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Protective effects of BMP-7 against tumor necrosis factor α-induced oligodendrocyte apoptosis
Accepted Manuscript Title: Protective effects of BMP-7 against tumor necrosis factor ␣-induced oligodendrocyte apoptosis Author: Xin Wang Jun-Mei Xu Ya-ping Wang Lin Yang Zhi-Jian Li PII: DOI: Reference:
S0736-5748(16)30003-X http://dx.doi.org/doi:10.1016/j.ijdevneu.2016.04.011 DN 2092
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
7-1-2016 19-4-2016 20-4-2016
Please cite this article as: Wang, Xin, Xu, Jun-Mei, Wang, Ya-ping, Yang, Lin, Li, Zhi-Jian, Protective effects of BMP-7 against tumor necrosis factor ␣-induced oligodendrocyte apoptosis.International Journal of Developmental Neuroscience http://dx.doi.org/10.1016/j.ijdevneu.2016.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Protective Effects of BMP-7 Against Tumor Necrosis Factor α- Induced Oligodendrocyte Apoptosis Xin Wang1,2, Jun-Mei Xu1,2, Ya-ping Wang1,2*, Lin Yang1,2, Zhi-Jian Li1,2 1
Department of Anesthesiology, Second Xiangya Hospital, Central South University,
Changsha, Hunan, China 2
Anesthesia Research Institute, Central South University, Changsha, Hunan, China
Corresponding Author: Ya-ping Wang，e-mail：[email protected]
Research highlights ► A phenomenon of BMP7 suppresses TNF-α-induced OLs apoptosis was revealed. ► The mechanism of BMP7 suppresses TNF-α-induced OLs apoptosis is not through influence of JNK pathway. ►The mechanism explains the cIAP1 expression level、the activity of caspase-3 and caspase-8 as important mediators of OLs survival after cellular stress and cytokine challenge.
Abstract Bone morphogenic protein-7 (BMP7) is a multifunctional cytokine with demonstrated neurogenic potential. Oligodendrocytes (OLs) death after spinal cord injury (SCI) contributes to demyelination of spared axons, even leading to a permanent neurological deficit. Therefore, therapeutic approaches to prevent OLs death after SCI should be considered. Since the effects of BMP7 on OLs after injury are largely unknown，we demonstrated the effects of BMP7 on TNF-α-induced OLs apoptosis in vitro. The effects of BMP7 on TNF-α-induced OLs apoptosis were verified by flow cytometry, spectrophotometry and western blotting on primary cultures from spinal cord of postnatal day 1 (P1) to P2 rats. As shown by flow cytometry, apoptosis rate was 25.6% for the control group, 59.0% for the TNF-α group, and 33.5% for the BMP7+TNF-α group; spectrophotometry showed caspase-3 and caspase-8 activity were significantly increased in the TNF-α group than in the control group, and BMP7 could reverse the increase. The involvement of cIAP1 in the protection of BMP7 was determined by western blotting and silencing cIAP1. In summary, our results demonstrated that BMP7 could potently inhibite TNF-α-induced OLs apoptosis and identified the cIAP1 expression level, the activity of caspase-3 and caspase-8 as important mediators of OLs survival after cellular stress and cytokine challenge.
Keywords: Spinal cord injury; oligodendrocyte; BMP7; TNF-α; apoptosis
Introduction SCI induces massive apoptotic cell death of neurons and OLs, which may result in axonal degeneration and demyelination and thereby lead to spinal cord dysfunction[1-3]. OLs death and associated demyelination have been postulated to contribute to chronic deficits after SCI[4-6]. At a contused SCI site, both neurons and glia die by necrosis and/or apoptosis soon after injury, and the spared rim of white matter exhibits demyelination[7,8]. In addition, a longer term secondary injury process appears to affect OLs associated with axons that have been damaged and are undergoing Wallerian degeneration remote from the lesion site, and it is particularly evidented in the dorsal columns rostral to the lesion[6,9]. This apoptotic OLs death can occur over many days or weeks at sites quite far from the injury[6,10]. Prevention of apoptosis after SCI could therefore potentially lead to spinal cord tissue repair and improve the motor function. Indeed, a number of experiments have attempted to suppress apoptotic cell death after SCI[11-13]. This cell death appears to be associated with microglial activation in the central nerves system (CNS), suggesting inflammatory cytokines and the production of oxidative stress may be involved. Furthermore, inflammatory cells and other reactive cells can produce a variety of cytokines, such as TNF-a, interleukin and interferon, etc., which regulate inflammatory reaction and further tissue damage[14-16]. TNF-α, a kind of inflammatory cytokines, could be generated by a variety of cells after SCI, including neurons, glial cells (astrocytes, OLs and microglia) and vascular endothelial cells. TNF-α is a potential trigger of neural cell injury in the spinal cord[17-19]. Rathore et al have studied that[20] TNF-α can increase the iron absorption and storage of astrocytes and microglia, and the functional recovery of CNS has been promoted by using antibodies or other cytokines to inhibit the effects of TNF-α after injury. TNF-α mediates several biologic and immunoregulatory responses in a variety of inflammatory diseases and trauma of the CNS including the spinal cord[21,22]. BMP7 is a member of the transforming growth factor-β superfamily, which has been documented to be a trophic factor for bone and cartilage[23]. Recent studies, however, indicated that the effects of BMP7 were not limited to the skeletal system.
BMP7 is expressed in fetal kidney, heart, teeth, eye, bone, intestine and in perinatal neuronal tissues (e.g., hippocampus, cortex and cerebellum)[24,25]. A growing body of research suggests that BMP7 exerts neuroprotective effects in the CNS. BMP7 selectively promotes dendritic growth in cultured sympathetic and CNS neurons — a property that distinguishes it from most other identified growth factors, which largely support axonal outgrowth[26]. BMP7 also induces neurorepair in stroke animals. Both BMP-7 and its receptors are expressed in the CNS[27] and can be upregulated after transient global cerebral ischemia[28,29]. Exogenous administration of BMP7 before methamphetamine administration reduced the induced increment in cerebral infraction, caspase-3 activation and behavioral deficits
[30]
. Pretreatment with BMP7 prior to
general hypoxia or ischemia reduced brain infarction volume and mortality in rats
[31]
.
