of CHOP, which is an ER stress-mediated transcription factor, in liver tissues using immunohistochemistry (IHC). The expression of CHOP in the nuclei of hepatocytes was significantly increased in the BDL-induced nontransplantation (NTx) group compared to the normal control (Con) group (*p < 0.05). Interestingly, the CHOP level was reduced, and CHOP translocation into the nucleus was significantly decreased in PD-MSCsPRL−1 (Fig. 1a and Supplementary Fig. 1a, *p < 0.05). In the NTx group, phosphorylated eIF2α (p-eIF2α) expression was increased at 1 and 2 weeks; however, the expression of PERK, ATF4 and CHOP decreased.
In contrast, the transplantation groups (Tx) showed decreased levels compared to the NTx group. Especially, the expression of PERK and p-eIF2α was significantly reduced in the PD-MSCPRL−1 group (Fig. 1b-e, *p < 0.05). Thapsigargin (TG) is an inhibitor of SERCA2b in hepatocyte-specific calcium channels on the ER membrane, resulting in cellular apoptosis (Fig. 1f). To induce ER stress in hepatocytes, we analyzed the expression of ER stress markers after 500 nM TG treatment for 24 h regardless of PD-MSC and PD-MSCPRL−1 cocultivation (Fig. 1g). The protein levels of ER stress markers in the WB-F344 cells treated with TG were substantially increased. However, their expression levels were significantly decreased after PD-MSC cocultivation. Specifically, PD-MSCPRL−1 cocultivation resulted in significantly reduced expression compared to that of naïve PD-MSCs (Fig. 1h and i, *p < 0.05). These findings indicated that PD-MSCsPRL−1 more efficiently decreased ER stress than naïve PD-MSCs in cirrhotic rat livers and hepatocytes.
PD-MSCsPRL-1 regulated calcium channels in a rat model of BDL and in hepatocytes
A calcium imbalance between the ER and mitochondria in the liver leads to the development of chronic metabolic diseases and impaired organelle function [24]. So, we confirmed the expression levels of calcium channels in the ER membrane (e.g., SERCA2b and IP3R) and mitochondria (e.g., VDAC1 and MCU) as well as ER-mitochondrial Ca2 + transfer factors (e.g., GRP75) in a rat model of BDL by Western blotting (Fig. 2a). The expression levels of SERCA2b, IP3R, and GRP75 were increased in the NTx group compared to the normal group. In contrast, all Tx groups showed decreased levels compared with the NTx group. In the PD-MSCPRL−1 group, the GRP75 levels were significantly decreased at 1 and 3 weeks (Fig. 2b-e, *p < 0.05). However, IP3R and GRP75 expression in the PD-MSCPRL−1 group was dramatically increased compared with that in the PD-MSC group (Supplementary Fig. 2a and b). Interestingly, the mRNA and protein levels of VDAC1 and MCU were increased in the PD-MSCPRL−1 Tx group (Fig. 2e and f, Supplementary Fig. 2c and d, *p < 0.05). Additionally, TG treatment induced a decrease in the expression levels of IP3R, VDAC1, and MCU, whereas the expression levels of GRP75 and CaM were increased in the WB-F344 hepatocyte cell line. With PD-MSCPRL−1 cocultivation, the expression levels of IP3R, VDAC1, MCU, and GRP75 were significantly changed compared to those with naïve PD-MSCs, although no significant change was found in the mRNA level between the treated group and the cocultivation group (Supplementary Fig. 2e-i, *p < 0.05). However, the CaM levels were decreased (Fig. 2g and h). Also, we confirmed the degree of colocalization with SERCA2b and ER Tracker by immunofluorescence (IF) staining (Fig. 2i). The expression of SERCA2b in the NTx group was increased; however, the expression was significantly decreased in the PD-MSCPRL−1 group compared to the NTx group and the PD-MSC group (Fig. 2j, *p < 0.05). These results indicated that PD-MSCsPRL−1 efficiently regulate calcium channels between the ER and mitochondria in cirrhotic rat livers and hepatocytes.
