Glutathione peroxidase-1 deficiency augments proinflammatory cytokine-induced redox signaling and human endothelial cell activation

Glutathione peroxidase-1 (GPx-1) is a crucial antioxidant enzyme, the deficiency of which promotes atherogenesis. Accordingly, we examined the mechanisms by which GPx-1 deficiency enhances endothelial cell activation and inflammation. In human microvascular endothelial cells, we found that GPx-1 deficiency augments intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by redox-dependent mechanisms that involve NFκB. Suppression of GPx-1 enhanced TNF-α-induced ROS production and ICAM-1 expression, whereas overexpression of GPx-1 attenuated these TNF-α-mediated responses. GPx-1 deficiency prolonged TNF-α-induced IκBα degradation and activation of ERK1/2 and JNK. JNK or NFκB inhibition attenuated TNF-α induction of ICAM-1 and VCAM-1 expression in GPx-1-deficient and control cells, whereas ERK1/2 inhibition attenuated only VCAM-1 expression. To analyze further signaling pathways involved in GPx-1-mediated protection from TNF-α-induced ROS, we performed microarray analysis of human microvascular endothelial cells treated with TNF-α in the presence and absence of GPx-1. Among the genes whose expression changed significantly, dual specificity phosphatase 4 (DUSP4), encoding an antagonist of MAPK signaling, was down-regulated by GPx-1 suppression. Targeted DUSP4 knockdown enhanced TNF-α-mediated ERK1/2 pathway activation and resulted in increased adhesion molecule expression, indicating that GPx-1 deficiency may augment TNF-α-mediated events, in part, by regulating DUSP4.     

Tumor necrosis factor-α-mediated suppression of dual-specificity phosphatase 4: crosstalk between NFκB and MAPK regulates endothelial cell survival

We investigated the effects of tumor necrosis factor-α (TNF-α) exposure on mitogen-activated protein kinase signaling in human microvascular endothelial cells. TNF-α caused a significant suppression of a dual specificity phosphatase, DUSP4, that regulates ERK1/2 activation. Thus, we hypothesized that suppression of DUSP4 enhances cell survival by increasing ERK1/2 signaling in response to growth factor stimulation. In support of this concept, TNF-α pre-exposure increased growth factor-mediated ERK1/2 activation, whereas overexpression of DUSP4 with an adenovirus decreased ERK1/2 compared to an empty adenovirus control. Overexpression of DUSP4 also significantly decreased cell viability, lessened recovery in an in vitro wound healing assay, and decreased DNA synthesis. Pharmacological inhibition of NFκB activation or a dominant negative construct of the inhibitor of κB significantly lessened TNF-α-mediated suppression of DUSP4 expression by 70–84 % and attenuated ERK activation, implicating NFκB-dependent pathways in the TNF-α-mediated suppression of DUSP4 that contributes to ERK1/2 signaling. Taken together, our findings show that DUSP4 attenuates ERK signaling and reduces cell viability, suggesting that the novel crosstalk between NFκB and MAPK pathways contributes to cell survival.     

Docosahexaenoic acid attenuates VCAM-1 expression and NF-κB activation in TNF-α-treated human aortic endothelial cells

This study was conducted to test the hypothesis that n-3 polyunsaturated fatty acids are able to down-regulate expression of adhesion molecules and nuclear factor-κB (NF-κB) activation in vascular endothelial cells, in addition to reducing atherosclerotic lesions in vivo. We report here that docosahexaenoic acid (DHA) reduces atherosclerotic lesions in the aortic arteries of apolipoprotein E knockout (apoE^-/-) mice. Consistent with the observation in animal study, DHA inhibited THP-1 cell adhesion to tumor necrosis factor α (TNF-α)-activated human aortic endothelial cells (HAECs). Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the cell surface of HAECs was determined by cell-surface enzyme-linked immunosorbent assay. DHA and eicosapentaenoic acid decreased VCAM-1 expression in a dose-dependent manner in TNF-α treated HAECs, while cis-linoleic acid and arachidonic acid did not have any significant effect on either VCAM-1 or ICAM-1 expression. Moreover, DHA significantly reduced VCAM-1 protein expression in the cell lysates of TNF-α-treated HAECs, as determined by Western blot analysis. In line with NF-κB signaling pathway, DHA suppressed the TNF-α-activated IκBα phosphorylation and degradation as well as IκB kinase-β phosphorylation. Subsequently, translocation of the NF-κB (p50/p65) and AP-1 (c-Fos/c-Jun) subunits was down-regulated by DHA in the nucleus of HAECs. These results suggest that DHA negatively regulates TNF-α-induced VCAM-1 expression through attenuation of NF-κB signaling pathway and AP-1 activation. This study provides evidence that DHA may contribute to the prevention of atherosclerosis and inflammatory diseases in vivo.     

