Introduction

Intracerebral hemorrhage (ICH) is a serious neurological crisis accounting for ~10–15% of all strokes where it is associated with high mortality and morbidity [1]. Astrocyte activation (also known as reactive astrogliosis) induced secondary injury has been reported to play a critical role in neurological deterioration [2, 3]. Moreover, the resulting glial scar formation from reactive astrogliosis impedes axonal regeneration [4]. Uncovering the mechanism of astrocyte activation will help improve the patient outcomes.

Being the most dominant glial cells in the central nervous system (CNS), astrocytes provide neurotrophic and structural support to neurons and help maintain the extracellular milieu homeostasis [5,35]. Indeed, YAP knockout mice develop reactive astrogliosis in the cortex supporting its critical involvement in brain development [47]. It is reported that adult neural stem cells in the subventricular zone are derived from embryonic radial glia [48]. While radial glia are rare in the adult CNS, Müller glia are specialized radial glial cells in the adult retina that can de-differentiate into readily dividing neural progenitors in response to injury [49].

Using a selective model of Hb administration, we noted that this globin can induce astrocytes to express high levels of Vimentin comparable to those in immature astrocytes and radial glia [38]. From the perspective of genesis and the morphology, Hb-induced Vimentin-positive astrocytes are not the typical radial glia, but they share some commonality. We propose that AMT may be a manifestation of Hb-induced de-differentiation of astrocyte, which can be considered as partial reversion from the terminally differentiated cells. It is reminiscent of the partial de-differentiation that astrocytes exhibit upon growth-factor-stimulation [16]. Proliferation of astrocytes is a prerequisite for glial scar formation [50]. Thus we propose that Hb-induced de-differentiation can prepare astrocytes for proliferation. Following this, the newly proliferated cells can undergo hypertrophy and get their processes overlapped and intertwined extensively to form the compact border which is referred to as a glial scar.

Derived from neuroectoderm, astrocytes are considered to be specialized epithelial cells in the CNS. Our proposed AMT model is analogous to the well-studied EMT. EMT was first described in embryonic development and is characterized by loss of epithelial cell polarity and cell–cell adhesion, yielding migratory properties [51]. EMT is widely involved in tissue repair, regeneration, and fibrosis [52]. ICH is a severe injury to the CNS where astrocytes undergo reactive astrogliosis analogous to fibrosis. We propose that the de-differentiation and AMT of astrocytes contribute to reactive astrogliosis by facilitating the proliferation and migration of reactive astrocytes.

Cx43-based intercellular gap junctions are the predominant cell adhesions in homeostatic astrocytes. The half-life of Cx43 in cultured cells is less than 5 h. Its degradation is quickly modulated by phosphorylation [23]. It is reported that pro-inflammatory cytokines promote Cx43 degradation via the ubiquitin proteasome system [53]. Glioma, which is characterized by the uncontrolled proliferation of poorly differentiated astrocytes, is an example of cell destabilization. It is reported that downregulation of Cx43 is related to the malignant transformation of glioma [24, 25]. In our research, Hb-induced astrocytes activation disrupted homeostasis. We found that Cx43 was downregulated with increased p-Cx43/Cx43 upon Hb stimulation. Cx43 phosphorylation leads to its conformational changes, channel closure, and degradation [23]. Based on Zhang’s work [53], we propose that downregulation of Cx43 in Hb-activated astrocytes is the result of inflammatory cytokines produced, but the mechanism needs further exploration. Because Cx43 was phosphorylated and downregulated in mitotic cells [23], we propose that downregulation of Cx43 may facilitate astrocyte detachment and proliferation.

The Hippo pathway is well-accepted as an important regulator of tissue homeostasis. Dysregulation of the Hippo pathway is highly relevant to tumor development [26]. As a key effector of the Hippo pathway, YAP is transcriptionally active when it translocates to the nucleus to form complexes with transcription factors. In our study, YAP nuclear translocation was observed in Hb-treated astrocytes as well as in the ICH brain. In addition, we found Hb treatment endowed astrocytes with increased proliferation and astroglial-mesenchymal phenotype switching; an effect arrested through co-administration of a YAP inhibitor. Our findings indicate that the astrocytic response to Hb exposure was at least partially YAP dependent. However, Huang and colleagues reported that YAP knockout mice developed reactive astrogliosis spontaneously [36], indicating that YAP may be a negative regulator of reactive astrogliosis. Their conclusion is different from ours possibly due to the highly distinct models of brain injury compared with genetic ablation.

