Histone methylation regulates Hif‐1 signaling cascade in activation of hepatic stellate cells

Liver fibrosis is characterized by deposition of excessive extracellular matrix (ECM). The major source of ECM is activated hepatic stellate cells (HSCs). Previously, we reported that hypoxia‐inducible factor‐1 (Hif‐1) regulates activation of HSCs through autophagy. In current work, human HSC cell line LX‐2 was used as cell model. It was determined that trimethylation of H3 histone on lysine 4 (H3K4me3) occurred in the Hif‐1 transcriptional complex. Inhibition of modifications of histone methylation suppressed Hif‐1 nuclear transport, autophagosome formation, and activation of LX‐2 cells. These data suggest that histone methylation modification plays an important role in the Hif‐1 signaling cascade regulating HSC activation.

Liver fibrosis is characterized by deposition of excessive extracellular matrix (ECM). The major source of ECM is activated hepatic stellate cells (HSCs). Previously, we reported that hypoxia-inducible factor-1 (Hif-1) regulates activation of HSCs through autophagy. In current work, human HSC cell line LX-2 was used as cell model. It was determined that trimethylation of H3 histone on lysine 4 (H3K4me3) occurred in the Hif-1 transcriptional complex. Inhibition of modifications of histone methylation suppressed Hif-1 nuclear transport, autophagosome formation, and activation of LX-2 cells. These data suggest that histone methylation modification plays an important role in the Hif-1 signaling cascade regulating HSC activation.
Liver fibrosis is a worldwide health issue due to the lack of effective treatment. All kinds of acute or chronic liver injuries, including viral hepatitis, nonalcoholic steatohepatitis, alcohol-induced liver damage, parasitemia, and autoimmune diseases, result in liver fibrosis and the end stage of liver fibrosis, cirrhosis [1,2]. Liver fibrosis is a reversible wound-healing process, which is characterized by deposition of excessive extracellular matrix (ECM) [3]. The major source of ECM is activated hepatic stellate cells (HSCs), which plays an important role in pathogenesis of liver fibrosis [4]. Activated HSCs are always accompanied by cytoskeleton reconstruction, expression of a-smooth-muscle actin (a-SMA) and vimentin, and loss of lipid droplets, which together facilitate the development of liver fibrosis [5][6][7].
Hypoxia-inducible factor-1 (Hif-1) is a heterodimeric transcription factor consisting of an oxygen-sensitive alpha subunit (Hif-1a) and a constitutive beta subunit (Hif-1b) that promotes cell survival by regulating the expression of essential genes under oxygen deprivation [8,9]. We previously reported that hypoxia-inducible factor-1 (Hif-1) acts as a main regulator in the activation of HSCs [5] and Hif-1 regulates autophagy to activate HSC [6]; however, the specific molecular mechanism is still vague.
translational modification refers to methylation, acetylation, ubiquitination, phosphorylation, and SUMOylation occurred in histone of nucleosome, which is one kind of epigenetic modification resulting from changes in gene expression and functions without alterations in the DNA sequence [10]. Nucleosomes are the basic unit of chromatin. Each of the two H2A, H2B and H3 and H4 subunits form a histone octamer and the 146 bp of DNA surrounds the histone octamer to form nucleosomes. Histone methylation modification, one kind of post-translational modification, commonly occurs in lysine or arginine residues of the N terminus of H3 and H4 histone in nucleosome. Lysine methylation usually occurs in H3 lysine 4 (H3K4), lysine 9 (H3K9), lysine 27 (H3K27), lysine 36 (H3K36), lysine 79 (H3K79), and H4 lysine 20 (H4K20). Arginine methylation usually occurs in H3 arginine 2 (H3R2), arginine 8 (H3R8), arginine 17 (H3R17), arginine 26 (H3R26), and H4 arginine 3 (H4R3). Depending on the number of methylated groups on the residue, they are divided into methylation (me1), dimethylation (me2), or trimethylation (me3). Among them, H3K4me3, H3K4 trimethylation, was found to especially exhibit high abundances near the transcriptional start site on promoter region of activated gene [11]. In recent years, the relatedness of hypoxia and histone methylation is gradually being recognized in cellular signal integration, which indicated that alteration of epigenetic homeostasis plays a role in gene regulatory switches under hypoxia [12,13].
In current work, using genomewide expression chip analysis, up-regulation of several genes regulating histone methylation was determined in hypoxia-induced HSC line, LX-2. The function of histone methylation modification was further explored in signaling cascade and biological functions of Hif-1, including Hif-1 nuclear transport, autophagy of HSC, and activation of HSC. It was determined that histone methylation modification of Hif-1 plays an important role in activation and autophagy of HSC, which provided a new perspective in the development of liver fibrosis.

