ML265 – Pci 32765 Chemical

Nuclear accumulation of pyruvate kinase M2 promotes liver regeneration via activation of signal transducer and activator of transcription 3
Kai Hua,c,1, Juanjuan Xua,1, Kerui Fana,1, Dan Zhoub, Longjiang Lia, Li Tanga, Xianwen Penga,
Li Zhanga,⁎, Yaping Wanga,⁎⁎
a Department of Pathophysiology, Chongqing Medical University, Chongqing, China
b Department of Pathology, Fuling Center Hospital of Chongqing City, Chongqing, China
c Department of Histology and Embryology, Chongqing Medical University, Chongqing, China

A R T I C L E I N F O

Keywords:
ML-265
Pyruvate kinase M2 Nuclear accumulation
Signal transducer and activator of transcription 3
Liver regeneration
A B S T R A C T

Aims: Pyruvate kinase M2 (PKM2), a unique isoform of the pyruvate kinases, not only acts as a crucial metabolic enzyme when it locates in the cytoplasm, but also plays important roles in tumor formation and growth when it accumulates in the nuclei. Our aim was to investigate the potential role of PKM2 in liver regeneration in mice insulted with carbon tetrachloride (CCl4).
Material and methods: The liver regeneration model was established by intraperitoneal injection of CCl4 for 48 h in male BALB/c mice. The expression of PKM2, phospho-STAT3, STAT3, proliferating cell nuclear antigen (PCNA) and Cyclin D1 were evaluated by western blot. The distribution of PKM2 was veriﬁed by immuno- ﬂuorescence staining. The degree of injured region was assessed by hematoxylin and eosin (HE) staining. The proliferation of liver cells was tested by Immunohistochemistry.
Key ﬁndings: The nuclear accumulation of PKM2 increased in the liver treated with CCl4, but treatment with ML- 265 signiﬁcantly suppressed CCl4-induced nuclear accumulation of PKM2. In addition, treatment with ML-265 suppressed the level of cyclin D1 and proliferating cell nuclear antigen (PCNA), reduced the count of Ki67- positive hepatocytes, and expanded the damaged region in histological examination. Meanwhile, treatment with ML-265 suppressed the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 by stattic made the same eﬀects as ML-265.
Signiﬁcance: These data uncovered the role of nuclear PKM2 in liver regeneration and the pro-proliferation eﬀects of nuclear PKM2 may be through targeting its downstream transcription factor STAT3.

⦁ Introduction

The liver has a very strong ability to regenerate in response to injury [1,2]. Liver regeneration is an indispensable process that most liver injury depends on for recovery [3,4]. The mechanisms in this process are very complicated and researches into the mechanism have been going on for decades [5–7]. Most of the studies show that various growth factors and cytokines participate in this process [3,8,9]. Recent studies have showed that the regeneration of some injured tissues was closely related with rapid metabolic reprogramming [10,11], which implicates that regulation of metabolism might be an new approach to promote liver regeneration.
Recent reports highlight the far-reaching eﬀects of PKM2 to

metabolic reprogramming [12,13]. PKM2 is encoded by PKM gene and they can display an active or inactive states [14]. The active state of PKM2 locates in the cytoplasm and serves as an rate-limiting enzyme to catalyze the ﬁnal step of glycolysis [15]. However, the inactive state of PKM2 locates in the nuclei and acts as a protein kinase to target its downstream gene [16]. The nuclear localization of PKM2 increased its protein kinase activity, which accelerates tumorigenesis via promoting the proliferation of tumor cells [17,18]. These results show that PKM2 might be an important regulator for tumor growth, but the roles of PKM2 in liver regeneration remains unknown.
In the present study, the potential roles of PKM2 on liver were in- vestigated in injured livers exposed to carbon tetrachloride (CCl4). CCl4-induced liver injury and subsequent repair is a well-established

⁎ Correspondence to: L. Zhang, Department of Pathophysiology, Chongqing Medical University, 1 Yixueyuan Road, Chongqing 400016, China.
⁎⁎ Correspondence to: Y. Wang, Department of Histology and Embryology, Chongqing Medical University, 1 Yixueyuan Road, Chongqing 400016, China.
E-mail addresses: [email protected] (L. Zhang), [email protected] (Y. Wang).
1 Kai Hu, Juanjuan Xu, and Kerui Fan contributed equally to this work.

https://doi.org/10.1016/j.lfs.2020.117561
Received 18 December 2019; Received in revised form 7 March 2020; Accepted 16 March 2020
Availableonline19March2020
0024-3205/©2020ElsevierInc.Allrightsreserved.

