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. 2015 Jul 2;35(8):1073–1079. doi: 10.1007/s10571-015-0215-5

Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1

Zhen Cao 1, Xue-Fei Wu 1, Yan Peng 1, Rui Zhang 2, Na Li 2, Jin-Yi Yang 3, Shu-Qin Zhang 1, Wan-Qin Zhang 1, Jie Zhao 2,✉, Shao Li 1,✉
PMCID: PMC11486269  PMID: 26134308

Abstract

Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.

Electronic supplementary material

The online version of this article (doi:10.1007/s10571-015-0215-5) contains supplementary material, which is available to authorized users.

Keywords: Astrocyte, Glial fibrillary acidic protein (GFAP), c-Jun, Scorpion venom heat-resistant peptide (SVHRP)

Introduction

Astrocytes play a pivotal role in the central nervous system (CNS) and have various essential functions in the healthy CNS. They respond to various types of CNS insults through a process called reactive astrogliosis, which has become a pathological hallmark of CNS structural lesions (Sofroniew and Vinters 2010). Astrogliosis is characterized by rapid synthesis of glial fibrillary acidic protein (GFAP) intermediate filaments. Moreover, increased protein expression of GFAP has been found in experimental models involving gliosis (Eng et al. 2000). In vitro and in vivo studies on the molecular profiles of substances that are upregulated during astrocyte activation document the complex and varied responses of astrocytes to injury (Eddleston and Mucke 1993). Increases in the mRNA and protein expression of GFAP have been shown in diseases including Alzheimer’s disease, scrapie and Creutzfeldt-Jakob disease, in other types of injuries including cerebrovascular accidents and stab wounds, and other types of lesions including experimental allergic encephalomyelitis, which is an animal model for multiple sclerosis (Eng and Ghirnikar 1994). Although the cellular and molecular mechanisms of reducing astrogliosis remain unclear, many drugs are likely to target transcription factors to decrease GFAP expression and exert neuroprotective effects. Scorpion venom heat-resistant peptide (SVHRP), an active component of scorpion venom, is extracted from Buthus martensii Karsch (BmK) in our laboratory with a patented method (Zhang et al. 2004). We found SVHRP could down-regulate GFAP expression in the astrocytes of the rat epileptic hippocampus (Jiang and Zhang 1999) or decrease GFAP expression in the hippocampus of Aβ1-40 -injected rats (Yu et al. 2009). However, the cellular and molecular mechanisms by which SVHRP regulates GFAP expression in astrocytes have not yet been elucidated. Our results show that SVHRP not only attenuates GFAP expression in vitro by reducing the expression of c-Jun/AP-1 but also reduces the interaction of c-Jun/AP-1 with the GFAP promoter. SVHRP could decrease GFAP expression, and this may be useful as a strategy for attenuating reactive gliosis through c-Jun/AP-1 in many CNS diseases.

Materials and Methods

Cell Culture and SVHRP Treatment

Cultured rat primary astrocytes were prepared from the brains of 1-day-old Sprague–Dawley (SD) rats, as described by McCarthy (McCarthy and de Vellis 1980) with some modifications. Briefly, tissues were triturated after removing the meninges and blood vessels. Cells were seeded onto 25-cm2 culture plates (5 × 105/plate) that were pre-coated with poly-d-lysine and grown at 37 °C/5 % CO2. The maintenance medium for mixed glial cell culture was high-glucose DMEM supplemented with 10 % FBS, 1 % l-glutamine, penicillin, and streptomycin (Invitrogen). The medium was changed after the first 3 days and three times per week thereafter. The mixed glial cells were cultured for 7–9 days. The mixed glial cells were then agitated on a rotary shaker at 240 rpm and 37 °C for 19 h. The suspended cells were removed, and fresh medium was added. The cells were resuspended in culture flasks, and purified astrocytes were replated in 6-well and 24-well plates (1 × 106/well) (corning). SVHRP was isolated from BmK venom with a patented method in our laboratory (Zhang et al. 2004). First, the crude venom was collected by electrical stimulation of the telson of scorpion BmK from Henan Province, China and the lyophilized crude venom was dissolved in ddH2O and heated at 100 °C for 4 h before centrifugation. Then the supernatant was loaded onto a Superdex Peptide 10/300GL Colum (ÄKTA avant 25) and separated by FPLC (fast protein liquid chromatograph). Fraction I (P1) from the Superdex Peptide 10/300GL Colum (ÄKTA avant 25) was collected and used for cell treatment. Result from reverse-phase HPLC using C18 column (Zorbax SB-C18 4.6 × 250 5μm) demonstrated that the purity of SVHRP was more than 99.5 %.