Atorvastatin suppresses LPS-induced rapid upregulation of Toll-like receptor 4 and its signaling pathway in endothelial cells

In the present study, we tested our hypothesis that atorvastatin exerts its anti-inflammation effect via suppressing LPS-induced rapid upregulation of Toll-like receptor 4 (TLR4) mRNA and its downstream p38, ERK, and NF-κB signaling pathways in human umbilical-vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). TLR4 mRNA expression and its downstream kinase activities induced by LPS alone or atorvastatin + LPS in endothelial cells were quantified using quantitative real-time PCR and enzyme-linked immunosorbent assay. Preincubation of LPS-stimulated endothelial cells with TLR4 siRNA was conducted to identify the target of the anti-inflammatory effects of atorvastatin. Atorvastatin incubation resulted in the reduction of LPS-induced TLR4 mRNA expression, ERK1/2 and P38 MAPK phosphorylation, and NF-κB binding activity. Pretreatment with MEK/ERK1/2 inhibitor PD98059 attenuated atorvastatin + LPS-induced NF-κB activity but had no effect on P38 MAPK phosphorylation. In contrast, pretreatment with P38 MAPK inhibitor SB203580 resulted in upregulation of atorvastatin + LPS-induced ERK1/2 phosphorylation but had no significant effects on NF-κB activity. On the other hand, blocking NF-κB with SN50 produced no effects on atorvastatin + LPS-induced ERK1/2 and P38 MAPK phosphorylation. Moreover, TLR4 gene silencing produced the same effects as the atorvastatin treatment. In conclusion, atorvastatin downregulated TLR4 mRNA expression by two distinct signaling pathways. First, atorvastatin stabilized Iκ-Bα, which directly inhibited NF-κB activation. Second, atorvastatin inactivated ERK phosphorylation, which indirectly inhibited NF-κB activation. Suppression of p38 MAPK by atorvastatin upregulates ERK but exerts no effect on NF-κB.     

Polysaccharide isolated from seeds of Plantago asiatica L. induces maturation of dendritic cells through MAPK and NF-κB pathway

Plantago species are used as traditional medicine in Asian and Europe. Polysaccharide isolated from the seeds of Plantago asiatica L. could stimulate maturation transformation of bone-marrow derived dendritic cells (DCs). We found that blocking p38, ERK1/2 and JNK MAPK signal transduction could significantly decreased the PLP-2 induced expression of MHC II, CD86 surface molecules on DCs. Blocking p38 and JNK signal also significantly inhibited the cytokine secretion of TNF-α and IL-12p70 as well, while blocking ERK1/2 signal only decreased the secretion of TNF-α. Meanwhile, DCs in the three MAPK signal-blocking groups showed dramatically attenuated effects on stimulating proliferation of T lymphocytes. Similarly, blocking signal transduction of NF-κB pathway also significantly impaired the phenotypic and functional maturation development of DCs induced by PLP-2. These data suggest that MAPK and NF-κB pathway mediates the PLP-induced maturation on DCs. Especially, among the three MAPK pathways, activation of JNK signal transduction is the most important for DCs development after PLP-2 incubation. And PLP-2 may activate the MAPK and NF-κB pathway by triggering toll-like receptor 4 on DCs.     

Propofol Administration Modulates AQP-4 Expression and Brain Edema After Traumatic Brain Injury

The increased intracranial pressure caused by brain edema following traumatic brain injury (TBI) always leads to poor patient prognosis. Aquaporin-4 (AQP-4) plays an important role in edema formation and resolution, which may provide a novel therapeutic target for edema treatment. In this present study, we found that propofol treatment, within a short time, after TBI significantly reduced brain edema in a controlled cortical injury rat model and suppressed in vivo expression of AQP-4. The ameliorating effect of propofol was associated with attenuated expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). In addition, the regulatory effect of propofol on AQP-4 expression was investigated in cultured astrocytes. Results showed that propofol could block the stimulatory effect of IL-1β and TNF-α on AQP-4 expression in cultured astrocytes. We also found that both NFκB and p38/MAPK pathways were involved in IL-1β and TNF-α-induced AQP-4 expression and that propofol functions as a dual inhibitor of NFκB and p38/MAPK pathways. In conclusion, treatment with propofol, within a short time, after TBI attenuates cerebral edema and reduces the expression of AQP-4. Propofol modulates acute AQP-4 expression by attenuating IL-1β and TNF-α expression and inhibiting IL-1β and TNF-α induced AQP-4 expression.     

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.     