Fig. 1. The nuclear accumulation of PKM2 increased in the liver treated with carbon tetrachloride (CCl4). The mice were treated with CCl4 for 24 h or 48 h and the liver samples were harvested. Nuclear (a, c) and cytoplasmic (b, d) PKM2 levels were detected by western blot analysis, values were expressed as the mean ± SD, n = 4, and (e, 48 h) the distribution of PKM2 were performed by immunoﬂuorescence. (⁎⁎P < 0.01, ⁎⁎⁎p < 0.001). experimental model for the investigation of liver regeneration [19]. The damaged liver in this model immediately triggers hepatocytes to pro- liferation [20]. In this model, the subcellular distribution of PKM2, the 2.2. Animals and experimental design Male BALB/c mice, 6 to 8 weeks old, were obtained from the de- role of nuclear PKM2 on liver regeneration, and the downstream target partment of Experimental Animal Center of Chongqing Medical were investigated. ⦁ Materials and methods ⦁ Reagents The PKM2 activating agent ML265, STAT3 inhibitor stattic, and the pyruvate kinase activity assay kit were the products of Cayman Chemical (Ann Arbor, MI, USA). The kit for the determination of nu- clear protein was the product of Genechem Co., Ltd. (Shanghai, China). The rabbit anti-mouse nuclear reference substance lamin B (D9V6H), PKM2 (D78A4), Ki67 (D3B5), PCNA (D3H8P), Cyclin D1(92G2), STAT3 (79D7), and phospho-STAT3 (D3A7) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary horse- radish peroxidase-conjugated goat anti-rabbit antibody was purchased from Proteintech (Wuhan, China). The BCA protein assay kit was the production of Thermo Scientiﬁc (Rockford, IL, USA) and the enhanced chemiluminescence (ECL) reagent was the product of Pierce Biotechnology (Rockford, IL, USA). University. All animals were housed in an environmentally controlled room with pathogen-free and a 12 h dark/light cycle (temperature: 20–25 °C, relative humidity: 50 ± 5%), and the mice were allowed free access to food and water. The experimental procedures were approved by Animal Care and Use Committee of Chongqing Medical University. Liver regeneration in mice was induced by intraperitoneal injection of CCl4 (0.8 ml/kg, dissolved in olive oil) [21]. The mice were sacriﬁced 48 h later after treatment with CCl4. To conﬁrm the pro-proliferation eﬀect of PKM2, the mice were treated with ML-265 (50 mg/kg, dissolved in DMSO, i.p.), 24 h later after CCl4 exposure. To explore the underlying me- chanism of PKM2, the mice were treated with stattic (a STAT3 in- hibitor, 5 mg/kg, dissolved in DMSO). At last, the liver was harvest for morphological analysis. ⦁ Immunoﬂuorescence assay To make the sections permeabilized, the ﬁxed cryosections were incubated in ice-cold 100% methanol for 10 min at −20 °C, and then Fig. 2. ML-265 suppressed the nuclear accumulation of PKM2 induced by carbon tetrachloride (CCl4). Mice with CCl4 induced liver injury were treated with ML-265 24 h after CCl4 administrated. The liver samples were harvested 48 h after CCl4 treatment. The levels of nuclear (a, c) and total (b, d) PKM2 were revealed by western blot analysis, data were expressed as the mean ± SD, n = 4, and the distribution of PKM2 were performed by immunoﬂuorescence, data were expressed as the mean ± SD, n = 8. (N. S·P > 0.05, ⁎P < 0.05, ⁎⁎⁎p < 0.001). Fig. 3. Suppression of the nuclear accumulation of PKM2 impaired carbon tetrachloride (CCl4)-induced liver proliferation. Mice were treated with ML-265 24 h after CCl4 exposure. The liver samples were collected 48 h after CCl4 treatment (a, b) PCNA and (a, c) cyclin D1 were determined by western blot analysis, data were expressed as the mean ± SD, n = 4. (d, f) Liver sections were stained with hematoxylin and eosin for morphological assessment of the degree of injured region and the representative liver sections of each group were shown. CV: central veins, and arrows indicate necrotic areas. (e, g) The proliferating cells were determined by immunohistochemical staining and the Ki67-positive cells showed a dark-brown nucleus. (f, g) The injured area and Ki67-positive cells were quantiﬁed by image-Pro Plus 6.0. Representative liver sections of each group were shown. IOD: Integrated option density. (N.S·P > 0.05, ⁎⁎P < 0.01, ⁎⁎⁎p < 0.001). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) rinsed in PBS for 5 min specimen blocked in blocking buﬀer containing 10% goat normal serum, 3% BSA, 0.3% Triton X-100, at 25 °C for 60 min. Then, the specimens were hatched with primary rabbit monoclonal antibodies PKM2 (1:200; Cell Signaling Technology), and Phosphate Buﬀered Saline (PBS) used for negative control overnight at 4 °C. And then, the specimens were incubated with FITC-conjugated secondary antibodies for 2 h at 37 °C. The stained sections were vi- sualized with a Leica ﬂuorescence microscope. ⦁ Immunohistochemical staining The liver samples were ﬁxed in formalin, followed by routine de- hydration. Then the samples were embedded in paraﬃn, and 4-μm sections were prepared. The sections from the liver samples were de- waxed in xylene for 2 h and then the sections were hydrated in diﬀerent concentration gradients of alcohol. After washing the section clean, their antigens were retrieved in citric acid for 20 min. Then, the sec- tions were incubated in 3% hydrogen for 30 min and then hatched with blocking buﬀer (containing 0.5% Triton X100 and 5% Bovine Serum Fig. 4. Inhibition of the nuclear accumulation of PKM2 suppressed carbon tetrachloride (CCl4)-induced phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). Mice were treated with ML-265 24 h after CCl4 exposure. The liver samples were collected 48 h after CCl4 treatment. The relative level of (a, b) phosphorylated STAT3 and (a, b) total STAT3 in the nuclear extracts were tested by western blot analysis, values were expressed as the mean ± SD, n = 4. (N.S·P > 0.05, ⁎P < 0.05, ⁎⁎P < 0.01). Albumin, room temperature) for 30 min. After these procedures, the sections were hatched with the primary antibodies Ki67 (1:400) over- night at 4 °C. After cleaning up the sections, they were hatched with the secondary antibody for 30 min and then covered with streptavidin- peroxidase for 30 min at 25 °C. And last, the sections were stained with 3, 3-diaminobenzidine for 3 min at room temperature and then the sections were counterstained with hematoxylin. After dehydration, the stained sections were examined using a light microscope. ⦁ Immunohistochemical analysis The images analysis of Immunohistochemistry was proceeded using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The brown staining area was quantiﬁed by the integral optical density (IOD) and the average value was calculated. A positive correlation was observed between the IOD value and the target protein stained in the sections [22]. ⦁ Hematoxylin and eosin (H&E) staining Liver tissues were ﬁxed in 4% paraformaldehyde, followed by a series of dehydrations, embedded in paraﬃn, sectioned at 4-μm thick- ness, and stained with H&E using standard methods. The histopatho- logical analysis of liver sections was performed under a light micro- scope (Olympus, Tokyo, Japan). ⦁ Western blot analysis The liver samples were minced with eye scissors in ice. Then the samples were homogenized in lysis buﬀer (RIPA lysis and nuclear Extraction kit) and clariﬁed by high speed centrifuge to obtain the total proteins or nuclear proteins. The protein samples were separated by electrophoresis on polyacrylamide–SDS gel. And then the separated proteins were transferred to nitrocellulose membrane. The membrane was then blocked with 5% nonfat milk in Tris-buﬀered saline and in- cubated with primary antibody against PKM2, phospho-STAT3, STAT3, proliferating cell nuclear antigen (PCNA), Cyclin D1, β-actin or lamin B overnight at 4 °C. After washing away the unbound antibodies, an anti- rabbit or anti-mouse horseradish peroxidase-conjugated secondary an- tibody was visualized using an enhanced chemiluminescence system (ECL, Advansta, USA) and the ChemiDoc Touch Imaging System (Bio- Rad). ⦁ Statistical analysis All quantiﬁed data were expressed as mean ± SD. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA). Comparisons among groups were performed by one-way ANOVA analysis, followed by the Turkey's post hoc test. p < 0.05 was considered to be statistically signiﬁcant. ⦁ Results ⦁ CCl4 induced upregulation of nuclear PKM2 The accumulation of nuclear PKM2 was investigated in the liver of CCl4 induced liver injury. The results implicated that treatment with CCl4 signiﬁcantly promoted the expression of nuclear PKM2 (Fig. 1a, b, c, d). We further veriﬁed the distribution of PKM2 by immuno- ﬂuorescence staining (Fig. 1e), and the results showed that CCl4 in- duced more PKM2 accumulation in the nucleus (cyan) than did it in control groups. ⦁ Inhibition of nuclear translocation of PKM2 suppressed liver proliferation The potential role of nuclear PKM2 in liver regeneration was ex- plored by preventing the nuclear accumulation of PKM2 with ML-265. ML-265 is a small molecule compound that prevent PKM2 distribute to nuclear. [23].In my study, the data showed that treatment of ML-265 reduced the accumulation of nuclear PKM2 (Fig. 2a, c) but did not change the expression of total PKM2 when it