Immunocytochemical/Immunofluorescent Staining

After astrocytes were treated with SVHRP for 24 h, the cells were fixed with 4 % paraformaldehyde (30 min), treated with 0.3 % hydrogen peroxide (10 min), and incubated with 5 % BSA solution (60 min). The diluted primary antibody (polyclonal anti-GFAP rabbit antibody, Dako, 1:1000) was incubated at 4 °C overnight, followed by incubation with the biotinylated secondary antibody for 1 h. Cells were then incubated with ABC reagents (1 h), and a diluted DAB solution until a color change was observed or Alexa Fluor 594 secondary antibody (Invitrogen, USA) was added for 1 h at 37 °C. GFAP staining was examined in high-power fields (×200) under a standard light microscope or a Leica DM 4000B microscope or Leica TCS SP5 microscope.

RNA Extraction and PCR

Cells were plated into 6-well plates (corning) at a density of 1 × 106 cells/well. After 2–3 days, SVHRP was diluted with maintenance medium and added to the cells for 24 h. Cells were rinsed twice with PBS and treated with the RNA extraction reagent Trizol (invitrogen). Total RNA was phenol–chloroform extracted and ethanol precipitated. Only RNA with a 260/280 ratio >1.8 was used for the reverse transcription reaction. cDNAs were prepared from 100 ng of total RNA using the Takara RT-PCR system (PrimeScript™ RT reagent Kit with gDNA Eraser) prior to their use as templates (2 μl) for PCR with the SYBR Green kit (SYBR® Premix Ex Taq™ II) and 0.4 μM forward and reverse primers. PCR was performed on a Rotor-Gene-Q Real-Time PCR System at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. GFAP, c-Jun, and c-Fos mRNA levels were measured through Rotor-Gene-Q Real-Time PCR Analysis System (Delta delta CT relative quantitation). Each sample is first normalized for the amount of template added (such as GFAP, c-Jun, c-Fos) by comparison with the normalizing gene (we use GAPDH). These normalized values are further normalized relative to a calibrator treatment that is the untreated control samples.

Western Blot Analysis

Cells were plated into 6-well plates (corning) at a density of 1 × 106 cells/well. After 2–3 days, cells were treated with SVHRP for 24 h; the proteins were extracted. Protein concentrations were determined by using the BCA protein assay kit according to the manufacturer’s introductions (Pierce, Rockford, IL, USA). Equal amounts of protein samples from each group were separated using 8–10 % SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Chemicon) for immunoblotting. Non-specific binding was blocked by incubation in 5 % non-fat milk in Tris-buffered saline containing 0.1 % Tween 20 (TBS-T) for 2 h at room temperature. Next, the membranes were incubated separately with the following primary antibodies: GFAP (polyclonal rabbit antibody, Dako, 1:1000), c-Jun (Monoclonal rabbit antibody, Cell Signaling, 1:600), c-Fos (Monoclonal rabbit antibody, Cell Signaling, 1:600), JunB (Monoclonal rabbit antibody, Cell Signaling, 1:600), and β-actin (Polyclonal rabbit antibody, Abcam, 1:2000). Western blot bands were scanned and quantified by densitometric analysis using the Molecular Imager Chemic Doc XR system (Bio-Rad). Three independent experiments were performed for each assay.

MTT Cell Viability Assay

MTT assay was mainly based on the standard protocol of Vybrant® MTT Cell Proliferation Assay Kit. Cells were plated into 96-well plates (corning) at a density of 5 × 104 cells/well. After 24 h SVHRP treatment, the medium was removed and replaced with 100 µl of fresh culture medium; 10 µl of 12 mM MTT solution was added into each well and incubated for 4 h at 37 °C. 100 µl of the SDS-HCl solution was added to each well and mixed thoroughly. the microplate was incubated at 37 °C for 4 h in a humidified chamber. Mix each sample and read absorbance at 570 nm. The assay was carried out with six replicates for each culture.

Dye Filling

Cells were plated into 24-well plates at a density of 1 × 106 cells/well. After SVHRP treatment for 24 h, cells with 250 µl of the membrane tracer dye (10 µg/ml Dio) were incubated for 20–30 min. The dye working solution was drained off, and the coverslips were washed three times with pre-warmed fresh medium for 30 min. The morphology of the cells after dye filling can be observed with Fluorescence microscope. Fluorescence emission: Ex/Em = 484/501 nm.

