Tauroursodeoxycholic acid reduces ER stress by regulating of Akt-dependent cellular prion protein

Although mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine, ischemia-induced endoplasmic reticulum (ER) stress induces low MSC engraftment and limits their therapeutic efficacy. To overcome this, we investigated the protective effect of tauroursodeoxycholic acid (TUDCA), a bile acid, on ER stress in MSCs in vitro and in vivo. In ER stress conditions, TUDCA treatment of MSCs reduced the activation of ER stress-associated proteins, including GRP78, PERK, eIF2α, ATF4, IRE1α, JNK, p38, and CHOP. In particular, TUDCA inhibited the dissociation between GRP78 and PERK, resulting in reduced ER stress-mediated cell death. Next, to explore the ER stress protective mechanism induced by TUDCA treatment, TUDCA-mediated cellular prion protein (PrPC) activation was assessed. TUDCA treatment increased PrPC expression, which was regulated by Akt phosphorylation. Manganese-dependent superoxide dismutase (MnSOD) expression also increased significantly in response to signaling through the TUDCA-Akt axis. In a murine hindlimb ischemia model, TUDCA-treated MSC transplantation augmented the blood perfusion ratio, vessel formation, and transplanted cell survival more than untreated MSC transplantation did. Augmented functional recovery following MSC transplantation was blocked by PrPC downregulation. This study is the first to demonstrate that TUDCA protects MSCs against ER stress via Akt-dependent PrPC and Akt-MnSOD pathway.

roles PrP C plays in the survival of transplanted stem cells may provide insights into the protection of MSCs and development of PrP C -targeted therapeutics.
Tauroursodeoxycholic acid (TUDCA) is an endogenous hydrophilic tertiary bile acid produces in humans at a low level. TUDCA is approved by the U.S. Food and Drug Administration for use in biliary cirrhosis, and it is used effectively for cholestatic liver diseases 11 . Recent studies have revealed that TUDCA has an ameliorating effect on several diseases, including neurodegenerative diseases, osteoarthritis, vascular diseases, and diabetes [12][13][14][15] . In addition, TUDCA regulates stem cell differentiation into various lineages such as adipogenic and osteogenic lineages 16,17 . Mechanistic studies indicate that TUDCA attenuates ER stress, prevents unfolded protein response dysfunction, and stabilizes mitochondria 18 . However, little is known about the molecular mechanism by which TUDCA protects cells from oxidative stress. In particular, the potential for TUDCA regulation of PrP C has not been investigated.
To clarify the effect of TUDCA on MSCs in ischemic conditions, we investigated whether TUDCA enhanced survival of MSCs in ischemia-induced ER stress conditions in vitro and in vivo. Results of this study reveal the mechanism by which TUDCA protects against oxidative stress by regulating Akt-dependent PrP C expression.

Results
ER stress induced cell death in a murine ischemic model. Ischemic-injured tissue induces ROS generation and oxidative stress, resulting in further induction of ER stress and transplanted-cell death 19,20 . To confirm ROS-mediated ER stress and cell death in ischemic tissue, ROS generation and cell apoptosis were assessed in ischemic-injured tissues, using a murine hindlimb ischemia model. At postoperative day 3, ROS levels were higher in ischemic-injured tissues than in normal tissues (Fig. 1a). In addition, TUNEL assay indicated that apoptotic cells were significantly higher in ischemic-injured tissues than in normal tissues ( Fig. 1b and c). To investigate ROS-mediated ER stress in ischemic conditions in vivo, the expression and activation of ER stress-associated proteins (78-kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2-alpha (eIF2α ), activating transcription factor 4 (ATF4), inositol-requiring protein 1 alpha (IRE1α ), c-Jun N-terminal kinase (JNK), p38, and CCAAT-enhancer-binding protein homologous protein (CHOP)) in normal and ischemic-injured tissues were determined by western blot analysis ( Fig. 1c-f). At postoperative day 3, ischemic-injured tissues exhibited significantly higher expression levels of ER stress markers (GRP78, ATF4, and CHOP) and phosphorylation of ER stress regulators (PERK, eIF2α , IRE1α , JNK, and p38) than those in normal tissues ( Fig. 1d and f). Moreover, cell death and apoptosis-associated proteins (BCL-2-associated X protein (Bax), cleaved caspase-3, and cleaved poly(ADP ribose) polymerase-1 (PARP-1)) were significantly higher in ischemic-injured tissues than in normal tissues ( Fig. 1g and h). Ischemia increased tissue death (Fig. 1g and h). These results indicate that ischemic conditions trigger ROS generation, resulting in cell apoptosis through the induction of ER stress.