The pre-stroke implantation of fetal kidney cells, a tissue with high levels of BMP7, into the rodent cortex reduced infarct volume and improved the functional recovery[32]. The protective effects of fetal kidney transplantation were attenuated after intracerebroventricular delivery of BMP antagonist noggin[33]. Intracisternal administration of BMP7 1 day after focal cerebral infarction induces an enhanced recovery of sensorimotor function in the impaired limbs[34]. BMP7 reduced the infarct size[35] and inhibited neuronal apoptosis[36] and improved functional recovery at delayed time points after stroke[37]. Moreover, post-stroke intracisternal or intracerebroventricular BMP7 injection partially improves motor function two weeks after ischemia in rats[38,39]. BMP7 overexpression by gene transfer was beneficial in both nerves and Schwann cells on functional recovery after sciatic nerve injury in rats[40]. In vitro, studies demonstrated that BMP7 had anti-oxidant[41] and antiapoptotic[42] properties. BMP7 also exerted neuroprotective effects in models of Parkinson’s disease and SCI[43,44]. However, the in vitro effects of BMP7 on TNF-α-mediated oligodendroglial apoptosis and its mechanisms have not been well elucidated. Considering the facts mentioned above and the role of OLs apoptosis in the pathophysiology of SCI, the present study was designed to explore the effects of BMP-7, as well as the underlying mechanisms by focusing on OLs apoptosis and TNF-α. In the early days, Michael S.
Beattie et al found out a SCI-mediated increase of caspase 3 activity in p75+ OLs from rats[9]. Anna Jurewicz et al demonstrated TNF-α-induced death of adult human OLs is mediated by c-jun NH2-terminal kinase-3[45]. And some recent studies found that TNF-α-induced OLs apoptosis included caspase-8 activity[46,47]. In our study, we investigated whether caspase-3、caspase-8 activity and the c-Jun N-terminal kinase (JNK) were involved in the regulation mechanism of TNF-α-induced OLs apoptosis. To our knowledge, this is the first experimental investigation of BMP7 on the protection of TNF-α-mediated oligodendroglial apoptosis.
Materials and Methods All animal care and surgical interventions were undertaken in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, 1996), and with the approval of the Central South University Institutional Animal Care and Use Committee and Institutional Biosafety Committee.
Primary cell culture Rat glial cultures were prepared from P1 to P2 rat pups of either sex using a modified shaking method[48]. Dissociated cells were filtered through a 70 um nylon mesh filter to remove the tissue blocks and then were plated on untreated T75 flasks (2.0×105/cm2) at 37℃for 40 min in DMEM/F12 medium containning 10% FBS. Non adherent cells were plated on 25 ug/ml poly-L-lysine-coated T75 flasks in DMEM/F12 medium containning 10% FBS to remove fibroblast cells. The flasks were rested in the first 3 days and then changed the media every 3 days. After grown for 8–9 days, cells were shaken at 37°C at 160 rpm for 2 hrs to remove the microglia cells. After changed the medium and incubated 2-4 hrs at 37°C, the cells were shaken at 37°C at 200 rpm for 18–20 hrs followed by an additional 30 min at 250 rpm. Non adherent cells were plated on untreated T75 flasks 30-60 min at 37°C to let the astrocytes stick wall, then the cell suspension was collected and filtered through a 20
um nylon mesh filter to remove astrocyte aggregates. The remaining cells (oligodendrocyte precursor cells, OPCs) were plated on poly-L-lysine-coated tissue culture dishes and grown in proliferation media: DMEM/F12 medium supplemented with 1% B27, 0.5% N2, 1% penicillin/streptomycin, 0.1 % BSA (all from Invitrogen, USA), 10 ng/ml platelet- derived growth factor AA (PDGFaa, PreproTech, USA), 20 ng/ml fibroblast growth factor basic (bFGF, PreproTech, USA) and 5 µg/ml insuline. To initiate differentiation, oligodendrocyte precursor cells (OPCs) were switched to DMEM/F12 medium supplemented with 1% B27, 0.5% N2, 1% P/S, 0.1% FBS, 10 ng/ml NT3 (PreproTech, USA), 30 ng/ml triidothyronine (T3, Sigma, USA), 5 µg/ml insulin and 5 ng/mL Ciliary Neurotrophic Factor (PreproTech, USA) was added afer 4 days of differentiation. Removal of bFGF and PDGFaa allowed OPCs to differentiate into OLs after 7–10 days.
Immunofluorescence The primary antibodies used in the study included: mouse monoclonal anti-A2B5 (1:200), mouse monoclonal anti-myelin basic protein (MBP, 1:20), rabbit polyclonal anti-glial fibrillary acid protein (GFAP, 1:1000; all from Millipore, USA). The secondary antibody were Alexa Fluor 488-conjugated goat anti-mouse IgM (1:1000; Invitrogen, USA), TRITIC-conjugated goat anti-rabbit IgG (1:50; KPL, USA), and FITC-conjugated goat anti-mouse IgG (1:100; sigma, USA). Cells were cultured on 12-well plates at a density of 2×105/ml were fixed with 4% paraformaldehyde for 30 min and washed 3 times in PBS, then permeabilized with 0.3% Triton X-100 for 10 min and blocked for 1 h with 10% natural goat serum in PBS. Cells were then incubated with primary antibodies overnight at 4℃, and incubated with secondary antibody for 1 h at RT (room temperature). After repeated washes, the samples were incubated with Hoechst 33342 (1:400; sigma, USA) for 10 min, covered with a coverslip and examined under the fluorescence microscope. Controls were performed with the appropriate species-specific non-immune sera and showed negligible background. Total cellular counts for each experimental were obtained in 10 fields under 20 objective from three culture wells. The results for each experimental
condition were verified a minimum of three independent times. For purified OPC cultures, the number of A2B5-positive cells relative to the total number of Hoechst 33342-positive cells were counted from the 10 randomly selected fields taken from three different coverslips. For OLs cultures, the number of MBP-positive cells relative to the total number of Hoechst 33342-positive cells were counted from the 10 randomly selected fields taken from three different coverslips.
Cell activity assay The impact of the different concentrations of (1,10,100,500,1000) ng/ml TNF-α (PreproTech, USA) treated on OLs during different time (24, 48, 72 hrs) and the effects of (10, 50, 200) ng/ml BMP7 (PreproTech, USA) on TNF-α treated OLs over different time (48,72 hrs) were determined using a Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China) according to the manufacturer’s instructions. OLs were seeded onto 25ug/ml PLL-coated 96-well plates at a density of 2×104 cells/100 ul and incubated in the presence of 1,10,100,500 and 1000ng/ml TNF-α for 24,48 and 72 hrs to detect the optimal concentration and action time TNF-α treated on OLs. The control group was added the same volume of DMEM/F12 medium and each group set 5 parallel holes. CCK-8 (10 ul) was added into 100 ul medium, and cells were then incubated for 0.5–4 hrs at 37℃. The absorbance was measured at 450 nm using a microplate reader (Model 680, Bio-Rad, USA) to reflect the number of living cells indirectly.
Apoptosis analysis by FACS OLs were stimulated in duplicate in 96-well microtiter culture plates, trypsinized (0.05%) at the indicated time points and washed with PBS containing 10% FBS to inactivate the trypsin. The double-staining procedure was carried out following the manufacturer’s instructions (Invitrogen, USA). Analysis with a FACS Calibur flow cytometer (Becton Dickinson, Erembodegem, Belgium). Data acquisition and analysis were carried out using Cell Quest software (Becton Dickinson).