Apoptosis Detection

Apoptosis detection was mainly based on the protocol of Sigma® Live/Dead Cell Double Staining Kit (04511). Cells were plated on 24-well plates at density of 5 × 105 cells/well and washed with PBS for three times after SVHRP treatment. Prepared assay solution was added into cells and incubated for 15 min at 37 °C. Calcein-AM (green) stains live cells, and PI (red) stains apoptotic cells. Fluorescence was detected using fluorescence microscope (Ex/Em = 490/515 nm).

Chromatin Immunoprecipitation

Cells were plated into 6-well plates (Corning) at a density of 1 × 106 cells/well. After 2–3 days, cells were treated with SVHRP for 24 h; the protein-DNA complexes were cross-linked with 1 % formaldehyde for 10 min. Cross-linking was quenched by adding 125 mM glycine, and cells were then washed with PBS, harvested and resuspended in 350 μl lysis buffer [50 mM Tris–HCl (pH 8.0); 10 mM EDTA; 1 % SDS] containing protease inhibitors (PMSF, Beyotime, PR China), and then incubated on ice at least 30 min. Next, we sonicated the lysates 12 times, for 5 s each time at 200 W output and with a 10-s refractory period between sonications. 50 μl supernatant was removed as input. Cell lysate is split into two halves (IgG and IP), and 300 μl lysate (5 × 106 cells) is diluted to 2 ml. Then the supernatants were incubated with 35 μl Protein A+G Agarose/Salmon Sperm DNA (Beyotime, PR China) under gentle agitation for 30 min at 4 °C to reduce nonspecific background. The soluble chromatin was collected by centrifugation and transferred to a new microcentrifuge tube, and 1 μg of antibody was added into 150 μl supernatant for incubation overnight at 4 °C. 35 μl Protein A+G Agarose/Salmon Sperm DNA were then added into two halves, respectively, and incubated for 1–2 h at 4 °C under gentle agitation. The pellets were successively washed for 5 min in 1 ml low salt immune complex wash buffer, 1 ml high salt immune complex wash buffer, 1 ml LiCl immune complex wash buffer (Beyotime, PR China), and 2 × 1 ml TE buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA]. The protein/DNA complexes were eluted in 500 µl elution buffer (1 % SDS, 0.1 M NaHCO3), and the cross-links were reversed by overnight incubation at 65 °C. The DNA was then extracted with phenol–chloroform. The purified DNA pellet was subjected to PCR. ChIP DNA (5 μl) segment which contain AP-1 banding site was amplified by PCR with the following primers: forward 5′-GCCTGGTCTGTAAGCTGGAA-3′ and reverse 5′-AATGGACTTCTCGGAAAGCA-3′.

Results

In previous studies, we found that in vivo treatment with SVHRP could inhibit the formation of glial scars in the hippocampus of epileptic rats (Jiang and Zhang 1999) and suppress astrocyte activation in Alzheimer’s disease (AD) rats (Yu et al. 2009). In the present study, we attempted to uncover the cellular and molecular mechanisms underlying this process in vitro. Using immunohistochemical staining, we observed that treatment with 20 μg/ml SVHRP for 24 h could attenuate GFAP expression in rat primary astrocyte cultures (Fig. 1a). Further studies using Western blotting (Fig. 1b, c) and real-time PCR (Fig. 1d) confirmed that GFAP expression was significantly decreased. GFAP is thought to play important roles in modulating astrocyte motility and shape by promoting the structural stability of astrocytic processes (Eng 1985). SVHRP treatment did not affect cell viability (Fig. 1e), cell morphology or survival (Fig. S), suggesting that reduced GFAP expression is not due to cell toxicity or effect on cell proliferation. The most crucial of elements in GFAP gene is a consensus AP-1 sequence, the binding site for the Fos and Jun families of transcription factors (Masood et al. 1993). Gao et al. reported that calcium influx triggered by scratch could activate the c-Jun/AP-1 signaling pathway to switch on GFAP expression (Gao et al. 2013).

Fig. 1.