Animals
BALB/c female mice, 8 weeks old, were obtained from the Wuhan Institute of Biological Products, Wuhan, China. The experiment was approved by the Committee on Animal Research of Tongji Medical College, Huazhong University of Science and Technology. Mice were randomly divided into two groups: the infected group and the control group. Oncomelania snails infected with Schistosoma japonicum were purchased from Hunan Province Institute of Parasitosis Control and Prevention, Yueyang, China. S. japonicum cercariae were shed from the snails. Each anaesthetized mouse in the infected group was percutaneously infected with 25 cercariae through the shaved abdomen [5,16,17]. The mice were sacrificed at 8 weeks postinfection, and samples of liver were collected.

Genomewide expression chips
Human HSC line, LX2, was cultured in normoxia or treated with 100 lM CoCl 2 for 8 h. Total RNA was extracted with TRIzol Reagent (15596-026; Invitrogen, Carlsbad, CA, USA) and further purified using Qiagen RNeasy Mini Kit (217004; QIAGEN, Stockach, Germany) according to manufacturer's instructions. RNA quality was assessed by formaldehyde agarose gel electrophoresis, and RNA was quantitated spectrophotometrically. Genomewide expression chip analysis was performed via technical support from GCBI (Shanghai, China). The samples were processed using Affymetrix GeneChip WT PLUS Reagent Kit (Affymetrix, Carlsbad, CA, USA), followed by Hybridization Wash and Stain Kit. Microarray expression profiles were collected using Affymetrix Human Transcriptome Array 2.0. Original CEL and files were analyzed by Affymetrix software programs Expression Console and Transcriptome Analysis Console. Genes with lower expression in CoCl 2treated cells than in normoxia cells with a fold change > 1.2 (P < 0.05) were selected as down-regulated ones, and those with higher expression in CoCl 2 -treated cells than in normoxia cells with a fold change > 1.2 (P < 0.05) were selected as up-regulated ones.

Western blot
Cells were collected at indicated time. Nuclear sample and cell lysates were separated using Nuclear and Cytoplasmic Protein Extraction Kit(P0028; Beyotime, Shanghai, China) and resolved in RIPA lysis buffer (P0013B; Beyotime). Protein concentration was valued using BCA Protein Assay Kit (P0011; Beyotime). Protein samples were then separated by SDS/PAGE and transferred onto polyvinylidene difluoride membrane (PVDF; Millipore, Burlington, MA, USA). After blocking in 5% BSA, membranes were incubated with primary antibodies

Immunoprecipitation
Cells were lysed in 4°C precooled RIPA buffer as described above, and 1 mg of cell lysate was incubated with 4 lg Hif-1a monoclonal antibody (ab16066; Abcam) or H3K4me3 antibody (39159; Active Motif) at 4°C overnight with continuous agitation. Protein A+G agarose (P2012; Beyotime) was added and incubated for additional 2 h at 4°C. The beads were washed five times with PBS. Precipitated proteins were eluted by boiling the beads in 29SDS/PAGE sample buffer for 5 min. The samples were analyzed by western blot with anti-H3K4me3 or anti-Hif-1a antibody.

Immunohistochemistry
The formalin-fixed and paraffin-embedded liver tissues were cut into 4-lm sections and then deparaffinized routinely. The slides were heated in 10 mM citrate buffer (pH 6.0) for antigen retrieval. After washing with PBS for three times, the slides were incubated with 3% H 2 O 2 at room temperature for 10 min and then incubated with monoclonal antibody to OGT (ab177941; Abcam) at 4°C overnight. The slides were washed with PBS and incubated with polyperoxidase-anti-rabbit IgG (Envision TM , DAKO, Beijing, China) at room temperature for 30 min. After washing, the slides were colored with 3, 3-diaminobenzidine and counterstained with hematoxylin.