Honokiol: an effective inhibitor of tumor necrosis factor-α-induced up-regulation of inflammatory cytokine and chemokine production in human synovial fibroblasts

In this study, we investigated the mechanisms underlying the anti-inflammatory effects of honokiol in tumor necrosis factor (TNF)-α-stimulated rheumatoid arthritis synovial fibroblasts (RASFs). RASFs pre-treated with honokiol (0-20 μM) were stimulated with TNF-α (20 ng/ml). The levels of prostaglandin E2 (PGE2), nitric oxide (NO), soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and Griess assay. In addition, protein expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and phosphorylated Akt, nuclear factor kappa B (NFκB), and extracellular signal-regulated kinase (ERK)1/2 were determined by western blot. The expression of NFκB-p65 was assessed by immunocytochemical analysis. TNF-α treatment significantly up-regulated the levels of PGE2, NO, sICAM-1, TGF-β1, MCP-1, and MIP-1α in the supernatants of RASFs, increased the protein expression of COX-2, iNOS, and induced phosphorylation of Akt, IκB-α, NFκB, and ERK1/2 in RASFs. TNF-α-induced expression of these molecules was inhibited in a dose-dependent manner by pre-treatment with honokiol. The inhibitory effect of honokiol on NFκB-p65 activity was also confirmed by immunocytochemical analysis. In conclusion, honokiol is a potential inhibitor of TNF-α-induced expression of inflammatory factors in RASFs, which holds promise as a potential anti-inflammatory drug.     

Docosahexaenoic acid inhibition of inflammation is partially via cross-talk between Nrf2/heme oxygenase 1 and IKK/NF-κB pathways

We examined the underlying mechanisms involved in n-3 docosahexaenoic acid (DHA) inhibition of inflammation in EA.hy926 cells. The present results demonstrated that pretreatment with DHA (50 and 100 μM) inhibited tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule 1 (ICAM-1) protein, mRNA expression and promoter activity. In addition, TNF-α-stimulated inhibitory kappa B (IκB) kinase (IKK) phosphorylation, IκB phosphorylation and degradation, p65 nuclear translocation, and nuclear factor-κB (NF-κB) and DNA binding activity were attenuated by pretreatment with DHA. DHA triggered early-stage and transient reactive oxygen species (ROS) generation and significantly increased the protein expression of heme oxygenase 1 (HO-1), induced nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, DHA inhibited Nrf2 ubiquitination and proteasome activity. DHA activated Akt, p38 and ERK1/2 phosphorylation, and specific inhibitors of respective pathways attenuated DHA-induced Nrf2 nuclear translocation and HO-1 expression. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of TNF-α-induced p65 nuclear translocation and ICAM-1 expression. Importantly, we show for the first time that HO-1 plays a down-regulatory role in NF-κB nuclear translocation, and inhibition of Nrf2 ubiquitination and proteasome activity are involved in increased cellular Nrf2 level by DHA. In this study, we show that HO-1 plays a down-regulatory role in NF-κB nuclear translocation and that the protective effect of DHA against inflammation is partially via up-regulation of Nrf2-mediated HO-1 expression and inhibition of IKK/NF-κB signaling pathway.     

Sulforaphane, a natural constituent of broccoli, prevents cell death and inflammation in nephropathy

Cisplatin (cis-diamminedichloroplatinum II, CIS) is a potent and widely used chemotherapeutic agent to treat various malignancies, but its therapeutic use is limited because of dose-dependent nephrotoxicity. Cell death and inflammation play a key role in the development and progression of CIS-induced nephropathy. Sulforaphane (SFN), a natural constituent of cruciferous vegetables such as broccoli, Brussels sprouts, etc., has been shown to exert various protective effects in models of tissue injury and cancer. In this study, we have investigated the role of prosurvival, cell death and inflammatory signaling pathways using a rodent model of CIS-induced nephropathy, and explored the effects of SFN on these processes. Cisplatin triggered marked activation of stress signaling pathways [p53, Jun N-terminal kinase (JNK), and p38-α mitogen-activated protein kinase (MAPK)] and promoted cell death in the kidneys (increased DNA fragmentation, caspases-3/7 activity, terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick-end labeling), associated with attenuation of various prosurvival signaling pathways [e.g., extracellular signal-regulated kinase (ERK) and p38-β MAPK]. Cisplatin also markedly enhanced inflammation in the kidneys [promoted NF-κB activation, increased expression of adhesion molecules ICAM and VCAM, enhanced tumor necrosis factor-α (TNF-α) levels and inflammatory cell infiltration]. These effects were significantly attenuated by pretreatment of rodents with SFN. Thus, the cisplatin-induced nephropathy is associated with activation of various cell death and proinflammatory pathways (p53, JNK, p38-α, TNF-α and NF-κB) and impairments of key prosurvival signaling mechanisms (ERK and p38-β). SFN is able to prevent the CIS-induced renal injury by modulating these pathways, providing a novel approach for preventing this devastating complication of chemotherapy.     