Analysis of caspase activities Caspase activities were measured using caspase-3 and caspase-8 activity assay kits (Beyotime, Shanghai, China) according to the manufacturer's instructions. The activities of caspase-3 and-8 were measured using substrate peptides acetyl-AspGlu-ValAsp
p-nitroanilide
(Ac-DEVD-pNA)
and
acetyl-Ile-Glu-Thr-Asp
p-
nitroanilide (Ac-IETD-pNA) respectively. The release of p-nitroanilide (pNA) was qualified by determining the absorbance with a microplate reader (Model 680, Bio-Rad, USA) at 405 nm.
Western blotting Cell samples were lysed in RIPA buffer (Millipore, USA) with 1% PMSF (sigma, USA). Protein samples were separated by SDS-PAGE (Beyotime, Shanghai, China) ( NuPAGE 10% Bis Tris Gel), transferred to PVDF membranes (Bio-Rad, USA), the membranes were blocked with 5% BSA in Tris-buffered saline (TBS) overnight, followed by incubation for 1h-2 hrs at RT with primary antibodies : JNK1, JNK2, JNK3, p-JNK, c-IAP1 (1:200, Santa Cruz, USA)；c-JUN, p-c-JUN (1:1000, Santa Cruz, USA), β-actin (1:5000, Santa Cruz, USA). Protein levels were quantified by densitometry and normalized to β-actin. Blots were washed in TBS and incubated with horse radish peroxidase-coupled secondary antibodies (1:5000, Santa Cruz, USA) for 1 h at RT then washed three times in TBS followed by ECL kit (Amersham Biosciences).
c-IAP1 knockdown Cells were seeded in a 6-well plate at a density of 4×105 cells /ml and were transfected with control siRNA or c-IAP1 siRNA (Santa Cruz，USA) following a Lipofectamine 2000 (Invitrogen, USA) protocol. After 48 hrs, the efficiency of transfection was determined by western blotting.
Statistical analysis All statistical analyses were performed using GraphPad Prism 5 (Graph Pad
Software, Inc., CA, USA). Results are expressed as means±standard deviation (SD) and data were analyzed using one way ANOVA with Bonferroni post hoc for multiple comparisons, or Student’s t-test for pairwise comparisons. A value of P < 0.05 was considered significant. All experiments below were replicated using cells from three different primary cultures. FACS analysis data were derived from counting at least 10000 events in each experiment. The means were compared using Student's t-test.
Results Primary cell culture In the purified OPCs, greater than 95% of these cells expressed the OPC specific markers A2B5 (Fig. 1C). To test their differentiation potential, OPCs appeared bipotential (Fig. 1D) induced to differentiate by withdrawing FGF2 and PDGFaa. When cultured in serum free medium lacking bFGF2 and PDGFaa for 3 days, a lot of cells developed the typical, highly process-bearing morphology of OLs (Fig. 1E). And over 6 days of differentiation, the cells developed into mature OLs with visible membrane sheets (Fig. 1F). We used MBP and GFAP as the marker proteins of mature OLs and astrocytes separately to investigate the mature OLs in cultures. The results showed that more than 95% of these cells were MBP+ cells (Fig. 1J). So OPCs from the rat spinal cord were bipotential with the capacity to differentiate into OLs in the absence of serum[49].
BMP7 suppresses TNF-α-induced OLs apoptosis More and more studies show BMP7 exerts neuroprotective effects in the CNS, since it can selectively promote dendritic growth in cultured sympathetic and CNS neurons[26] and can induce neurorepair besides improve functional recovery in stroke animals[27-29]. Some vitro studies demonstrated that BMP7 had anti-oxidant[41] and anti-apoptotic[42] properties. BMP7 also exerted neuroprotective effects in models of Parkinson’s disease and SCI[43,44]. In this part, we examined whether BMP7 would protect OLs against apoptosis induced by TNF-α in vitro. First, we measured the
effects of TNF-α(1,10,100,500,1000) ng/ml on OLs viability at 24 h，48h and 72h using the CCK-8 assay. The results showed the cell viability of TNF-α (100, 500, 1000) ng/ml treated after 48hrs and 72hrs were significantly reduced compared with the control group. There was no significant difference oberserved in any time points between TNF-α (1 ng/ml, 10 ng/ml) treatment group and the control group (Fig. 2A). So, we chose the concentration 100 ng/ml of TNF-α treated OLs for 48hrs as the proper condition. Further, we measured the cell viability after co-administration with (100 ng/ml) TNF-α and (10, 50, 200) ng/ml BMP7 at 24 h and 48 h using the CCK-8 assay. We found that (50, 200) ng/ml BMP7 could effectively ameliorated the cytotoxic effects of TNF-α after 48hrs (Fig. 2B). The results suggested BMP7 altered the cytotoxic effects on OLs. And the graph of flow cytometric analysis (Fig. 2C) indicated the percentage of apoptotic cells (the early and the late apoptosis, Q2 and Q3 ) was higher in the TNF-α group (59.0%) than in the control group (25.6%). The ratio of apoptotic cells fell to 33.5% after co-incubation with BMP7 (50 ng/ml) compared with exposure to only TNF-α. Together, our results suggested that BMP7 could suppresse TNF-α-induced OLs apoptosis.
BMP7 suppresses TNF-α-induced OLs apoptosis is not through influence of JNK pathway In this part, we measured caspase-3, caspase-8 activity and the relative protein expression levels of the JNK signaling in OLs. The results from spectrophotometry showed that caspase-3 and caspase-8 activity were increased in the TNF-α group than in control group, and BMP7 could reverse the increase (Fig. 3A and B). However, immunoblot analysis indicated there were no significant differences about the expression of JNK1,JNK2,JNK3 and c-JUN between the experimental group and the control group, meanwhile, the expression of p-JNK and pc-JUN were significantly increased both in the TNF-α group and the BMP7+TNF-α group when compared with the control group seperately (Fig. 3C to H). The results indicated that TNF-α may induce JNK and c-JUN activation to mediate OLs apoptosis, but the protection of BMP7 against TNF-α may not through influencing the JNK pathway. Together, the
results shown the protective effects of BMP7 on TNF-α-induced apoptosis was due to inhibition of caspase-3 and caspase-8 activity, but not the JNK signaling pathway.