Fig. 1

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SVHRP attenuates GFAP protein and mRNA expression in primary astrocyte cultures. a Immunocytochemistry and immunofluorescence. b, c Western blotting (c densitometric quantification of GFAP/actin). d Real-time PCR. e Survival of cultured primary astrocytes after SVHRP treatment. Numerical data are shown as the mean ± SEM. t test: *P < 0.05, **P < 0.01 compared with the control group. n = 4, scale bars are 20 μm

We hypothesized that SVHRP treatment decreased GFAP expression by affecting c-Jun/AP-1. Next, we performed chromatin immunoprecipitation to ascertain whether SVHRP reduces the association of c-Jun with the GFAP promoter. After SVHRP treatment, the binding between c-Jun and the GFAP promoter was reduced (Fig. 2a). The ChIP analysis shows that the localization of c-Jun to the GFAP promoter was significantly decreased in primary astrocyte cultures (Fig. 2b). This demonstrates that SVHRP resulted in reduced association between c-Jun and the GFAP promoter. Moreover, c-Jun expression levels were detected by Western blot and real-time PCR (Fig. 2c–e). Similar to the changes in GFAP expression, SVHRP induced an apparent downregulation of c-Jun expression compared with the vehicle control group, as shown by real-time PCR (Fig. 2c) and Western blot (Fig. 2d, e). The AP-1 family of transcription factors consists of homodimers and heterodimers of Jun and Fos (Karin et al. 1997). Interestingly, SVHRP reduced the association of c-Jun and the GFAP promoter; however, it did not affect the binding of the c-Fos component (Fig. 3a). The ChIP assays show that the binding of c-Fos/AP-1 with GFAP promoter was not changed (Fig. 3b). However, SVHRP decreased the expression of c-Fos at both the mRNA and protein levels (Fig. 3c–e). These results suggest that SVHRP may act on c-Jun homodimers binding to the GFAP promoter and may also reduce the expression of both c-Jun and c-Fos at the mRNA and protein levels. Collectively, these data suggest that SVHRP can depress GFAP expression via the c-Jun/AP-1 signaling pathway in astrocytes.

Fig. 2.

Fig. 2

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Decreases in the ability of c-Jun to bind to the GFAP promoter and of c-Jun expression at the protein and mRNA levels in primary astrocytes after treatment with the indicated concentrations of SVHRP. a ChIP demonstrating the change in c-Jun binding to the GFAP promoter. b Quantification of the band densities from a. c Real-time PCR. d Western blot. e Densitometric quantification of c-Jun/actin. Numerical data are shown as the mean ± SEM. t test: *P < 0.05, **P < 0.01 compared with the control group. n = 4

Fig. 3.

Fig. 3

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The ability of c-Fos to bind the GFAP promoter remained unchanged, but the expression of both the protein and mRNA decreased in primary astrocytes after treatment with the indicated concentrations of SVHRP. a ChIP showing c-Fos binding to the GFAP promoter. b Quantification of the band densities from a. c Real-time PCR. d Western blot. e Densitometric quantification of c-Fos/actin. Numerical data are shown as the mean ± SEM. t test: *P < 0.05, **P < 0.01 compared with the control group. n = 4

Discussion

Astrocytes are involved in a wide range of neurological disorders in the CNS, including trauma, ischemia, and neurodegeneration. In response to CNS lesions, astrocytes undergo a characteristic change in morphology, i.e., the hypertrophy of their cellular processes, in a phenomenon called reactive gliosis. A well-known characteristic of reactive astrocytes is the increased production of intermediate filaments (IFs) as a result of the increased expression of GFAP (Pekny and Pekna 2004). In the CNS, seizure activity caused by many different stimuli can induce the expression of AP-1 transcription factors in the brain, particularly in the hippocampus (Dragunow and Robertson 1987; Morgan et al. 1987; White and Gall 1987; Sonnenberg et al. 1989a, b; Sakura-Yamashita et al. 1991; Pennypacker et al. 1993). Meanwhile, one of the most important phenomena of seizure is the emergence of gliosis. To date, experimental models involving gliosis have shown increased protein expression or immunostaining of GFAP, the hallmark of astrocytes. As a traditional Chinese medicine, BmK is used to treat immune-related diseases, such as osteoporosis, in Korea and China. SVHRP is an active component of the scorpion venom extracted from BmK (Zhang et al. 2004). Our present study reveals that SVHRP decreases GFAP expression in astrocytes in vitro. This is consistent with our previous finding that SVHRP could inhibit GFAP expression in astrocytes of the epileptic hippocampus (Jiang and Zhang 1999) or decrease GFAP expression in the hippocampal area of Aβ1–40-injected rats (Yu et al. 2009). Our results suggest that SVHRP may act on astrocyte directly to modulate GFAP expression and related function. It is clear that different molecular, morphological, and functional changes in reactive astrocytes are specifically controlled by inter- and intra-cellular signaling mechanisms that reflect the specific contexts of the stimuli to produce specific and gradated responses to reactive astrogliosis. The various intracellular signaling pathways associated with STAT3, NF-κB, SOCS3, Nrf2, cAMP, and Olig2 are implicated in mediating different aspects or different degrees of reactive astrogliosis, such as GFAP up-regulation (Sofroniew 2009).