PD-MSCsPRL-1 regulate calcium influx in rat hepatocytes
To further analyze the concentration of calcium in hepatocytes, we confirmed the mRNA and protein levels of SERCA2b by qRT-PCR and Western blotting, respectively. Their mRNA expression levels were increased in the TG-treated group compared to the normal group but decreased in the cocultivation group (Fig. 3a). Although the protein expression was reduced after TG treatment, the expression of SERCA2b was significantly increased in cocultivated PD-MSCs and PD-MSCsPRL−1 (Fig. 3b and c, *p < 0.05). In particular, the mRNA expression of SERCA2b and STIM1, which are calcium sensors in the ER, was significantly increased in the PD-MSCsPRL−1 (Supplementary Fig. 3a and b). To investigate the change in calcium in the ER, cytoplasm, and mitochondria of hepatocytes, we analyzed these organelles using calcium biosensors, including ER-LAR-GECO for the ER, Y-GECO1 for the cytoplasm, and mito-GEM-GECO1 for the mitochondria (Fig. 3d). The influx of ER Ca2 + in the TG-treated WB-F344 cells declined, whereas the influx of cytoplasmic and mitochondrial Ca2 + was enhanced. Interestingly, PD-MSCPRL−1 cocultivation induced an enhanced ER Ca2 + concentration while decreasing the influx of cytoplasmic and mitochondrial Ca2 + compared to that of PD-MSCs (Fig. 3e-g, *p < 0.05). These results indicated that PD-MSCPRL−1 cocultivation modulates calcium influx in ER stress-induced hepatocytes.
PRL-1 regulates EGFR-PI3K-CaM calcium signaling in the BDL-injured rat liver
To further investigate calcium signaling by PRL-1, we analyzed the expression of EGFR in hepatocytes injured by pentamidine regardless of cocultivation and recombinant PRL-1 treatment. As shown in Fig. 4a, PRL-1 binds to EGFR and activates PI3K-p110α expression. PIP2 is split into IP3, and released IP3 binds to IP3R on the ER membrane. These signaling pathways release Ca2 + and regulate the cellular response via the Ca2+/CaM complex (Fig. 4a). We performed an RTK dot blot assay and identified which RTK was phosphorylated among 49 different RTKs (Fig. 4b). Compared to PD-MSCs, PD-MSCsPRL−1 strongly activated phosphorylated EGFR after recombinant PRL-1 treatment. In addition, recombinant PRL-1 and pentamidine inhibition treatment reduced the activity of phosphorylated EGFR (Fig. 4c, *p < 0.05). The expression of PI3K, which is a downstream factor of EGFR through PRL-1, was increased in the PD-MSCPRL−1 group compared to the NTx and PD-MSC groups, whereas the CaM levels were strongly decreased at 1, 3, and 5 weeks (Fig. 4e and f, *p < 0.05). Additionally, endogenous CaM levels in cirrhotic livers in the NTx group were significantly increased; however, their expression levels were significantly reduced in the PD-MSCPRL−1 group compared to the naïve PD-MSC group (Fig. 4g and h, *p < 0.05). These results suggest that PRL-1 interacts with EGFR-PI3K-CaM and regulates calcium levels in BDL-injured rat livers.
Hepatic regenerative effects of PD-MSCsPRL-1 in a rat model of BDL
Previously, we reported that administration of PD-MSCsPRL−1 improved hepatic functions in a rat model of BDL [20]. In the NTx group, the collagen accumulation in liver tissues was significantly increased compared to that in the normal group. However, the amount of Sirius red-positive areas significantly declined in all Tx groups. In particular, antifibrotic effects due to decreased collagen deposition were dramatically observed in the PD-MSCPRL−1 group compared to the PD-MSC group (Fig. 5a and b, *p < 0.05). In the blood chemistry analysis, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were decreased in the transplantation groups compared to the NTx group, whereas the albumin (ALB) level was significantly increased (Fig. 5c-f, *p < 0.05). In particular, the PD-MSCPRL−1 group exhibited substantially decreased ALT and AST and increased ALB levels. To further confirm the proliferation of hepatocytes in the livers of BDL-induced rats by transplantation, we examined proliferating cell nuclear antigen (PCNA) by IHC (Fig. 5g). Compared to that of naïve PD-MSCs, the positive signal was significantly enhanced in the PD-MSCPRL−1 groups (Fig. 5h, *p < 0.05). Additionally, the expression of hepatic ALB and proliferation markers (e.g., CDK4 and cyclin D1) in the PD-MSCPRL−1 group was remarkably higher than that in the PD-MSC group (Fig. 5i). The correlation coefficient between ALB and cyclin D1 expression was R2 = 0.8819 (Fig. 5j). These results suggest that PD-MSCsPRL−1 promote hepatic regeneration in a BDL-injured rat model.