c-IAP1 is included in the protection BMP7exert on TNF-α-induced OLs apoptosis Ai Ing Lim et al[50] have made a research recently on the effection and the related regulating mechanism of BMP7 on human serum albumin (HSA)-induced chemokine synthesis in proximal tubular epithelial cells (PTECs) in vitro. The study showed BMP7 repressed PTECs apoptosis by upregulating the expression level of cIAP1. Since the protective mechanism of BMP7 against TNF-α did not inclue JNK passway. To determine whether the factor cIAP1 was involved in the protection exerted by BMP7, we compared the expression of cIAP1 in OLs between the TNF-α group and the BMP7+TNF-α group by western blotting. The result suggested the expression of cIAP1 was obviously decreased in the TNF-α treated group compared with the control group，while，significantly increased in the BMP7+TNF-α treated group compared with the TNF-α treated group (Fig. 4A and B). This results shown BMP7 raised the cIAP1 level which had been reduced by TNF-α in OLs. In order to further find out the function of cIAP1, we planned to silence the expression of cIAP1. We used western blotting to exam the transfect efficiency of cIAP1 siRNA, the result showed the target protein expression was significantly decreased in cIAP1 siRNA transfection group when compared with the control group (Fig. 4C and D). To determine whether the factor cIAP1 was involved in the inhibition of the caspase activity exerted by BMP7, through silencing the expression of cIAP1, there were increases in caspase-3 and caspase-8 activity in OLs (Fig. 4E and F). The results demonstrated BMP7 could reduce the caspase activity resulting from TNF-α-induced cytotoxicity through the effection of cIAP1. To find out if the factor cIAP1 was involved in the protection of BMP7 against TNF-α-induced OLs apoptosis, we transfected the cells with cIAP1 siRNA. In the absence of cIAP1, the graph of flow cytometric analysis indicated that the percentage of apoptotic cells (the early and the late apoptosis, Q2 and Q3 ) in the cIAP1 siRNA group (62.6%) was higher than the control group (28.78%) by Annexin V/PI assay (Fig. 4G). Together, these results demonstrated that the presence of the
increased cIAP1 level is crucial for BMP7-induced OLs survival after TNF-α administration.
Discussion CNS demyelinating diseases，such as SCI[51], multiple sclerosis[46], periventricular leukomalacia[52] and so on，are initiated by over secretion of pro-inflammatory cytokines such as IFN-γ, IL-1β, and TNF-a. These cytokines cause the loss of OLs and neuronal axons which give rise to subsequent neurological disabilities in individuals. After SCI, a large portion of proximal OLs undergoes apoptotic death during an extended period of time, with up to one-half of the population ultimately lost from the immediate area of injury[53]. The result of this process is chronic demyelination[54]. OLs are sensitive to TNF-a mediated cytotoxicity in vitro and in vivo. Administration of TNF-a induced apoptotic cell death of primary OLs in vitro [45,55]
. In vivo, elevated levels of TNF-a are found in multiple sclerosis plaques and in
experimental autoimmune encephalomyelitis lesions[56]. Transgenic mice expressing TNF-a in the CNS showed increased OLs apoptosis and primary demyelination[57]. Inhibition of TNF-a decreased apoptotic cell death and improved functional recovery after SCI[58]. TNF-a-induced OLs apoptosis was regulated through caspase-8 and caspase-3 activiation[51] or mediated by the JNK pathway[45] in vitro. TNF-a activates the JNK pathway as part of their apoptotic signals when they bind their cognate receptors[45,59], both in vivo and in vitro. The cellular functions of TNF-α are mediated by TNF receptor 1 (TNFR1) and TNFR2. TNFR1contains a death domain localized within the cytoplasmic segment. TNF-a mediated apoptosis occurs as a result of TNF-a binding to the TNFR1, which recruits FAS-associated death domain (FADD). FADD recruits and activates caspases-8 and -10 within a death-inducing signaling complex, caspases-8 and -10 activate apoptotic death through the same effector caspases as the intrinsic pathway，which results in the cleavage and activation of caspase-3 and caspase-7 and subsequent apoptosis. TNFR2 lacks a DD, but has a cytoplasmic motif that binds TRAFs (TNFR-associated factors). These receptors trigger several intracellular signaling pathways, including the I-kB
kinase, JNK, and p38 or p42/44 mitogen-activated protein kinase cascades[47]. In our study, OLs treated with TNF-a could result in activated caspase-8 and caspase-3 and increased the number of active JNK (p-JNK) and c-JUN (pc-JUN). And the presence of BMP7 in TNF-a treated OL cultures significantly protected OLs against caspases activation and cells apoptosis. Although, BMPs have been reported as potent inhibitors for myelin regeneration while promote astrogliosis[60]. Interestingly, the temporal competence of the BMP pathway to promote astrocyte fate might depend on the presence of specific Smad co-factors, such as the pro-neural bHLH[61]. Activation of the BMP receptor, BRIa, can activate NFkB, p38, Smad 1/5/8, and JNK pathways through TAB1 and TAK1 complex[62]. In our study, BMP7 was more effective at maintaining oligodendrocyte survival through the caspase inhibition than influencing the JNK pathway. These results suggested that BMP7 did not inhibit the JNK pathway activation but may work by the other signaling pathway to protect OLs from the cytotoxic of TNF-a. As we know, upon binding to ligated TNFR1, TRADD recruits the secondary adaptors RIP1, TRAF2, or TRAF5. TRAF2 can recruits ancillary proteins that modulate signaling through each TNFR, i.e., cIAP1 and 2, and TRAF1. CIAP1 and cIAP2 are antiapoptotic proteins existing in the cytoplasm, and have the activity of the ubiquitin-protein ligase to inhibit cell apoptosis[47]. Some researchers[50] have made a study recently on the effection and the related regulating mechanism of BMP7 on HSA-induced chemokine synthesis in PTECs in vitro. The study showed the cytoplasmic expression of cIAP1, tumor necrosis factor receptor-associated factor TRAF2 and TRAF3 were increased after BMP7 treatment. To find out whether the factor cIAP1 was involved in the protection of BMP7 against TNF-a -induced OLs apoptosis, OLs were incubated with TNF-a and BMP7, the western blotting results showed the expression of cIAP1 was significantly increased in the BMP7+TNF-a treated group compared with the TNF-a treated group. After silencing the expression of cIAP1, cells apoptosis rate was higher (62.6%) in the siRNA cIAP1 group when compared with the control group (28.78%) detected by flow cytometry. And there were increases in active caspase-3 and caspase-8 examed by spectrophotometry. In the
presence of cIAP1, BMP7 was as effective as the caspases inhibitors in protecting the cultures from TNF-a cytotoxicity. In the absence of cIAP1, BMP7 was less effective at the caspases inhibition. These results demonstrated that TNF-a induced caspase-8 and downstream caspase activation in OLs and BMP7 could reduce the caspases activity resulting from TNF-a -induced cytotoxicity through the effection of cIAP1. In summary, our results demonstrated that BMP7 could potently inhibite TNF-a -induced OLs apoptosis and identified the cIAP1 expression level together with the caspase-3 and caspase-8 activity as the important mediators of OLs survival after cellular stress and cytokine challenge.
Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Acknowledgments The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by National Science Foundation of China (#81070994).
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S0736-5748(16)30003-X http://dx.doi.org/doi:10.1016/j.ijdevneu.2016.04.011 DN 2092
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
7-1-2016 19-4-2016 20-4-2016
Please cite this article as: Wang, Xin, Xu, Jun-Mei, Wang, Ya-ping, Yang, Lin, Li, Zhi-Jian, Protective effects of BMP-7 against tumor necrosis factor ␣-induced oligodendrocyte apoptosis.International Journal of Developmental Neuroscience http://dx.doi.org/10.1016/j.ijdevneu.2016.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Protective Effects of BMP-7 Against Tumor Necrosis Factor α- Induced Oligodendrocyte Apoptosis Xin Wang1,2, Jun-Mei Xu1,2, Ya-ping Wang1,2*, Lin Yang1,2, Zhi-Jian Li1,2 1
Department of Anesthesiology, Second Xiangya Hospital, Central South University,
Changsha, Hunan, China 2
Anesthesia Research Institute, Central South University, Changsha, Hunan, China
Corresponding Author: Ya-ping Wang，e-mail：[email protected]
Research highlights ► A phenomenon of BMP7 suppresses TNF-α-induced OLs apoptosis was revealed. ► The mechanism of BMP7 suppresses TNF-α-induced OLs apoptosis is not through influence of JNK pathway. ►The mechanism explains the cIAP1 expression level、the activity of caspase-3 and caspase-8 as important mediators of OLs survival after cellular stress and cytokine challenge.
Abstract Bone morphogenic protein-7 (BMP7) is a multifunctional cytokine with demonstrated neurogenic potential. Oligodendrocytes (OLs) death after spinal cord injury (SCI) contributes to demyelination of spared axons, even leading to a permanent neurological deficit. Therefore, therapeutic approaches to prevent OLs death after SCI should be considered. Since the effects of BMP7 on OLs after injury are largely unknown，we demonstrated the effects of BMP7 on TNF-α-induced OLs apoptosis in vitro. The effects of BMP7 on TNF-α-induced OLs apoptosis were verified by flow cytometry, spectrophotometry and western blotting on primary cultures from spinal cord of postnatal day 1 (P1) to P2 rats. As shown by flow cytometry, apoptosis rate was 25.6% for the control group, 59.0% for the TNF-α group, and 33.5% for the BMP7+TNF-α group; spectrophotometry showed caspase-3 and caspase-8 activity were significantly increased in the TNF-α group than in the control group, and BMP7 could reverse the increase. The involvement of cIAP1 in the protection of BMP7 was determined by western blotting and silencing cIAP1. In summary, our results demonstrated that BMP7 could potently inhibite TNF-α-induced OLs apoptosis and identified the cIAP1 expression level, the activity of caspase-3 and caspase-8 as important mediators of OLs survival after cellular stress and cytokine challenge.
Keywords: Spinal cord injury; oligodendrocyte; BMP7; TNF-α; apoptosis
Introduction SCI induces massive apoptotic cell death of neurons and OLs, which may result in axonal degeneration and demyelination and thereby lead to spinal cord dysfunction[1-3]. OLs death and associated demyelination have been postulated to contribute to chronic deficits after SCI[4-6]. At a contused SCI site, both neurons and glia die by necrosis and/or apoptosis soon after injury, and the spared rim of white matter exhibits demyelination[7,8]. In addition, a longer term secondary injury process appears to affect OLs associated with axons that have been damaged and are undergoing Wallerian degeneration remote from the lesion site, and it is particularly evidented in the dorsal columns rostral to the lesion[6,9]. This apoptotic OLs death can occur over many days or weeks at sites quite far from the injury[6,10]. Prevention of apoptosis after SCI could therefore potentially lead to spinal cord tissue repair and improve the motor function. Indeed, a number of experiments have attempted to suppress apoptotic cell death after SCI[11-13]. This cell death appears to be associated with microglial activation in the central nerves system (CNS), suggesting inflammatory cytokines and the production of oxidative stress may be involved. Furthermore, inflammatory cells and other reactive cells can produce a variety of cytokines, such as TNF-a, interleukin and interferon, etc., which regulate inflammatory reaction and further tissue damage[14-16]. TNF-α, a kind of inflammatory cytokines, could be generated by a variety of cells after SCI, including neurons, glial cells (astrocytes, OLs and microglia) and vascular endothelial cells. TNF-α is a potential trigger of neural cell injury in the spinal cord[17-19]. Rathore et al have studied that[20] TNF-α can increase the iron absorption and storage of astrocytes and microglia, and the functional recovery of CNS has been promoted by using antibodies or other cytokines to inhibit the effects of TNF-α after injury. TNF-α mediates several biologic and immunoregulatory responses in a variety of inflammatory diseases and trauma of the CNS including the spinal cord[21,22]. BMP7 is a member of the transforming growth factor-β superfamily, which has been documented to be a trophic factor for bone and cartilage[23]. Recent studies, however, indicated that the effects of BMP7 were not limited to the skeletal system.
BMP7 is expressed in fetal kidney, heart, teeth, eye, bone, intestine and in perinatal neuronal tissues (e.g., hippocampus, cortex and cerebellum)[24,25]. A growing body of research suggests that BMP7 exerts neuroprotective effects in the CNS. BMP7 selectively promotes dendritic growth in cultured sympathetic and CNS neurons — a property that distinguishes it from most other identified growth factors, which largely support axonal outgrowth[26]. BMP7 also induces neurorepair in stroke animals. Both BMP-7 and its receptors are expressed in the CNS[27] and can be upregulated after transient global cerebral ischemia[28,29]. Exogenous administration of BMP7 before methamphetamine administration reduced the induced increment in cerebral infraction, caspase-3 activation and behavioral deficits
[30]
. Pretreatment with BMP7 prior to
general hypoxia or ischemia reduced brain infarction volume and mortality in rats
[31]
.
The pre-stroke implantation of fetal kidney cells, a tissue with high levels of BMP7, into the rodent cortex reduced infarct volume and improved the functional recovery[32]. The protective effects of fetal kidney transplantation were attenuated after intracerebroventricular delivery of BMP antagonist noggin[33]. Intracisternal administration of BMP7 1 day after focal cerebral infarction induces an enhanced recovery of sensorimotor function in the impaired limbs[34]. BMP7 reduced the infarct size[35] and inhibited neuronal apoptosis[36] and improved functional recovery at delayed time points after stroke[37]. Moreover, post-stroke intracisternal or intracerebroventricular BMP7 injection partially improves motor function two weeks after ischemia in rats[38,39]. BMP7 overexpression by gene transfer was beneficial in both nerves and Schwann cells on functional recovery after sciatic nerve injury in rats[40]. In vitro, studies demonstrated that BMP7 had anti-oxidant[41] and antiapoptotic[42] properties. BMP7 also exerted neuroprotective effects in models of Parkinson’s disease and SCI[43,44]. However, the in vitro effects of BMP7 on TNF-α-mediated oligodendroglial apoptosis and its mechanisms have not been well elucidated. Considering the facts mentioned above and the role of OLs apoptosis in the pathophysiology of SCI, the present study was designed to explore the effects of BMP-7, as well as the underlying mechanisms by focusing on OLs apoptosis and TNF-α. In the early days, Michael S.