Further we noted that knock down of Cx43 triggers YAP nuclear translocation. It is reported that cell adhesion and tight junction related proteins such as Angiomotin (AMOT), protein tyrosine phosphatase non-receptor type 14 (PTPN14), and α-catenin contribute to sequestering YAP at cell junctions [26]. We conducted immunofluorescence double staining and co-immunoprecipitation studies to examine the physical association between Cx43 and YAP. The results confirmed our hypothesis. Whether they are binding directly or mediated by other proteins remains to be determined.

Taken together, we propose that YAP is sequestered to astroglial gap junctions through direct or indirect interactions with Cx43. Upon Hb stimulation, Cx43 undergoes phosphorylation and degradation. This liberates YAP and allows its nuclear translocation where it can transcriptionally activate and promote the expression of genes involved in cell proliferation and AMT (Figure S4).

To our knowledge, this is the first study to report astroglial-mesenchymal phenotype switching during Hb-induced astrocyte activation. Activated astrocytes acquire enhanced proliferation and mesenchymal features, which may contribute to reactive astrogliosis. Cx43 downregulation-facilitated YAP nuclear translocation is a novel mechanism involved in this process. Cx43–YAP interaction may represent a potentially important therapeutic target in the management of ICH.

Materials and methods

ICH mouse model

All experimental procedures were approved by the Shanghai Jiao Tong University experimental ethics committee. 10-week-old male mice were anesthetized with a single intraperitoneal dose of ketamine (100 mg/kg) and xylazine (10 mg/kg). The mice were secured in a stereotactic frame (RWD Life Science co., Shenzhen, China) and subjected to ICH using collagenase IV (Sigma-Aldrich, MO, USA) as previously described [54] with modification. A 1 mm diameter burr hole was drilled 2.2 mm lateral to the midline and 0.5 mm anterior to bregma. The needle was advanced 3 mm into the right striatum. A total of 0.075 U collagenase IV dissolved in 0.4 μL phosphate buffer (PBS) was injected over 90 s using a microinfusion pump (WPI, Sarasota, FL). The needle was left in place for 5 min to avoid the reflux. After withdrawal of the needle, the burr hole was sealed with bone wax and the scalp was sutured. The animals were allowed to recover on a 37 °C heating pad after operation.

After being deeply anesthetized, mice were perfused with saline followed by fixation with 4% paraformaldehyde (PFA) in 0.1 mol/L PBS at 12 h, 1d, 3d, 7d, and 14d postoperation. Brains were dehydrated in 30% sucrose after an overnight postfixation in 4% PFA. Brain cryosections (20 μm) were prepared and then subjected to immunofluorescence staining. For Western blotting analysis, the mice were killed at 12 h, 1d, 3d, 7d, and 14d postoperation. The peri-lesion area was collected and subjected to lysate preparation and Western blotting analysis.

Astrocyte cultures

Primary astrocyte cultures were prepared from newborn Sprague Dawley rats (SLAC, Shanghai, China), as described previously [55]. In brief, after brain cortices were dissected, the meninges, blood vessels, and hippocampus were removed under a microscope. The remaining cortical tissue was trypsinized for 10 min at 37 °C, and dissociated by gentle trituration. The cell pellet was collected by centrifugation, resuspended in culture medium (90% Dulbecco modified Eagle medium (DMEM), 10% fetal bovine serum, 100 U/mL penicillin G, and 100 mg/mL streptomycin sulfate) and plated onto poly-d-lysine-coated 75 cm2 flasks, maintained at 37 °C in 95% humidity with 5% CO2. The culture medium was refreshed every 4 days. When the cells reached confluence, microglia and oligodendrocyte progenitors were removed by shaking the flask at 220 rpm for 1 h at 37 °C. The pure secondary astrocytes (over 99%), as verified by immunofluorescence staining (GFAP for astrocytes, Iba-1 for microglia), were used in the experiments.

RNA interference experiment

Cy3 labeled negative control siRNA (si-NC) and Cx43 siRNA (si-Cx43) were purchased from Genepharm (Shanghai, China). The sequences are as follows: si-NC sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, si-NC antisense: 5′-ACGUGACACGUUCGGAGAATT-3′, si-Cx43 sense: 5′-ACAUCAUUGAGCUCUUCUATT-3′, si-Cx43 antisense: 5′-UAGAAGAGCUCAAUGAUGUTT-3′. Cells were seeded at a density of 5 × 105 cells per dish in 6 cm-dish or 2 × 104 cells per well in 24-well plate, for Western blotting analysis or immunofluorescence staining, respectively. Twenty-four hours later, the cells were transfected with the siRNAs with Lipoectamine 2000 according to the manufacturer’s instructions. The medium was replaced 24 h later to remove the excessive transfection complex. 72 h after transfection, the cells were harvested and used for subsequent analysis.