Statistical analysis
All data are expressed as mean AE SD. Differences between experimental and control groups were assessed by one-way ANOVA using GraphPad Prism 5, *P < 0.05, **P < 0.01.
H3K4me3 histone methylation modification occurred in Hif-1 transcriptional complex in hypoxia-induced LX-2 human stellate cells Among histone methylation modification, H3K4me3, trimethylation of histone H3 at lysine 4, was found to exhibit high abundances and especially enrich near the transcriptional start site on promoter region of activated gene. We therefore detected H3K4me3 histone methylation modification in LX-2 cells under hypoxia using western blot and immunoprecipitation. In nuclear sample, Hif-1a and histone H3K4me3 was gradually increased as cells were induced with CoCl 2 ( Fig. 2A,B). Consistent with our previous report, in cytoplasm of hypoxic LX-2 cells, Hif-1a expression was also increased ( Fig. 2A) [5,6]. Anti-Hif-1a or anti-H3K4me3 antibody was, respectively, added to the cell lysate to determine the occurrence of histone H3K4me3 methylation modification in Hif-1 transcriptional complex with co-immunoprecipitation. It was shown that H3K4me3 histone methylation modification occurred in Hif-1 transcription complex in hypoxic LX-2 cells (Fig. 2C). As a transcriptional regulator, Hif-1 forms transcriptional complex in cells to exert its biological regulatory function. The above result suggested the formation of Hif-1 transcription complex in hypoxic LX-2 cells undergoes histone methylation modification, at least H3K4me3.

Inhibition of histone methylation modification affects Hif-1a nuclear translocation in hypoxiainduced LX-2 cells
Under hypoxia stimulation, degradation of Hif-1a was suppressed. Hif-1a forms heterodimer with Hif-1b and translocates into the nucleus. Hif-1 transcription complex binds with promoters of target genes to induce gene expression. To further explore the role of histone methylation modification in Hif-1 nuclear transport, methylation inhibitor MTA was used to inhibit histone methylation modification [14,15] in LX-2 cells and the nucleation of Hif-1a was detected with western blot and immunofluorescence staining. It was determined that CoCl 2 -induced hypoxia led to nuclear transport of Hif-1a, while inhibition of histone methylation modification led to suppress of nuclear translocation of Hif-1a (Fig. 3A,B). Collectively, the results indicated that in hypoxia-stimulated HSC, histone methylation plays an important role in Hif-1a nuclear transport.

Inhibition of histone methylation modification blocks autophagy in hypoxia-induced LX-2 cells
It was previously reported that Hif-1 regulates HSCs activation by autophagy. Histone methylation modification was inhibited by MTA, and autophagy markers, LC-3B and P62, were detected with western blot and immunofluorescence staining. It was shown that hypoxia stimulation led to increase in 14-kDa lipidated LC-3B and formation of autophagosome indicated by P62 (Fig. 4A-C), which indicated the occurrence of autophagy. However, as cells were treated with MTA, autophagy induced by hypoxia treatment was significantly inhibited (Fig. 4A-C), which suggested that histone methylation modification contributes to autophagy in LX-2.

Deficiency of histone methylation modification impedes the activation of hypoxia-induced LX-2 cells
In order to investigate the relationship between histone methylation modification and activation in hypoxiainduced LX-2, we used methylation inhibitor MTA to inhibit histone methylation and observed whether activation of HSC was impacted. LX2 cells were pretreated with 1 mM MTA for 16 h and then stimulated by CoCl 2 . In CoCl 2 -treated LX2 cells, increase in a-SMA, vimentin, and cytoskeleton rearrangement indicated the activation of cells (Fig. 5A-C). As CoCl 2 -treated LX2 cells were pretreated with MTA, the expression of a-SMA in HSCs was significantly inhibited (Fig. 5A,B). In addition, MTA treatment inhibited vimentin expression and cytoskeleton rearrangement (Fig. 5C), indicating that histone methylation was involved in hypoxia-induced LX-2 activation.