compared with the CCl4 groups (Fig. 2b, d). In addition, the distribution of PKM2 were de- termined by immunoﬂuorescence staining, and the results showed that nuclear PKM2 (Fig. 2e) were increased in CCl4 groups compared with ML-265 treatment groups. Afterwards, the degree of liver regeneration was investigated. The data implicated that treatment with ML-265 suppressed the expression level of PCNA (Fig. 3a, b) and cyclin D1 (Fig. 3a, c), which were the indicators of cell proliferation. The samples from ML-265 treated groups had larger injured region surrounding the central veins compared to CCl4 groups (Fig. 3d, f), including more se- vere centrilobular necrosis, more hepatocyte ballooning and much more inﬁltration of inﬂammatory cells, and there was more intense of anti-Ki67 (Fig. 5e, g) staining in the nuclei of CCl4 groups than in those of CCl4 treatment groups. ⦁ STAT3 contributed to the pro-proliferation activities of nuclear PKM2 We speculated that STAT3 may participate in the pro-proliferation eﬀect of PKM2. Our data showed that nuclear STAT3 (Fig. 4a, b) and phosphorylated STAT3 (Try 705, Fig. 4a, b) signiﬁcantly increased in CCl4 treatment groups than that in the control groups. However, sup- pression of PKM2 nuclear accumulation with ML-265 apparently de- creased the level and activity of nuclear STAT3 (Fig. 4a, b). To conﬁrm whether STAT3 could promote liver regeneration, a STAT3 inhibitor stattic was administrated in the experimental animals. The results im- plicated that inhibition of STAT3 depressed the expression level of PCNA (Fig. 5a, b) and cyclin D1 (Fig. 5a, c). Treatment with stattic results in larger injured region surrounding the central veins compared to the CCl4 treatment (Fig. 5d, f) including more severe centrilobular necrosis, more hepatocyte ballooning and much more inﬁltration of inﬂammatory cells, a. In addition, there was more intense anti-Ki67 Fig. 5. Suppression of signal transducer and activator of transcription 3 depressed the regeneration of injured liver induced by carbon tetrachloride (CCl4). The mice were treated with stattic 24 h after carbon tetrachloride (CCl4) administration. The liver samples were collected 48 h after carbon tetrachloride (CCl4) treatment. The expression level of (a, b) PCNA and (a, c) cyclin D1 were determined by western blot analysis, values were expressed as the mean ± SD, n = 4. (d, f) hematoxylin and eosin staining used for histological examination of the degree of injured region, and the representative liver sections of each group were shown, CV: central veins, and arrows indicate necrotic areas. (e, g) immunohistochemical staining of the Ki67-positive cells showed a dark-brown nucleus. (f, g) The injured area and Ki67- positive cells were quantiﬁed by image-Pro Plus 6.0. Representative liver sections of each group were shown. IOD: Integrated option density. (N. S·P > 0.05,
⁎P < 0.05, ⁎⁎⁎p < 0.001). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) (Fig. 5e, g) staining in the nuclei of CCl4 groups than in those of stattic treatment groups. ⦁ Discussion Tissue regeneration is usually associated with rapid metabolic re- programming [10,11,26]. Recent studies also found that PKM2 plays an important role in the process of metabolic reprogramming [13,27]. In the present study, we ﬁrst examined the level of PKM2 in nucleus and cytoplasm by western blot and the result showed that the level of nu- clear PKM2 and cytoplasmic PKM2were both increased in mice with CCl4 insulted groups. In the next step, the increased PKM2 in mice treated with CCl4 were further veriﬁed by immunoﬂuorescence staining. These results showed that PKM2 involved in CCl4-induced liver injury. To explore the role of PKM2 in liver regeneration, the potential signiﬁcance of nuclear PKM2 was proved by pharmacological prevention of PKM2 nuclear accumulation with ML265. ML-265 is a small molecule compound that prevent PKM2 distribute to nuclear and the data revealed by western blot and immunoﬂuorescence implicated that treatment of ML-265 reduced the accumulation of nuclear PKM2 but did not change the expression of total PKM2 in the mice insulted with carbon tetrachloride (CCl4). These data showed that ML-265 prevented the distribution of nuclear PKM2. Recent reports also show that ML-265 inhibited the distribution of nuclear PKM2 [23,24]. In addition, we found that the lessened accumulation of nuclear PKM2 by ML-265 were associated with down-regulation of PCNA, cyclinD1, and Ki67. These data suggested that inhibition of PKM2 nuclear accumu- lation suppressed liver regeneration. Previous studies also have showed

