Unconjugated
Synaptic loss induced by beta-amyloid (Aβ) plays a critical role in the pathophysiology of Alzheimer's disease (AD), but the mechanisms underlying this process remain unknown. In this study, we found that oridonin (Ori) rescued synaptic loss induced by Aβ1-42 in vivo and in vitro and attenuated the alterations in dendritic structure and spine density observed in the hippocampus of AD mice. In addition, Ori increased the expression of PSD-95 and synaptophysin and promoted mitochondrial activity in the synaptosomes of AD mice. Ori also activated the BDNF/TrkB/CREB signaling pathway in the hippocampus of AD mice. Furthermore, in the Morris water maze test, Ori reduced latency and searching distance and increased the number of platform crosses in AD mice. These data suggest that Ori might prevent synaptic loss and improve behavioral symptoms in Aβ1-42-induced AD mice.
Here we show that A-kinase anchoring protein 95 (AKAP95) and connexin 43 (Cx43) dynamically interact during cell cycle progression of lung cancer A549 cells. Interaction between AKAP95 and Cx43 at different cell cycle phases was examined by tandem mass spectrometry(MS/MS), confocal immunofluorescence microscopy, Western blot, and co-immunoprecipitation(Co-IP). Over the course of a complete cell cycle, interaction between AKAP95 and Cx43 occurred in two stages: binding stage from late G1 to metaphase, and separating stage from anaphase to late G1. The binding stage was further subdivided into complex binding to DNA in interphase and complex separating from DNA in metaphase. In late G1, Cx43 translocated to the nucleus via AKAP95; in anaphase, Cx43 separated from AKAP95 and aggregated between two daughter nuclei. In telophase, Cx43 aggregated at the membrane of the cleavage furrow. After mitosis, Cx43 was absent from the furrow membrane and was located in the cytoplasm. Binding between AKAP95 and Cx43 was reduced by N-(2-[P-Bromocinnamylamino]-ethyl)-5-isoquinolinesulfonmide (H89) treatment and enhanced by Forskolin. dynamic interaction between AKAP95 and Cx43 varies with cell cycle progression to regulate multiple biological processes.
Excessive βAR stimulation is an independent factor in inducing pathological cardiac hypertrophy. Here, we report miR-145 regulates both expression and localization of GATA6, thereby protecting the heart against cardiomyocyte hypertrophy induced by isoproterenol (ISO). The protective activity of miR-145 was associated with down-regulation of ANF, BNP and β-MHC expression, a decreased rate of protein synthesis, inhibited cardiomyocyte growth and the modulation of several signaling pathways including ERK1/2, JNK and Akt-GSK3β. The anti-hypertrophic effect was abrogated by exogenous over-expression of transcription factor GATA6 which was further identified as a direct target of miR-145. In addition, GSK3β antagonists, LiCl and TDZD8, restored the nuclear accumulation of GATA6, which was attenuated by miR-145 Finally, we observed a dynamic pattern of miR-145 expression in ISO-treated NRCMs and in the hearts of TAC mice. Together, our results identify miR-145 as an important regulator in cardiac hypertrophy.
Hyperforin, a lipophilic constituent of medicinal herb St John's wort, has been identified as the main active ingredient of St John's wort extract for antidepressant action by experimental and clinical studies. Hyperforin is currently known to activate transient receptor potential canonical (subtype) 6 (TRPC6) channel, increase the phosphorylated CREB (p-CREB), and has N-methyl-D-aspartate receptor-antagonistic effect that convert potential neuroprotective effects in vitro. However, the protective effects of hyperforin on ischemic stroke in vivo remain controversial and its neuroprotective mechanisms are still unclear. This study was designed to examine the effects of intracerebroventricular (i.c.v.) injection of hyperforin on transient focal cerebral ischemia in rats. Hyperforin, when applied immediately after middle cerebral artery occlusion (MCAO) onset, significantly reduced infarct volumes and apoptotic cells, and also increased neurologic scores at 24 hours after reperfusion accompanied by elevated TRPC6 and p-CREB activity and decreased SBDP145 activity. When MEK or CaMKIV activity was specifically inhibited, the neuroprotective effect of hyperforin was attenuated, and we observed a correlated decrease in CREB activity. In conclusion, our results clearly showed that i.c.v. injection of hyperforin immediately after MCAO onset blocked calpain-mediated TRPC6 channels degradation, and then to stimulate the Ras/MEK/ERK and CaMKIV pathways that converge on CREB activation, contributed to neuroprotection.