Beattie et al found out a SCI-mediated increase of caspase 3 activity in p75+ OLs from rats[9]. Anna Jurewicz et al demonstrated TNF-α-induced death of adult human OLs is mediated by c-jun NH2-terminal kinase-3[45]. And some recent studies found that TNF-α-induced OLs apoptosis included caspase-8 activity[46,47]. In our study, we investigated whether caspase-3、caspase-8 activity and the c-Jun N-terminal kinase (JNK) were involved in the regulation mechanism of TNF-α-induced OLs apoptosis. To our knowledge, this is the first experimental investigation of BMP7 on the protection of TNF-α-mediated oligodendroglial apoptosis.
Materials and Methods All animal care and surgical interventions were undertaken in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, 1996), and with the approval of the Central South University Institutional Animal Care and Use Committee and Institutional Biosafety Committee.
Primary cell culture Rat glial cultures were prepared from P1 to P2 rat pups of either sex using a modified shaking method[48]. Dissociated cells were filtered through a 70 um nylon mesh filter to remove the tissue blocks and then were plated on untreated T75 flasks (2.0×105/cm2) at 37℃for 40 min in DMEM/F12 medium containning 10% FBS. Non adherent cells were plated on 25 ug/ml poly-L-lysine-coated T75 flasks in DMEM/F12 medium containning 10% FBS to remove fibroblast cells. The flasks were rested in the first 3 days and then changed the media every 3 days. After grown for 8–9 days, cells were shaken at 37°C at 160 rpm for 2 hrs to remove the microglia cells. After changed the medium and incubated 2-4 hrs at 37°C, the cells were shaken at 37°C at 200 rpm for 18–20 hrs followed by an additional 30 min at 250 rpm. Non adherent cells were plated on untreated T75 flasks 30-60 min at 37°C to let the astrocytes stick wall, then the cell suspension was collected and filtered through a 20
um nylon mesh filter to remove astrocyte aggregates. The remaining cells (oligodendrocyte precursor cells, OPCs) were plated on poly-L-lysine-coated tissue culture dishes and grown in proliferation media: DMEM/F12 medium supplemented with 1% B27, 0.5% N2, 1% penicillin/streptomycin, 0.1 % BSA (all from Invitrogen, USA), 10 ng/ml platelet- derived growth factor AA (PDGFaa, PreproTech, USA), 20 ng/ml fibroblast growth factor basic (bFGF, PreproTech, USA) and 5 µg/ml insuline. To initiate differentiation, oligodendrocyte precursor cells (OPCs) were switched to DMEM/F12 medium supplemented with 1% B27, 0.5% N2, 1% P/S, 0.1% FBS, 10 ng/ml NT3 (PreproTech, USA), 30 ng/ml triidothyronine (T3, Sigma, USA), 5 µg/ml insulin and 5 ng/mL Ciliary Neurotrophic Factor (PreproTech, USA) was added afer 4 days of differentiation. Removal of bFGF and PDGFaa allowed OPCs to differentiate into OLs after 7–10 days.
Immunofluorescence The primary antibodies used in the study included: mouse monoclonal anti-A2B5 (1:200), mouse monoclonal anti-myelin basic protein (MBP, 1:20), rabbit polyclonal anti-glial fibrillary acid protein (GFAP, 1:1000; all from Millipore, USA). The secondary antibody were Alexa Fluor 488-conjugated goat anti-mouse IgM (1:1000; Invitrogen, USA), TRITIC-conjugated goat anti-rabbit IgG (1:50; KPL, USA), and FITC-conjugated goat anti-mouse IgG (1:100; sigma, USA). Cells were cultured on 12-well plates at a density of 2×105/ml were fixed with 4% paraformaldehyde for 30 min and washed 3 times in PBS, then permeabilized with 0.3% Triton X-100 for 10 min and blocked for 1 h with 10% natural goat serum in PBS. Cells were then incubated with primary antibodies overnight at 4℃, and incubated with secondary antibody for 1 h at RT (room temperature). After repeated washes, the samples were incubated with Hoechst 33342 (1:400; sigma, USA) for 10 min, covered with a coverslip and examined under the fluorescence microscope. Controls were performed with the appropriate species-specific non-immune sera and showed negligible background. Total cellular counts for each experimental were obtained in 10 fields under 20 objective from three culture wells. The results for each experimental
condition were verified a minimum of three independent times. For purified OPC cultures, the number of A2B5-positive cells relative to the total number of Hoechst 33342-positive cells were counted from the 10 randomly selected fields taken from three different coverslips. For OLs cultures, the number of MBP-positive cells relative to the total number of Hoechst 33342-positive cells were counted from the 10 randomly selected fields taken from three different coverslips.
Cell activity assay The impact of the different concentrations of (1,10,100,500,1000) ng/ml TNF-α (PreproTech, USA) treated on OLs during different time (24, 48, 72 hrs) and the effects of (10, 50, 200) ng/ml BMP7 (PreproTech, USA) on TNF-α treated OLs over different time (48,72 hrs) were determined using a Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China) according to the manufacturer’s instructions. OLs were seeded onto 25ug/ml PLL-coated 96-well plates at a density of 2×104 cells/100 ul and incubated in the presence of 1,10,100,500 and 1000ng/ml TNF-α for 24,48 and 72 hrs to detect the optimal concentration and action time TNF-α treated on OLs. The control group was added the same volume of DMEM/F12 medium and each group set 5 parallel holes. CCK-8 (10 ul) was added into 100 ul medium, and cells were then incubated for 0.5–4 hrs at 37℃. The absorbance was measured at 450 nm using a microplate reader (Model 680, Bio-Rad, USA) to reflect the number of living cells indirectly.
Apoptosis analysis by FACS OLs were stimulated in duplicate in 96-well microtiter culture plates, trypsinized (0.05%) at the indicated time points and washed with PBS containing 10% FBS to inactivate the trypsin. The double-staining procedure was carried out following the manufacturer’s instructions (Invitrogen, USA). Analysis with a FACS Calibur flow cytometer (Becton Dickinson, Erembodegem, Belgium). Data acquisition and analysis were carried out using Cell Quest software (Becton Dickinson).