Scientific RepoRts | 6:39838 | DOI: 10.1038/srep39838 Bax, cleaved caspase-3, and cleaved PARP-1 increased with ROS-mediated ER stress ( Fig. 3a and b). To confirm TUDCA protection against ER stress-induced apoptosis, MSCs were pre-treated with TUDCA (100 μ M), and then apoptosis-associated protein levels were measured in H 2 O 2 -induced ER stress conditions (Fig. 3c). Under ER stress, treatment with TUDCA significantly increased the expression of BCL-2 and significantly decreased the expression of Bax, cleaved caspase-3, and cleaved PARP-1, compared with that of untreated cells (Fig. 3d). These findings indicate that treatment with TUDCA protects MSCs from ER stress-induced apoptosis by inhibiting the dissociation of GRP78 and PERK and regulating the apoptosis-associated signaling pathway.  TUDCA mediates ER stress resistance via the expression of Akt-dependent PrP C . A previous study revealed that PrP C promotes post-ischemic neuronal survival and neurogenesis in brain ischemia 8 . In considering how TUDCA protects against ER stress, we hypothesized that the TUDCA-related Akt signaling pathway regulates PrP C and MnSOD. First, we analyzed the response of the Akt signaling pathway to treatment of human adipose tissue-derived MSCs with TUDCA. In a western blot analysis, treatment of MSCs with TUDCA increased phosphorylation of Akt within 30 min of treatment (Fig. 4a). To determine whether TUDCA plays a role in the regulation of Akt-mediated protein expression, the expression levels of PrP C and MnSOD were investigated after treatment with TUDCA for various times (0, 6, 12, and 24 h). After 24 h of treatment, the expression levels of PrP C and MnSOD dramatically increased, and expression of these proteins was suppressed by the use of an Akt inhibitor ( Fig. 4b and c). To further explore whether TUDCA-related PrP C expression ameliorates cell death in ROS-induced ER stress, a cell viability assay was performed under oxidative stress conditions using PRNP siRNA, which is an siRNA targeting the human PrP gene (Fig. 4d). In H 2 O 2 -induced ER stress conditions, treatment of MSCs with TUDCA significantly enhanced cell viability compared with that in the non-treatment group, while pre-treatment with PRNP siRNA significantly decreased cell viability compared with that in the TUDCA treatment and non-treatment groups (Fig. 4e). In addition, flow cytometric analysis of PI and Annexin V indicated that downregulation of PrP C increased cell death in H 2 O 2 -induced ER stress conditions (Fig. 4f). These findings suggest that the protective effect of TUDCA on cells under ER stress is mediated by the Akt-PrP C and MnSOD pathways, and that this mechanism may be PrP C -dependent.
TUDCA-treated MSCs enhance functional recovery in murine hindlimb ischemia via PrP C . To assess whether TUDCA-treated MSCs increase neovascularization in vivo, blood perfusion and tissue repair were investigated following transplantation of PBS, untreated MSCs (MSC), TUDCA-treated MSCs (TUDCA), TUDCA-treated MSCs pretreated with PRNP-specific siRNA (TUDCA + siPRNP), and TUDCA-treated MSCs pretreated with scramble siRNA into hindlimb ischemia mice. Blood perfusion was analyzed by LDPI at postoperative days 0, 3, 7, 14, 21, and 28 (Fig. 5a). The blood perfusion ratio was significantly greater in the TUDCA-treated MSC group than in the other groups (Fig. 5b). Moreover, transplantation of TUDCA-treated MSCs resulted in a reduction in limb loss and foot necrosis ( Fig. 5c and d), and transplantation of TUDCA-treated MSCs pretreated with PRNP-specific siRNA decreased functional recovery. To evaluate the anti-oxidative effect of TUDCA-treated MSCs in ischemic conditions, the expression of MnSOD in ischemic-injured sites was assessed by immunohistochemistry after transplantation of MSCs. Prior to the in vivo studies, catalase activity of MSCs in ischemic conditions was assessed in vitro to determine whether treatment of MSCs with TUDCA augmented MnSOD activity. Treatment with TUDCA significantly increased catalase activity, but this activity was significantly decreased by PrP C protein inhibition (Fig. 6a). At postoperative day 1, immunohistochemistry for MnSOD indicated that the expression of MnSOD in ischemic-injured sites was higher in TUDCA-treated transplanted MSCs than that in other groups (Fig. 6b). To confirm apoptosis of transplanted MSCs in ischemic sites, immunohistochemistry for HNA and cleaved caspase-3 was performed at postoperative day 3 (Fig. 6c). Apoptosis of transplanted MSCs was significantly lower in the TUDCA-treated group than in other groups (Fig. 6d). To investigate neovascularization by transplanted MSCs, immunohistochemistry for CD31 or α -SMA was performed at postoperative day 28 (Fig. 6e-h). Capillary density and arteriole density were significantly higher in transplanted TUDCA-treated MSCs than in the other groups (Fig. 6e-h). Inhibition of PrP C protein significantly decreased the expression of MnSOD, cell survival, and vascular formation. These data indicated that TUDCA-treated MSCs facilitated vascular repair and functional recovery in ischemic-injured tissues, and that TUDCA-mediated PrP C plays a pivotal role in the functionality of transplanted MSCs in these tissues.