Cell viability assay

Cell viability was determined using the Cell Counting Kit-8 (CCK-8) (Beyotime, Jiangsu, China) assay following the manufacturer’s instructions. Briefly, cells were plated into 96-well plates at a density of 1 × 104 cells per well in 100 μL culture medium. Twenty-four hours later, the cells were exposed to 0, 10, 20, 30, or 50 μM Hb (Beyotime, Jiangsu, China) for 24 h, then 10 μL CCK-8 solution was added into each well. After incubating for 4 h at 37°C with 5% CO2, absorbance at 450 nm was measured using a microplate reader (BioTek, VT, USA).

Cell proliferation assessment

Astrocytes were seeded onto the 15mm-diameter coverslips at a density of 5 × 104, and 24 h later, the cells were treated with or without 25 μM Hb for 24 h. In additional experiments, 2.5 or 5 μM verteporfin (VP) (Cat# SML0534, Sigma-Aldrich, MO, USA) were administered with Hb. Then the cells were fixed in 4% PFA for 10 min and then subjected to Ki67 immunofluorescence staining. Fluorescence images were acquired using a confocal laser-scanning microscope (Leica, Solms, Germany). The average percentage of Ki67-positive cells was calculated from 10 random visual fields from each sample.

Total RNA extraction and quantitative real-time PCR analysis

Astrocytes were plated into 6-well plates at a density of 5 × 105 cells per well, after treatment with or without 25 μM Hb for 6 or 24 h, total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA). Reverse transcription was performed using PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Dalian, China). IL-1β, IL-6, TNF-α, and Cx43 mRNA expression was quantified using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Dalian, China). The oligonucleotide primers used were as follows:

Gene name

Forward (5′-3′)

Reverse (5′-3′)

IL-1β

GTCTGACCCATGTGAGCTG

GCCACAGGGATTTTGTCGTT

IL-6

TACCCCAACTTCCAATGCTC

GGTTTGCCGAGTAGACCTCA

TNF-α

TGATCGGTCCCAACAAGGA

TGCTTGGTGGTTTGCTACGA

Connexin43

CTCGCCTATGTCTCCTCCTG

TTTGCTCTGCGCTGTAGTTC

GAPDH

GATGGTGAAGGTCGGTGTGA

TGAACTTGCCGTGGGTAGAG

The data were normalized to the internal reference glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The fold-change values were calculated by normalizing to control samples. PCR amplification was performed for 40 cycles, data were collected using SDS software (Applied Biosystems, CA, USA).

Immunofluorescence staining

The brain cryosections and the coverslips seeded with astrocytes were immunofluorescence stained following the routine procedure. The primary antibodies used were as follow: rabbit anti-YAP polyclonal antibody (1:100, Cat# sc-15407, Santa Cruz Biotechnology, CA, USA), rabbit anti-Connexin43 polyclonal antibody (1:300, Cat# SAB4501175, Sigma-Aldrich, MO, USA), goat anti-GFAP polyclonal antibody (1:100, Cat# sc-6170, Santa Cruz Biotechnology, CA, USA), mouse anti-Vimentin monoclonal antibody (1:300, Cat# MAB3400, Millipore, MA, USA), rabbit anti-Ki67 polyclonal antibody (1:500, Cat# ab15580, Abcam, UK), rabbit anti-Adolase-C polyclonal antibody (1:200, Cat# ab200049, Abcam, UK), rabbit anti-Iba-1 polyclonal antibody (1:200, Cat# 019-19741, WAKO, Japan), rabbit anti-NeuN polyclonal antibody (1:500, Cat# ab177487, Abcam, UK), rat anti-E-Cadherin polyclonal antibody (1:200, Cat# ab11512, Abcam, UK), rabbit anti-N-Cadherin polyclonal antibody (1:200, Cat# ab76057, Abcam, UK). The secondary antibodies used (1:300, Invitrogen, CA, USA): Alexa Fluor 488 donkey anti-rabbit IgG (Cat# A21206), Alexa Fluor 594 donkey anti-rabbit IgG (Cat# A21207), Alexa Fluor 594 donkey anti-rat IgG (Cat# A21209), Alexa Fluor 647 donkey anti-rabbit IgG (Cat# A31573), Alexa Fluor 647 donkey anti-mouse IgG (Cat# A31571), Alexa Fluor 488 donkey anti-goat IgG (Cat# A11055), and Alexa Fluor 594 donkey anti-mouse IgG (Cat# A21203). Nuclei were counterstained with DAPI (1:5000, Invitrogen, CA, USA). Fluorescence images were captured using a confocal laser-scanning microscope (Leica, Solms, Germany). The Image J Just Another Colocalization Plugin (JACoP) was applied for quantitative colocalization analyses [56].