BACKGROUND:
KIF23 (kinesin family member 23) is a kinesin-like motor protein and plays an important role in cytokinesis. In search for genes associated with hepatocellular carcinoma (HCC) by cDNA microarray, we found that KIF23 was upregulated in HCC tissues. At present, much less is known about its expression and functions in tumor cells. In this work, we aimed to investigate the expression of KIF23 in HCC and the correlation between its expression and clinical features.
METHODS:
Total RNA was extracted from 16 HCC and paired adjacent non-cancerous tissues. The expressions of the two KIF23 splice variants (KIF23 V1 and KIF23 V2) in normal and HCC tissues were determined by reverse transcriptase polymerase chain reaction (RT-PCR). Polyclonal antibody specific to KIF23 V1 was prepared, and the specificity of the antibody was confirmed by siRNA knockdown and Western blotting experiments. KIF23 protein expression in HCC was examined by immunohistochemistry staining with anti-KIF23 V1 or anti-KIF23 (commercially available for recognizing both KIF23 V1 and V2) antibodies, respectively. Univariate and Multivariate Cox regression analyses were used to determine the correlation between KIF23 protein expression and overall survival of HCC patients.
RESULTS:
The two splicing variants of KIF23 mRNA were not detected in normal liver tissue by RT-PCR, but they were aberrantly expressed in HCC tissues. Immunohistochemistry staining with anti-KIF23 V1 antibody revealed that KIF23 V1 was mainly distributed in the nucleus, whereas the positive staining signals were predominantly in the cytoplasm when using anti-KIF23 antibody, suggesting that KIF23 V2 might localize in the cytoplasm of HCC cells. KIF23 V1 protein was detected in 57.6% (83/144) HCC patients and the mean H-score was 42, while KIF23 V2 was detected in 94.4% (135/143) HCC samples and the mean H-score was 68. Follow-up study showed that HCC patients with expression of KIF23 V1 had a longer 5-year survival (p=0.0052), however, expression of KIF23 V2 protein did not associate with 3- and 5-year survival.
CONCLUSION:
In this study we show for the first time that KIF23 V1 and V2 have different localizations in HCC cells. Furthermore, KIF23 V1 protein expression might be a marker of longer overall survival in HCC patients.
AIM:
Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro.
METHODS:
The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis.
RESULTS:
Aconine (0.125, 0.25 μmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, β3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion.
CONCLUSION:
These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.
AKAP95 in lung cancer tissues showed higher expression than in paracancerous tissues. AKAP95 can bind with cyclin D and cyclin E during G1/S cell cycle transition, but its molecular mechanisms remain unclear. To identify the mechanism of AKAP95 in cell cycle progression, we performed AKAP95 transfection and silencing in A549 cells, examined AKAP95, cyclin E1 and cyclin E2 expression, and the interactions of AKAP95 with cyclins E1 and E2. Results showed that over-expression of AKAP95 promoted cell growth and AKAP95 bound cyclin E1 and E2, low molecular weight cyclin E1 (LWM-E1) and LWM-E2. Additionally AKAP95 bound cyclin E1 and LMW-E2 in the nucleus during G1/S transition, bound LMW-E1 during G1, S and G2/M, and bound cyclin E2 mainly on the nuclear membrane during interphase. Cyclin E2 and LMW-E2 were also detected. AKAP95 over-expression increased cyclin E1 and LMW-E2 expression but decreased cyclin E2 levels. Unlike cyclin E1 and LMW-E2 that were nuclear located during the G1, S and G1/S phases, cyclin E2 and LMW-E1 were expressed in all cell cycle phases, with cyclin E2 present in the cytoplasm and nuclear membrane, with traces in the nucleus. LMW-E1 was present in both the cytoplasm and nucleus. The 20 kDa form of LMW-E1 showed only cytoplasmic expression, while the 40 kDa form was nuclear expressed. The expression of AKAP95, cyclin E1, LMW-E1 and -E2, might be regulated by cAMP. We conclude that AKAP95 might promote cell cycle progression by interacting with cyclin E1 and LMW-E2. LMW-E2, but not cyclin E2, might be involved in G1/S transition. The binding of AKAP95 and LMW-E1 was found throughout cell cycle.
