Maslinic acid suppresses osteoclastogenesis and prevents ovariectomy‐induced bone loss by regulating RANKL‐mediated NF‐κB and MAPK signaling pathways

Activation of NF‐κB and MAPK/activator protein 1 (AP‐1) signaling pathways by receptor activator NF‐κB ligand (RANKL) is essential for osteoclast activity. Targeting NF‐κB and MAPK/AP‐1 signaling to modulate osteoclast activity has been a promising strategy for osteoclast‐related diseases. In this study we examined the effects of maslinic acid (MA), a pentacyclic triterpene acid that is widely present in dietary plants, on RANKL‐induced osteoclastogenesis, osteoclast function, and signaling pathways by in vitro and in vivo assay systems. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, MA inhibited RANKL‐induced osteoclastogenesis in a dose‐dependent manner within nongrowth inhibitory concentration, and MA decreased osteoclastogenesis‐related marker gene expression, including TRACP, MMP9, c‐Src, CTR, and cathepsin K. Specifically, MA suppressed osteoclastogenesis and actin ring formation at early stage. In ovariectomized mice, administration of MA prevented ovariectomy‐induced bone loss by inhibiting osteoclast activity. At molecular levels, MA abrogated the phosphorylation of MAPKs and AP‐1 activity, inhibited the IκBα phosphorylation and degradation, blocked NF‐κB/p65 phosphorylation, nuclear translocation, and DNA‐binding activity by downregulating RANK expression and blocking RANK interaction with TRAF6. Together our data demonstrate that MA suppresses RANKL‐induced osteoclastogenesis through NF‐κB and MAPK/AP‐1 signaling pathways and that MA is a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis. © 2011 American Society for Bone and Mineral Research.     

Signal peptide mutations in RANK prevent downstream activation of NF‐κB

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF‐κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF‐κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF‐κB occurred in HEK293 cells overexpressing wild‐type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild‐type RANK demonstrated ligand‐dependent activation of NF‐κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild‐type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins. © 2011 American Society for Bone and Mineral Research     

Hypoxia suppresses serum deprivation‐induced degradation of the nucleus pulposus cell extracellular matrix through the JNK and NF‐κB pathways

Intervertebral disc (IVD) degeneration is associated with the imbalance between anabolism and catabolism of the nucleus pulposus (NP) extracellular matrix (ECM). Serum deprivation (SD) has been reported to exacerbate IVD degeneration; however, the effect of SD on ECM metabolism is not fully understood. Hypoxia plays important roles in maintaining the physiological functions of IVD cells; however, whether hypoxia has any effect on NP ECM production under conditions of SD is still unclear. In the current study, we established an in vitro SD model by exposing NP cells to serum‐free medium. SD decreased the expression of aggrecan and collagen II, as well as the production of sulfated glycosaminoglycan (sGAG) in a time‐dependent manner. However, hypoxia abolished SD‐mediated down‐regulation of aggrecan and collagen II expression via JNK1/2 activation. Moreover, hypoxia abolished SD‐induced MMP‐3 and MMP‐13 expression by inhibiting NF‐κB activation, p65 translocation, and MMP‐3 and MMP‐13 promoter activity. These results indicated that, hypoxia maintained ECM production under conditions of SD. This effect was elicited in part through JNK1/2‐mediated up‐regulation of matrix gene expression and down‐regulation of MMP expression, through the inhibition of NF‐κB. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2059–2066, 2017.

     

miR-34b-5p promotes renal cell inflammation and apoptosis by inhibiting aquaporin-2 in sepsis-induced acute kidney injury

ObjectiveThis study was designed to uncover the mechanism of miR-34b-5p-mediated aquaporin-2 (AQP2) in sepsis-induced injury using human renal tubular epithelial cells (HK-2).MethodsSerum levels of miR-34b-5p, TNF-α, IL-1β, IL-6, serum creatinine (SCr), and blood urea nitrogen (BUN) in septic patients with acute kidney injury (AKI) and healthy controls were detected. Lipopolysaccharide (LPS) was used to induce sepsis in HK-2 cells. LPS-induced HK-2 cells were transfected with miR-34b-5p inhibitor, miR-34b-5p mimic, pcDNA3.1-AQP2, si-AQP2, miR-34b-5p inhibitor + si-NC, or miR-34b-5p inhibitor + si-AQP2. The expressions of miR-34b-5p, AQP2, Bax, Bcl-2, cleaved caspase-3, TNF-α, IL-1β, and IL-6 in HK-2 cells were detected. TUNEL staining revealed the apoptosis of HK-2 cells. Dual-luciferase reporter assay verified the binding between miR-34b-5p and AQP2.ResultsThe expression of miR-34b-5p and the inflammatory responses were augmented in septic AKI patients. miR-34b-5p was up-regulated and AQP2 was down-regulated in LPS-induced HK-2 cells. miR-34b-5p inhibition or AQP2 overexpression ameliorated apoptosis and inflammation in LPS-induced HK-2 cells. In contrast, overexpressing miR-34b-5p deteriorated LPS-induced injury in HK-2 cells. AQP2 was a downstream target of miR-34b-5p. AQP2 silencing abolished the suppressive effects of miR-34b-5p inhibition on LPS-induced apoptosis and inflammatory response in HK-2 cells.ConclusionmiR-34b-5p inhibits AQP2 to promote LPS-induced injury in HK-2 cells.     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

TNF‐α–induced NF‐κB signaling reverses age‐related declines in VEGF induction and angiogenic activity in intervertebral disc tissues

We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF‐α induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF‐α–induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF‐α–induced VEGF using organ disc cultures with wild type, TNF receptor 1‐null (TNF‐RI^null), or TNF receptor 2‐null (TNF‐RII^null) mice. VEGF induction was inhibited when we used TNF‐RI^null‐derived disc tissues. NF‐κB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF‐α induced VEGF expression in disc cells primarily through the NF‐κB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF‐α stimulation. TNF‐α treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF‐κB inhibitors or anti‐VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF‐κB pathway, both of which are induced by TNF‐α. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patient's age. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:229–235, 2009     

Jiedu Huoxue decoction improves inflammation in rat type III prostatitis: The importance of the NF‐κB signalling pathway

This study aimed to investigate the mechanism of Jiedu Huoxue decoction (JDHXD) in type III prostatitis based on the NF‐κB signalling pathway. Twenty‐six Sprague–Dawley male rats were divided into blank control, model, positive (Prostate Plus), low‐dose JDHXD, medium‐dose JDHXD and high‐dose JDHXD groups. Type III prostatitis rat model was established and confirmed with HE staining. NF‐кB P50 and NF‐κB P65 expression was detected with immunohistochemistry. NF‐κB mRNA expression was detected with qRT‐PCR. Protein expression of NF‐κB and its inhibitor Iκ‐Bα was detected with Western blot. Compared to the model group, a decrease in glandular hyperplasia and inflammation, and in NF‐кB P50 and NF‐κB P65 expression in the medium‐ and high‐dose JDHXD groups was observed. NF‐κB mRNA expression was significantly increased in the model group compared to control (p < 0.05), and significantly decreased in the JDHXD treatment groups compared to model group (p < 0.05). Protein expression of NF‐κB was significantly increased in the model and low‐dose JDHXD groups compared to control(p < 0.05), and significantly decreased in the medium‐ and high‐dose JDHXD groups compared to model group (p < 0.05). Protein expression of Iκ‐Bα was vice versa. JDHXD could be a potential treatment for type III prostatitis via its regulation of NF‐κB and Iκ‐Bα expression.