that nuclear localization of PKM2 is a crucial molecular event in cancer progression [17,28,29] and central nervous system repair [30], These data suggested that nuclear PKM2 accumulation might be a critical even for cell proliferation.
STAT3 is known to function in diﬀerent biological process and mediates the expression of various genes that involved in cell growth and proliferation in response to diverse stimuli [31]. Knockout of Stat3 in muscle stem cells damages their proliferation and self-renewal ability upon injury [25]. Conditional deletion of STAT3 results in abnormal morphology of repair cells and regeneration tracks in never system after injury [32]. Pharmacological inhibition of STAT3 suppresses bone marrow stromal cells proliferation and diﬀerentiation [33]. In the present study, the role of STAT3 was investigated by STAT3 inhibitor stattic, a small molecule that inhibits the function of the SH2 domain of STAT3 in a dose-dependent manner, preventing phosphorylation of STAT3 at Tyr705 and, subsequently, activity [34,35]. Our data showed that stattic suppressed the expression of PCNA and cyclin D1, reduced the count of Ki67 positive cells. These data suggested that STAT3 may be a crucial regulator in liver regeneration.
PKM2 is one of the four pyruvate kinase isoforms that locates in both cytoplasmic and nucleus. The cytoplasmic PKM2 exhibit enzy- matic active in glucose metabolism. Under certain pathophysiological conditions, nuclear PKM2 act as a protein kinase regulate the pro- liferation related genetic transcription [16], and STAT3 is an important transcription factor extensively involved in cell proliferation [25]. Previous reports, including from our own lab, has implicated that STAT3 was the target of nuclear PKM2 [16,37,38]. Nuclear PKM2 function as a protein kinase phosphorylate STAT3 at Y705 promoted tumor cells proliferation [16,39]. Our ﬁndings showed in line with previous studies that inhibition of nuclear PKM2 suppressed the activity of nuclear STAT3. These data showed that the enhanced phosphoryla- tion STAT3 in the nuclei might contribute to the pro-proliferation eﬀect of nuclear PKM2.
In addition, what physiological state contributed for the nuclear translocation of PKM2 after treatment with CCl4 is still unknown. Reports showed that increased ROS concentration lead to the oxidation of PKM2 at cysteine residue C358, which induce the tetramer-to-dimer alteration [40,41]. Maybe the increased ROS concentration leading to the nuclear accumulation of PKM2. The typical feature of the model induced by CCl4 is highly expression of inﬂammatory factor TNF-α and
IL-6,which promote the cell proliferation though binding to its re-
ceptors [42].In order to avoid the inﬂuence of ML-265 on the in- ﬂammatory factors, the ML-265 were treated 24 h later after the ex- posure of CCl4.

⦁ Conclusion

In summary, this study found that nuclear PKM2 increased in mice with CCl4-induced liver injury, and the nuclear accumulation of PKM2 promoted the liver regeneration. The nuclear PKM2 function as a pro- tein kinase promoted liver regeneration via phosphorylating activation its downstream gene STAT3.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2020.117561.

Author contributions

Kai Hu contributed to data curation, investigation and writing
-original draft. Juanjuan Xu contributed to data curation and formal analysis. Kerui Fan contributed to the data validation, visualization and software analysis. Longjiang Li, contributed to the experiment super- vision. Li Tang contributed to the article conceptualization. Dan Zhou provided resources in this research. Xianwen Peng contributed to the data validation and provided the methodology in the research. Li Zhang contributed to the writing-review & editing. Yaping Wang contributed to the study conceptualization and supervision.
Funding

This work is supported by National Natural Science Foundation of China (approval number of projects:81871606).

Declaration of competing interest

All the authors who have taken part in this study declared that they have no conﬂicts of interest to this manuscript.

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