Discussion
Recent preclinical animal studies and clinical trials have shown that autologous and allogeneic MSCs from various sources transplanted into ischemic-injured sites localize to injured tissues 21 . However, transplanted MSCs can die in ischemic tissues, largely as a result of pathophysiological conditions such as low oxygen, high ROS levels, and inflammation 22 . This study is the first to demonstrate that TUDCA effectively protects MSCs against ER stress-related cell death and improves functional recovery of vessels through an Akt-dependent PrP C signaling cascade in vivo and in vitro.
Our findings indicate that ischemic injury induces ROS generation, and that GRP78, PERK, eIF2α , and ATF4 are subsequently activated, resulting in the induction of ER stress mediated-apoptosis cascades. ER stress   is caused by nutrient deprivation, hypoxic injury, redox and glycosylation reactions, and disturbances in calcium mobilization 23 . GRP78, an ER chaperone, plays a pivotal role in cell survival and death via interactions with PERK, which regulates eIF2α and the ATF4 signaling pathway 24 . In non-stress conditions, GRP78 binds to PERK, causing it to remain inactive, but ER stress causes GRP78 to dissociate from PERK, and this activation of PERK leads to eIF2α and ATF4 activation, resulting in cell death 25 . In addition, we found that TUDCA inhibits the activation of ER stress-mediated pro-apoptotic mediators, such as IRE1α , JNK, p38, and CHOP, under oxidative stress conditions. IRE1, as an ER transmembrane sensor, triggers ER stress-associated apoptosis through the decay of anti-apoptotic miRNA 26 . IRE1 also regulates the determination of cell fate via phosphorylation of JNK under ER stress conditions 27 . ER stress increases the activation of JNK and p38 28 . Furthermore, the apoptosis-related transcription factor CHOP, which promotes the expression of apoptotic genes such as cell surface death receptor 5 and BH3-only protein BIM, is regulated by the PERK-eIF2α -ATF4 pathway under ER stress conditions, resulting in the induction of apoptosis 29 . Our results show that TUDCA inhibits the dissociation of GRP78 from PERK during ER stress, preventing the activation of eIF2α and ATF4. This suggests that TUDCA protects cells from ER stress by regulating the binding of GRP78 to PERK.
Our results show that TUDCA facilitates expression of PrP C in MSCs. PrP C has a protective effect on cells in conditions of hypoxia, ischemia, and excitotoxicity [30][31][32] . PrP C promotes long-term neuroprotection and angiogenesis in the ischemic brain 8,33 . PrP C deficiency increases sensitivity to oxidative stress and aggravates brain ischemia [34][35][36] . In particular, downregulation of PrP C increases phosphorylation of extracellular signal-regulated kinases 1/2 and reduces the activation of Akt, resulting in an increase in caspase-3 activity 36 . In addition, enhancement of Akt-mediated MnSOD expression promotes protection of MSCs against oxidative stress in vitro and in vivo 37 . Akt is a central cell signaling molecule downstream to cytokines, growth factors, and several stimulations 38 . Various stimuli induce the phosphorylation of Akt, thus activating it to regulate cellular functions such as survival, growth, proliferation, angiogenesis, metabolism, glucose uptake, migration, and invasion 38 . Under ER stress conditions, TUDCA decreases the activity of protein tyrosine phosphatase 1B, resulting in activation of the PI3K-Akt signal pathway and subsequently, the inhibition of ER stress 39 . This study, for the first time, showed that TUDCA decreased apoptosis signaling and increased cell viability under ischemic conditions via regulation of Akt-dependent PrP C expression. TUDCA-induced phosphorylation of Akt enhanced the expression of both PrP C and MnSOD, thus augmenting the catalase activity. However, inhibition of the Akt pathway blocked TUDCA-induced expression of PrP C and MnSOD. Interestingly, knockdown of PrP C did not protect against ER stress-mediated cell death. These findings indicate that TUDCA protects MSCs against ER stress through the Akt-dependent PrP C signaling pathway and Akt-dependent MnSOD expression and suggest that activation of PrP C is a key mechanism underlying TUDCA-mediated ER stress protection.