In particular, activator protein 1 (AP-1) has been demonstrated to play a critical role in GFAP gene (Masood et al. 1993) and transfection a negative c-Jun expression vector drastically inhibited expression of GFAP in a glioma cell line (Gopalan et al. 2006). Gennadij et al. observed astroglial activation measured by GFAP was impaired in c-Jun knockout mice (Raivich et al. 2004). Whereas mutation of the conserved consensus AP-1 site had little effect on GFAP transgene expression (Yeo et al. 2013). The AP-1 family of transcription factors consists of homodimers and heterodimers of Jun and Fos (Karin et al. 1997). Jun-Fos heterodimers have higher stability than Jun homodimers, which accounts for their increased DNA binding activity and c-Fos, itself, does not seem to dimerize and bind to the AP-1 site (Smeal et al. 1989). Our study demonstrated that SVHRP reduced the association of c-Jun in GFAP promoter and this may be responsible for the decrease in GFAP expression. However, SVHRP did not cause a significant decrease in c-Fos binding. One explanation is that SVHRP mainly affects the binding of homodimers of c-Jun on GFAP promoter and has less effect on fos-jun heterodimer, or c-Jun homodimer is more important in the regulation of GFAP. Meanwhile, other groups have demonstrated that c-Jun and c-Fos are relevant to cell survival (Shaulian and Karin 2002) and cell proliferation and differentiation. (Raivich and Behrens 2006). GFAP is also thought to play important roles in modulating astrocyte motility and shape by promoting the structural stability of astrocytic processes (Eng 1985). Whereas in this study, we have not seen changes of astrocyte morphology or cell viability after SVHRP treatment (Fig. 1e, supplementary). However, we cannot exclude the possibility that SVHRP may affect astrocyte motility or morphology under in vivo conditions.

In summary, this study demonstrates that SVHRP attenuates GFAP expression in cultures of primary astrocytes. The decrease in GFAP expression in astrocytes treated with SVHRP may be caused by the impairment of the association between c-Jun/AP-1 and the GFAP promoter and decreased expression of c-Jun/AP-1.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10571_2015_215_MOESM1_ESM.tif (4.1MB, tif)

Supplementary material 1 (TIFF 4247 kb). Fig. S Cell morphology and survival of cultured primary astrocytes after SVHRP treatment remained unchanged. (a) Morphology of cultured primary astrocytes demonstrated by dye filling with the membrane tracer after SVHRP or vehicle treatment. (b) Live (green) and apoptotic (red) primary astrocytes stained with calcein-AM/PI after SVHRP or vehicle treatment. n = 3, scale bars are 10 μm

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (NSFC, 81371223 and 81371437) and the Research Fund for the Doctoral Program of Higher Education of China (20122105110010).

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

BmK

Buthus martensii Karsch

SVHRP

Scorpion venom heat-resistant peptide

GFAP

Glial fibrillary acidic protein

AP-1

Activator protein 1

Contributor Information

Jie Zhao, Phone: +86-411-8611-0005, Email: dlzhaoj@163.com.

Shao Li, Phone: +86-411-8611-0352, Email: lishao89@hotmail.com.

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Associated Data

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Supplementary Materials

10571_2015_215_MOESM1_ESM.tif (4.1MB, tif)

Supplementary material 1 (TIFF 4247 kb). Fig. S Cell morphology and survival of cultured primary astrocytes after SVHRP treatment remained unchanged. (a) Morphology of cultured primary astrocytes demonstrated by dye filling with the membrane tracer after SVHRP or vehicle treatment. (b) Live (green) and apoptotic (red) primary astrocytes stained with calcein-AM/PI after SVHRP or vehicle treatment. n = 3, scale bars are 10 μm

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