The therapeutic effectiveness of moracins as 2-arylbenzofuran derivatives against airway inflammation was examined. Moracin M, O, and R were isolated from the root barks of Morus alba, and they inhibited interleukin (IL)-6 production from IL-1β-treated lung epithelial cells (A549) at 101-00μM. Among them, moracin M showed the strongest inhibitory effect (IC50=8.1μM). Downregulation of IL-6 expression by moracin M was mediated by interrupting the c-Jun N-terminal kinase (JNK)/c-Jun pathway. Moracin derivatives inhibited inducible nitric oxide synthase (iNOS)-catalyzed NO production from lipopolysaccharide (LPS)-treated alveolar macrophages (MH-S) at 50-100μM. In particular, moracin M inhibited NO production by downregulating iNOS. When orally administered, moracin M (20-60mg/kg) showed comparable inhibitory action with dexamethasone (30mg/kg) against LPS-induced lung inflammation, acute lung injury, in mice with that of dexamethasone (30mg/kg). The action mechanism included interfering with the activation of nuclear transcription factor-κB in inflamed lungs. Therefore, it is concluded that moracin M inhibited airway inflammation in vitro and in vivo, and it has therapeutic potential for treating lung inflammatory disorders.
Paeonol and danshensu is the representative active ingredient of traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizae, respectively. Paeonol and danshensu combination (PDSS) has putative cardioprotective effects in treating ischemic heart disease (IHD). However, the evidence for the protective effect is scarce and the pharmacological mechanisms of the combination remain unclear. The present study was designed to investigate the protective effect of PDSS on isoproterenol (ISO)-induced myocardial infarction in rats and to elucidate the potential mechanism. Assays of creatine kinase-MB, cardiac troponin I and T and histopathological analysis revealed PDSS significantly prevented myocardial injury induced by ISO. The ISO-induced profound elevation of oxidative stress was also suppressed by PDSS. TUNEL and caspase-3 activity assay showed that PDSS significantly inhibited apoptosis in myocardia. In exploring the underlying mechanisms of PDSS, we found PDSS enhanced the nuclear translocation of Nrf2 in myocardial injured rats. Furthermore, PDSS increased phosphorylated PI3K and Akt, which may in turn activate antioxidative and antiapoptotic signaling events in rat. These present findings demonstrated that PDSS exerts significant cardioprotective effects against ISO-induced myocardial infarction in rats. The protective effect is, at least partly, via activation of Nrf2/HO-1 signaling and involvement of the PI3K/Akt cell survival signaling pathway.
Inflammasome NLRP3 plays a crucial role in the process of colitis and colitis--associated colon cancer. Even though much is known regarding the NLRP3 inflammasome that regulates pro-inflammatory cytokine release in innate immune cells, the role of NLRP3 in non-immune cells is still unclear. In this study, we showed that NLRP3 was highly expressed in mesenchymal-like colon cancer cells (SW620), and was upregulated by tumor necrosis factors-α (TNF-α) and transforming growth factor-β1 (TGF-β1) respectively, during EMT in colon cancer epithelial cells HCT116 and HT29. Knockdown of NLRP3 retained epithelial spindle-like morphology of HCT116 and HT29 cells and reversed the mesenchymal characteristic of SW620 cells, indicated by the decreased expression of vimentin and MMP9 and increased expression of E-cadherin. In addition, knockdown of NLRP3 in colorectal carcinoma cells displayed diminished cell migration and invasion. Interestingly, during the EMT process induced by TNF-α or TGF-β1, the cleaved caspase-1 and ASC speck were not detected, indicating that NLRP3 functions in an inflammasome-independent way. Further studies demonstrated that NLRP3 protein expression was regulated by NF-κB signaling in TNF-α or TGF-β1-induced EMT, as verified by the NF-κB inhibitor Bay 11-7082. Moreover, NLRP3 knockdown reduced the expression of Snail1, indicating that NLRP3 may promote EMT through regulating Snail1. In summary, our results showed that the NLRP3 expression, not the inflammasome activation, was required for EMT in colorectal cancer cells.