Finally, this study showed that TUDCA-treated MSCs enhanced functional recovery and neovascularization in a murine hindlimb ischemia model. Blood flow ratio, limb salvage, expression levels of MnSOD, transplanted cell survival, and vessel repair were all increased following transplantation of TUDCA-treated MSCs and were mediated by PrP C expression. PrP C knockout mice showed severe renal dysfunction and structural damage following renal ischemia/reperfusion injury 40 . Our results indicate that TUDCA-treated MSCs have enhanced bioactivities, and that transplantation of TUDCA-treated MSCs could be used in stem cell-based therapeutics for ischemic diseases. In summary, this study, for the first time, demonstrated that TUDCA protects MSCs against ROS-mediated ER stress through the Akt-dependent PrP C signaling pathway, which suggests that activation of PrP C is a crucial mechanism for TUDCA-mediated MSC protection. These findings also suggest that TUDCA-treated MSCs may offer new therapeutics for ischemic disease, and that understanding the regulation of PrP C may provide important insights survival mechanisms of transplanted cells that will facilitate successful cell engraftment.

Methods
Human MSC cultures. Human adipose tissue-derived MSCs were obtained from the American Type Culture Collection (Manassas, VA, USA). MSCs were free of hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and syphilis) and negative for mycoplasma. The supplier certified that the MSCs expressed MSC surface markers (CD73 and CD105) and showed adipogenic and osteogenic differentiation potential when cultured with specific differentiation media. MSCs were cultured in alpha-Minimum Essential Medium (α -MEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL) and 100 U/mL penicillin/streptomycin (Gibco BRL). MSCs were incubated in a humidified incubator at 37 °C and 5% CO 2 . Murine hindlimb ischemia model. To induce ischemia and oxidative stress and to assess neovascularization, a previously described hindlimb ischemia model was used with minor modifications 41,42 . Ischemia was induced by ligation of the proximal femoral artery and boundary vessels of the mice. No later than 6 h after surgery, SCIENTIfIC REPORTS | 6:39838 | DOI: 10.1038/srep39838 PBS, MSCs, TUDCA-treated MSCs, and TUDCA-treated MSCs with scramble or human PrP gene (PRNP) small interfering RNA (siRNA) were injected intramuscularly into the ischemic thigh area (5 × 10 5 cells/80 μ L PBS per mouse; n = 5 for each group). Cells were injected into four ischemic sites. Blood perfusion was investigated by measuring the ratio of blood flow in the ischemic (left) limb to that in the non-ischemic (right) limb on postoperative days 0, 3, 7, 14, 21, and 28 using laser Doppler perfusion imaging (LDPI; Moor Instruments, Wilmington, DE).

Dihydroethidium (DHE
Immunoprecipitation. MSCs were lysed with a lysis buffer (1% Triton X-100 in 50 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 5 mM EDTA, 2 mM Na 3 VO 4 , 2.5 mM Na 4 PO 7 , 100 mM NaF, and protease inhibitors). Cell lysates (300 μ g) were mixed with anti-GRP78 antibody (Santa Cruz Biotechnology). The samples were incubated for 4 h, mixed with Protein A/G PLUS-Agarose Immunoprecipitation Reagent (Santa Cruz Biotechnology), and then incubated for an additional 12 h. The beads were washed four times, and the bound protein was released from the beads by boiling in SDS-PAGE sample buffer for 5 min. The precipitated proteins were analyzed by western blotting with anti-PERK antibody (Santa Cruz Biotechnology).
Inhibition of PrP C expression by RNA interference. MSCs (2 × 10 5 ) were seeded in 60-mm dishes and were transfected with siRNA in serum-free Opti-MEM (Gibco BRL) using Lipofectamine 2000, following the manufacturer's instructions (Thermo Fisher Scientific). At 48 h after transfection, total protein was extracted and gene expression was determined by western blot analysis. The siRNA used to target PRNP and a scrambled sequence were synthesized by Bioneer (Daejeon, Korea).