Preparation of cell extracts and Western blotting analysis

Astrocytes were plated into six-well plates at a density of 5 × 105 cells per well, and treated with 0, 10, 15, and 25 μM Hb for 24 h, or with 25 μM Hb for 0, 6, 12, and 24 h. In additional experiments, cells treated with 25 μM Hb were simultaneously treated with or without 2.5 or 5 μM VP. The cells were collected and whole cell lysates were prepared using RIPA lysis buffer (Millipore, MA, USA) supplemented with protease inhibitor cocktail (Roche, Basel, Swiss). Nucleus and cytoplasmic protein was extracted from cell cultures using a nuclear and cytoplasmic protein extraction kit (Beyotime, Jiangsu, China). Protein concentrations were determined with the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, MA, USA). Equal amounts of protein (30 μg) were separated by standard sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrotransferred onto PVDF membranes. The blots were blocked with 5% non-fat milk and incubated overnight at 4 °C with the appropriate primary antibodies: rabbit anti-Connexin 43 polyclonal antibody (1:1000, Cat# SAB4501175, Sigma, MO, USA), mouse anti-GFAP monoclonal antibody (1:3000, Cat# MAB3402, Millipore, MA, USA), mouse anti-Vimentin monoclonal antibody (1:3000, Cat# MAB3400, Millipore, MA, USA), rabbit anti-E-cadherin polyclonal antibody (1:1000, Cat# YT1454, Immunoway, TX, USA), rabbit anti-N-cadherin polyclonal antibody (1:1000, Cat# YT2988, Immunoway, TX, USA), rabbit anti-SLUG polyclonal antibody (1:1000, Cat# YM3371, Immunoway, TX, USA), rabbit anti-YAP monoclonal antibody (1:1000, Cat# 14074, Cell Signaling Technology, MA, USA), rabbit anti-Phospho-YAP(S127) monoclonal antibody (1:1000, Cat# 13008, Cell Signaling Technology, MA, USA), rabbit anti-histone-H3 monoclonal antibody (1:1000, Cat# 4499, Cell Signaling Technology, MA, USA), and rabbit anti-GAPDH monoclonal antibody (1:2000, Cat# 5174, Cell Signaling Technology, MA, USA). Subsequently, the membranes were washed thrice, then incubated with corresponding horseradish peroxidase conjugated secondary antibody for 1 h at room temperature. After washing, the membranes were incubated with ECL solution (ThermoFisher Scientific, MA, USA) for 3 min. The signal was detected using the Tanon image system (Shanghai, China). Densitometric analysis of the bands was performed using ImageJ 1.6.0 (NIH, MD, USA).

Co-immunoprecipitation of Cx43 and YAP

Astrocyte cultures at 100% confluence were lysed with IP lysis buffer containing protease inhibitors (Beyotime, Jiangsu, China) and subsequently centrifuged at 12,000g for 10 min at 4 °C. The supernatant was used for co-immunoprecipitation. The protein concentration was estimated with Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, MA, USA). After preclearing with 20 μL washed protein A/G agarose (Cat# sc-2003, Santa Cruz Biotechnology, CA, USA), the lysate (1 mg protein) was incubated with 1 μg mouse anti-Cx43 monoclonal antibody (Cat# C8093, Sigma, MO, USA), or normal mouse IgG (Cat# sc-2025, Santa Cruz Biotechnology, CA, USA) for 6 h at 4 °C. Then 20 μL washed protein A/G agarose was added to the complex, and rotated for 1 h at 4 °C. The agarose was collected by centrifugation at 1000g for 2 min. After washing thrice with 1 mL IP lysis buffer, the protein bound to the agarose was eluted by boiling in 20 μL 2 × loading buffer and analyzed by Western blotting using rabbit anti-YAP monoclonal antibody (1:1000, Cat# 14074, Cell Signaling Technology, CA, USA) to evaluate the association between Cx43 and YAP protein. Mouse anti-YAP monoclonal antibody (Cat# MAB8094, R&D system, MN, USA) was used for additional immunoprecipitation and the precipitated complex was detected with rabbit anti-Connexin 43 polyclonal antibody (1:1000, Cat# SAB4501175, Sigma, MO, USA) for a further verification.

Statistical analysis

All data are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Statistical significance was evaluated using Student’s t-test or One-way ANOVA followed by Post Hoc Least-Significant Difference (LSD) tests. p-values < 0.05 were considered to be significant. Statistical analysis was performed using SPSS 18.0 (IBM, NY, USA). Statistical charts were drawn using GraphPad Prism 5 (GraphPad Software, CA, USA).