Analysis of caspase activities Caspase activities were measured using caspase-3 and caspase-8 activity assay kits (Beyotime, Shanghai, China) according to the manufacturer's instructions. The activities of caspase-3 and-8 were measured using substrate peptides acetyl-AspGlu-ValAsp
p-nitroanilide
(Ac-DEVD-pNA)
and
acetyl-Ile-Glu-Thr-Asp
p-
nitroanilide (Ac-IETD-pNA) respectively. The release of p-nitroanilide (pNA) was qualified by determining the absorbance with a microplate reader (Model 680, Bio-Rad, USA) at 405 nm.
Western blotting Cell samples were lysed in RIPA buffer (Millipore, USA) with 1% PMSF (sigma, USA). Protein samples were separated by SDS-PAGE (Beyotime, Shanghai, China) ( NuPAGE 10% Bis Tris Gel), transferred to PVDF membranes (Bio-Rad, USA), the membranes were blocked with 5% BSA in Tris-buffered saline (TBS) overnight, followed by incubation for 1h-2 hrs at RT with primary antibodies : JNK1, JNK2, JNK3, p-JNK, c-IAP1 (1:200, Santa Cruz, USA)；c-JUN, p-c-JUN (1:1000, Santa Cruz, USA), β-actin (1:5000, Santa Cruz, USA). Protein levels were quantified by densitometry and normalized to β-actin. Blots were washed in TBS and incubated with horse radish peroxidase-coupled secondary antibodies (1:5000, Santa Cruz, USA) for 1 h at RT then washed three times in TBS followed by ECL kit (Amersham Biosciences).
c-IAP1 knockdown Cells were seeded in a 6-well plate at a density of 4×105 cells /ml and were transfected with control siRNA or c-IAP1 siRNA (Santa Cruz，USA) following a Lipofectamine 2000 (Invitrogen, USA) protocol. After 48 hrs, the efficiency of transfection was determined by western blotting.
Statistical analysis All statistical analyses were performed using GraphPad Prism 5 (Graph Pad
Software, Inc., CA, USA). Results are expressed as means±standard deviation (SD) and data were analyzed using one way ANOVA with Bonferroni post hoc for multiple comparisons, or Student’s t-test for pairwise comparisons. A value of P < 0.05 was considered significant. All experiments below were replicated using cells from three different primary cultures. FACS analysis data were derived from counting at least 10000 events in each experiment. The means were compared using Student's t-test.
Results Primary cell culture In the purified OPCs, greater than 95% of these cells expressed the OPC specific markers A2B5 (Fig. 1C). To test their differentiation potential, OPCs appeared bipotential (Fig. 1D) induced to differentiate by withdrawing FGF2 and PDGFaa. When cultured in serum free medium lacking bFGF2 and PDGFaa for 3 days, a lot of cells developed the typical, highly process-bearing morphology of OLs (Fig. 1E). And over 6 days of differentiation, the cells developed into mature OLs with visible membrane sheets (Fig. 1F). We used MBP and GFAP as the marker proteins of mature OLs and astrocytes separately to investigate the mature OLs in cultures. The results showed that more than 95% of these cells were MBP+ cells (Fig. 1J). So OPCs from the rat spinal cord were bipotential with the capacity to differentiate into OLs in the absence of serum[49].
BMP7 suppresses TNF-α-induced OLs apoptosis More and more studies show BMP7 exerts neuroprotective effects in the CNS, since it can selectively promote dendritic growth in cultured sympathetic and CNS neurons[26] and can induce neurorepair besides improve functional recovery in stroke animals[27-29]. Some vitro studies demonstrated that BMP7 had anti-oxidant[41] and anti-apoptotic[42] properties. BMP7 also exerted neuroprotective effects in models of Parkinson’s disease and SCI[43,44]. In this part, we examined whether BMP7 would protect OLs against apoptosis induced by TNF-α in vitro. First, we measured the
effects of TNF-α(1,10,100,500,1000) ng/ml on OLs viability at 24 h，48h and 72h using the CCK-8 assay. The results showed the cell viability of TNF-α (100, 500, 1000) ng/ml treated after 48hrs and 72hrs were significantly reduced compared with the control group. There was no significant difference oberserved in any time points between TNF-α (1 ng/ml, 10 ng/ml) treatment group and the control group (Fig. 2A). So, we chose the concentration 100 ng/ml of TNF-α treated OLs for 48hrs as the proper condition. Further, we measured the cell viability after co-administration with (100 ng/ml) TNF-α and (10, 50, 200) ng/ml BMP7 at 24 h and 48 h using the CCK-8 assay. We found that (50, 200) ng/ml BMP7 could effectively ameliorated the cytotoxic effects of TNF-α after 48hrs (Fig. 2B). The results suggested BMP7 altered the cytotoxic effects on OLs. And the graph of flow cytometric analysis (Fig. 2C) indicated the percentage of apoptotic cells (the early and the late apoptosis, Q2 and Q3 ) was higher in the TNF-α group (59.0%) than in the control group (25.6%). The ratio of apoptotic cells fell to 33.5% after co-incubation with BMP7 (50 ng/ml) compared with exposure to only TNF-α. Together, our results suggested that BMP7 could suppresse TNF-α-induced OLs apoptosis.
BMP7 suppresses TNF-α-induced OLs apoptosis is not through influence of JNK pathway In this part, we measured caspase-3, caspase-8 activity and the relative protein expression levels of the JNK signaling in OLs. The results from spectrophotometry showed that caspase-3 and caspase-8 activity were increased in the TNF-α group than in control group, and BMP7 could reverse the increase (Fig. 3A and B). However, immunoblot analysis indicated there were no significant differences about the expression of JNK1,JNK2,JNK3 and c-JUN between the experimental group and the control group, meanwhile, the expression of p-JNK and pc-JUN were significantly increased both in the TNF-α group and the BMP7+TNF-α group when compared with the control group seperately (Fig. 3C to H). The results indicated that TNF-α may induce JNK and c-JUN activation to mediate OLs apoptosis, but the protection of BMP7 against TNF-α may not through influencing the JNK pathway. Together, the
results shown the protective effects of BMP7 on TNF-α-induced apoptosis was due to inhibition of caspase-3 and caspase-8 activity, but not the JNK signaling pathway.