Omentin inhibits TNF-α-induced expression of adhesion molecules in endothelial cells via ERK/NF-κB pathway

In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-α-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-α-activated signal pathway of nuclear factor-κB (NF-κB) by preventing NF-κB inhibitory protein (IκBα) degradation and NF-κB/DNA binding activity. Omentin pretreatment significantly inhibited TNF-α-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-α-induced NF-κB activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-α. These results suggest that omentin may inhibit TNF-α-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-κB pathway.     

Sophocarpine exert protective effect against ox-LDL-induced endothelial damage via regulating NF-κB signaling pathway

ABSTRACT

Oxidized low-density lipoprotein (ox-LDL) was known to induce endothelial cell injury to the progression of atherosclerosis (AS). Sophocarpine (SPC), a compound of sophora alkaloids isolated from the plant Sophora alopecuroides, has been shown to exhibit various pharmacological activities. This study was designed to investigate the protective effect of SPC on ox-LDL-induced endothelial cells and explored its underlying mechanism. Our results show that SPC pre-incubation ameliorated ox-LDL-mediated HAECs cytotoxicity, DNA fragmentation, and apoptosis in a dose-dependent manner. Moreover, SPC significantly downregulated the mRNA or protein expression level of pro-inflammatory mediators (TGF-β, IL-6, IL-1β, TNF-α) and pro-inflammatory vascular adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Mechanistically, SPC pre-treatment downregulated IκBα expression and inhibited translocation of NF-κB in ox-LDL-mediated HAECs, overexpression of NF-κB p65 counteracted the cytoprotective and anti-apoptotic effect of SPC, suggesting that its action is dependent on NF-κB signaling pathway. Collectively, SPC suppresses ox-LDL-induced HAECs injury by inhibiting the NF-κB signaling pathway.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1β, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.     

Indoxyl sulfate induces leukocyte-endothelial interactions through up-regulation of E-selectin

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.     

Quercetin inhibits IL-1 beta-induced ICAM-1 expression in pulmonary epithelial cell line A549 through the MAPK pathways

Quercetin is a herbal flavonoid derived from various foods of plant origin and widely used as a major constituent of nutritional supplements. Quercetin has been shown to have anti-inflammatory properties and can play a role in anti-inflammatory procedure. Intercellular adhesion molecule-1 (ICAM-1) is one of the important pro-inflammatory factors, especially in early phage of inflammation. However, the mechanisms regulating ICAM-1 expression by quercetin in human A549 cells were still unclear. In this study, the inhibitory effect of quercetin on ICAM-1 expression by interleukin-1 beta (IL-1 beta)-stimulated A549 cells was investigated, and the roles of mitogen-activated protein kinases (MAPK) pathways were explored. Quercetin attenuated IL-1 beta-induced expression of ICAM-1 mRNA and protein in a dose-dependent manner. The experiment suggested that quercetin actively inhibited inhibitory protein of nuclear factor-kappa B (I kappa B) degradation, and nuclear factor-kappa B (NF-kappa B) activity. The c-fos and c-jun, components of activator protein-1 (AP-1), were mediated by MAPK pathways. ERK and p38 were involved in the c-fos mRNA expression, and JNK was involved in the c-jun mRNA expression. The inhibitory effect of quercetin on ICAM-1 expression was mediated by the sequential attenuation of the c-fos and c-jun mRNA expressions. These inhibitory effects were partially inhibited by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a specific inhibitors of extracellular signal-regulated kinase (ERK), and SP600125, a specific inhibitor of c-Jun-N-terminal kinase (JNK). Taken together, these results suggest that quercetin negatively modulating ICAM-1 partly dependent on MAPK pathways. Binwu Ying and Ting Yang have contributed equally to this work.     

ABD56 causes osteoclast apoptosis by inhibiting the NFkappaB and ERK pathways

We have previously shown that the biphenylcarboxylic acid butanediol ester (ABD56) inhibits osteoclast formation and activity in vitro and in vivo. However, the mechanism of action of this compound is unknown. ABD56 inhibited osteoclast formation and caused osteoclast apoptosis, but had no effects on osteoblasts or macrophages. As the NFκB and MAPK pathways are essential for osteoclast formation and survival, we studied the effects of ABD56 on these pathways. ABD56 caused phosphorylation of p38, JNK and nuclear translocation of c-jun in osteoclasts. ABD56-induced apoptosis was prevented by the caspase inhibitor zVAD-fmk but was not prevented by the p38- or JNK-inhibitors. ABD56 completely abolished RANKL-induced IκB and ERK1/2 phosphorylation. Increasing the amount of RANKL partially rescued ABD56-induced apoptosis, indicating that the apoptosis is most probably due to the inhibition of survival signals such as ERK and NFκB, rather than activation of the p38 or Jnk MAPK pathways.