Discussion
Our previous work has reported that hypoxia-inducible factor 1, Hif-1, regulates phenotypic transformation and activation of HSCs by autophagy, when the liver is stimulated by inflammation or various factors that form a local hypoxic micro-environment [5,6]. In recent years, more and more genes were determined as target genes of Hif-1, including vegf (vascular endothelial growth factor), pgk-1 (phosphoglycerate kinase 1), ldha (lactate dehydrogenase A), and glut-1 (glucose transport-1) [18][19][20]. Activities of Hif-1 target genes vary according to different physiological or pathological circumstances. In current work, preliminary study of Hif-1 potential target genes in HSC was screened using genomewide expression chips. Among differential genes in normoxia and CoCl 2 -treated hypoxia-induced LX-2 cells, bnip3, p4 ha1, glut1, gsy1, and stc1 were previously determined as target genes of Hif-1. Genes such as ogt, kdm3a, and kdm2a were reported to be involved in different forms of histone methylation modification [21][22][23]. The increased expression of OGT was further confirmed at mRNA and protein level in hypoxiainduced LX-2 cells, and also in tissue samples of liver fibrosis infected by S. japonicum.
Recently, it was reported that OGT regulates H3K4me3 histone methylation modification [21,24]. OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) catalyzes the GlcNAc glycosylation of serine/ threonine hydroxyl group on the protein surface [25]. O-GlcNAc glycosylation is a special post-translational modification of proteins [26]. OGT regulates the subcellular localization and enzymatic activity of TET3, which converts 5mC to 5-hydroxymethylcytosine [27,28]. OGT catalyzes the O-GlcNAcylation of TET3 and promotes TET3 nuclear export, which consequently inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3 [29]. Studies have shown that the interaction of TET2 and TET3 promotes the occurrence of H3K4me3 in the promoter region of target genes and enhances expression of corresponding genes. When expression of either TET2/3 or OGT is inhibited, H3K4me3 histone methylation will be suppressed, resulting in a reduction in expression of target genes [21]. Furthermore, complex interaction of OGT and Hif-1 was reported in research of cancer, which indicates that OGT regulates Hif-1 signaling to catalyze O-GlcNAcylation reprogramming cancer cell metabolic and survival response [30]. In current work, it was firstly determined that OGT increasingly expressed in hypoxia-induced HSCs and in tissues of liver fibrosis. The detailed role of OGT in Hif-1 signaling cascade and in development of liver fibrosis is worthy further research.
In this work, research from histone methylation modification was investigated to reveal the mechanism and function of Hif-1 to HSC activation, as Hif-1 acts as a master transcriptional factor. H3K4me3, trimethylation of histone H3 at lysine 4, is an important marker of histone methylation modification in chromatin, which is involved in activation of gene expression. As previously reported, hypoxia induces H3K4me3 histone methylation modification in cells [12]. We determined that H3K4me3 histone methylation modification occurred in Hif-1 transcriptional complex in hypoxiainduced LX-2 cells. As previously reported, Hif-1 regulates activation of HSC via autophagy [6]. Nuclear transport of Hif-1a molecule, and autophagy and activation of HSC were apparently inhibited in hypoxiainduced LX-2 cells, as H3K4me3 histone methylation was inhibited by MTA, suggesting that histone methylation modification plays an important role in Hif-1 signaling cascade to regulate cell activities.
Autophagy is an evolutionarily conserved process through autophagosome-dependent lysosomal degradation of cytoplasmic components, which is essential to scavenge the toxic accumulation of abnormal protein aggregates and organelles, to sustain metabolism, as cells are lack of nutrients and oxygen. As liver is injured, a large number of lipid droplets in HSCs gradually reduced or even disappeared. It is generally believed that autophagy has a crucial function in lipid droplet degradation (known as lipophagy) [31][32][33]. Studies have shown that epigenetic modifications regulate the occurrence of autophagy at transcriptional and post-transcriptional level and play an important role in biological functions of autophagy [34]. Study of epigenetic modification in autophagy is a hot topic in the current research. The mechanisms of epigenetic modification in Hif-1 regulating HSC activation via autophagy to degrade lipid droplet remain further research.
In summary, our studies demonstrated that histone methylation modification plays a pivotal role in HSCs autophagy and activation. Inhibition of H3K4me3 histone methylation modification affected Hif-1 nuclear transport, ultimately affected autophagy and activation of HSCs. Mechanisms of histone methylation modification in Hif-1 signaling activities, including regulation of autophagy and activation of HSCs, are worthy researched, to deeply understand the mechanism of activation of HSC and development of liver fibrosis, and to provide new therapeutic interventions for liver disease.