c-IAP1 is included in the protection BMP7exert on TNF-α-induced OLs apoptosis Ai Ing Lim et al[50] have made a research recently on the effection and the related regulating mechanism of BMP7 on human serum albumin (HSA)-induced chemokine synthesis in proximal tubular epithelial cells (PTECs) in vitro. The study showed BMP7 repressed PTECs apoptosis by upregulating the expression level of cIAP1. Since the protective mechanism of BMP7 against TNF-α did not inclue JNK passway. To determine whether the factor cIAP1 was involved in the protection exerted by BMP7, we compared the expression of cIAP1 in OLs between the TNF-α group and the BMP7+TNF-α group by western blotting. The result suggested the expression of cIAP1 was obviously decreased in the TNF-α treated group compared with the control group，while，significantly increased in the BMP7+TNF-α treated group compared with the TNF-α treated group (Fig. 4A and B). This results shown BMP7 raised the cIAP1 level which had been reduced by TNF-α in OLs. In order to further find out the function of cIAP1, we planned to silence the expression of cIAP1. We used western blotting to exam the transfect efficiency of cIAP1 siRNA, the result showed the target protein expression was significantly decreased in cIAP1 siRNA transfection group when compared with the control group (Fig. 4C and D). To determine whether the factor cIAP1 was involved in the inhibition of the caspase activity exerted by BMP7, through silencing the expression of cIAP1, there were increases in caspase-3 and caspase-8 activity in OLs (Fig. 4E and F). The results demonstrated BMP7 could reduce the caspase activity resulting from TNF-α-induced cytotoxicity through the effection of cIAP1. To find out if the factor cIAP1 was involved in the protection of BMP7 against TNF-α-induced OLs apoptosis, we transfected the cells with cIAP1 siRNA. In the absence of cIAP1, the graph of flow cytometric analysis indicated that the percentage of apoptotic cells (the early and the late apoptosis, Q2 and Q3 ) in the cIAP1 siRNA group (62.6%) was higher than the control group (28.78%) by Annexin V/PI assay (Fig. 4G). Together, these results demonstrated that the presence of the
increased cIAP1 level is crucial for BMP7-induced OLs survival after TNF-α administration.
Discussion CNS demyelinating diseases，such as SCI[51], multiple sclerosis[46], periventricular leukomalacia[52] and so on，are initiated by over secretion of pro-inflammatory cytokines such as IFN-γ, IL-1β, and TNF-a. These cytokines cause the loss of OLs and neuronal axons which give rise to subsequent neurological disabilities in individuals. After SCI, a large portion of proximal OLs undergoes apoptotic death during an extended period of time, with up to one-half of the population ultimately lost from the immediate area of injury[53]. The result of this process is chronic demyelination[54]. OLs are sensitive to TNF-a mediated cytotoxicity in vitro and in vivo. Administration of TNF-a induced apoptotic cell death of primary OLs in vitro [45,55]
. In vivo, elevated levels of TNF-a are found in multiple sclerosis plaques and in
experimental autoimmune encephalomyelitis lesions[56]. Transgenic mice expressing TNF-a in the CNS showed increased OLs apoptosis and primary demyelination[57]. Inhibition of TNF-a decreased apoptotic cell death and improved functional recovery after SCI[58]. TNF-a-induced OLs apoptosis was regulated through caspase-8 and caspase-3 activiation[51] or mediated by the JNK pathway[45] in vitro. TNF-a activates the JNK pathway as part of their apoptotic signals when they bind their cognate receptors[45,59], both in vivo and in vitro. The cellular functions of TNF-α are mediated by TNF receptor 1 (TNFR1) and TNFR2. TNFR1contains a death domain localized within the cytoplasmic segment. TNF-a mediated apoptosis occurs as a result of TNF-a binding to the TNFR1, which recruits FAS-associated death domain (FADD). FADD recruits and activates caspases-8 and -10 within a death-inducing signaling complex, caspases-8 and -10 activate apoptotic death through the same effector caspases as the intrinsic pathway，which results in the cleavage and activation of caspase-3 and caspase-7 and subsequent apoptosis. TNFR2 lacks a DD, but has a cytoplasmic motif that binds TRAFs (TNFR-associated factors). These receptors trigger several intracellular signaling pathways, including the I-kB
kinase, JNK, and p38 or p42/44 mitogen-activated protein kinase cascades[47]. In our study, OLs treated with TNF-a could result in activated caspase-8 and caspase-3 and increased the number of active JNK (p-JNK) and c-JUN (pc-JUN). And the presence of BMP7 in TNF-a treated OL cultures significantly protected OLs against caspases activation and cells apoptosis. Although, BMPs have been reported as potent inhibitors for myelin regeneration while promote astrogliosis[60]. Interestingly, the temporal competence of the BMP pathway to promote astrocyte fate might depend on the presence of specific Smad co-factors, such as the pro-neural bHLH[61]. Activation of the BMP receptor, BRIa, can activate NFkB, p38, Smad 1/5/8, and JNK pathways through TAB1 and TAK1 complex[62]. In our study, BMP7 was more effective at maintaining oligodendrocyte survival through the caspase inhibition than influencing the JNK pathway. These results suggested that BMP7 did not inhibit the JNK pathway activation but may work by the other signaling pathway to protect OLs from the cytotoxic of TNF-a. As we know, upon binding to ligated TNFR1, TRADD recruits the secondary adaptors RIP1, TRAF2, or TRAF5. TRAF2 can recruits ancillary proteins that modulate signaling through each TNFR, i.e., cIAP1 and 2, and TRAF1. CIAP1 and cIAP2 are antiapoptotic proteins existing in the cytoplasm, and have the activity of the ubiquitin-protein ligase to inhibit cell apoptosis[47]. Some researchers[50] have made a study recently on the effection and the related regulating mechanism of BMP7 on HSA-induced chemokine synthesis in PTECs in vitro. The study showed the cytoplasmic expression of cIAP1, tumor necrosis factor receptor-associated factor TRAF2 and TRAF3 were increased after BMP7 treatment. To find out whether the factor cIAP1 was involved in the protection of BMP7 against TNF-a -induced OLs apoptosis, OLs were incubated with TNF-a and BMP7, the western blotting results showed the expression of cIAP1 was significantly increased in the BMP7+TNF-a treated group compared with the TNF-a treated group. After silencing the expression of cIAP1, cells apoptosis rate was higher (62.6%) in the siRNA cIAP1 group when compared with the control group (28.78%) detected by flow cytometry. And there were increases in active caspase-3 and caspase-8 examed by spectrophotometry. In the
presence of cIAP1, BMP7 was as effective as the caspases inhibitors in protecting the cultures from TNF-a cytotoxicity. In the absence of cIAP1, BMP7 was less effective at the caspases inhibition. These results demonstrated that TNF-a induced caspase-8 and downstream caspase activation in OLs and BMP7 could reduce the caspases activity resulting from TNF-a -induced cytotoxicity through the effection of cIAP1. In summary, our results demonstrated that BMP7 could potently inhibite TNF-a -induced OLs apoptosis and identified the cIAP1 expression level together with the caspase-3 and caspase-8 activity as the important mediators of OLs survival after cellular stress and cytokine challenge.
Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Acknowledgments The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by National Science Foundation of China (#81070994).
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