Liver fibrosis is one of the major causes of morbidity and mortality worldwide and lacks efficient therapy. Recent studies suggest the curcumin protects liver from fibrosis. However, curcumin itself is in low bioavailable concentration when administered orally, and the protective mechanism remains poorly understood. The current study aimed to investigate whether a more stable derivative of curcumin, C66, protects against CCl4-inudced liver fibrosis and examine the underlying mechanism involving cannabinoid receptor (CB receptor). At a dose lower than curcumin itself, C66 displayed a superior anti-fibrotic effect. C66 significantly reduced collagen deposition, pro-inflammatory cytokine expression, and liver enzyme activities. Mechanistic study revealed that C66 treatment decreased CCl4-induced cannabinoid receptor 1 (CB1 receptor) expression and increased cannabinoid receptor 2 (CB2 receptor) expression, along with an inhibition of JNK/NF-κB-mediated inflammatory signaling. In conclusion, this curcumin derivative attenuates liver fibrosis likely involving a CB/JNK/NF-κB-mediated pathway.
Dp71 is one of the most ubiquitously expressed isoforms of dystrophin, the pathological genes of DMD. In order to find whether the alteration of Dp71 can affect the phenotypes of cell other than PC12, an A549 cell line with stably transfected Dp71 siRNA plasmids was set up and named A549-Dp71AS cell. It is demonstrated for the first time that the A549-Dp71AS cell line displayed decreased invasion capabilities, reduced migration ability, decreased proliferation rate, and lessened clonogenic formation. Cisplatin-induced apoptosis was also increased in A549-Dp71AS cell line via enhancing the Caspase 3, Caspase 8, and Caspase 9 activities. Knocking down Dp71 expression can significantly inhibit the A549 xenograft tumor growth in nude mice. The A549-Dp71AS cells and xenograft tumor tissues displayed reduced lamin B1, Bcl-2, and MMP2 protein expression, which accounts for the reduced malignancy of A549-Dp71AS cells in vivo and in vitro.
Accumulating evidences demonstrated that statins impaired glucose utilization. This study was aimed to investigate whether PXR was involved in the atorvastatin-impaired glucose utilization. Rifampicin/PCN served as PXR activator control. Glucose utilization, glucose uptake, protein levels of GLUT2, GCK, PDK2, PEPCK1 and G6Pase in HepG2 cells were measured. PXR inhibitors, PXR overexpression and PXR siRNA were applied to verify the role of PXR in atorvastatin-impaired glucose utilization in cells. Hypercholesterolemia rats induced by high fat diet feeding, orally received atorvastatin (5 and 10 mg/kg), pravastatin (10 mg/kg) for 14 days, or intraperitoneally received PCN (35 mg/kg) for 4 days. Results showed that glucose utilization was markedly inhibited by atorvastatin, simvastatin, pitavastatin, lovastatin and rifampicin. Neither rosuvastatin nor pravastatin showed the similar effect. Atorvastatin and pravastatin were selected for the following study. Atorvastatin and rifampicin significantly inhibited glucose uptake and down-regulated GLUT2 and GCK expressions. Similarly, overexpressed PXR significantly down-regulated GLUT2 and GCK expressions and impaired glucose utilization. Ketoconazole and resveratrol attenuated the impaired glucose utilization by atorvastatin and rifampicin in both parental and overexpressed PXR cells. PXR knockdown significantly up-regulated GLUT2 and GCK proteins and abolished the decreased glucose consumption and uptake by atorvastatin and rifampicin. Animal experiments showed that atorvastatin and PCN significantly elicited postprandial hyperglycemia, leading to increase in glucose AUC. Expressions of GLUT2 and GCK in rat livers were markedly down-regulated by atorvastatin and PCN. In conclusion, atorvastatin impaired glucose utilization in hepatocytes via repressing GLUT2 and GCK expressions, which may be partly due to PXR activation.
Toll-like receptor-4 (TLR4) signaling has been implicated in innate immunity and acute inflammation following acute lung injury (ALI). As such, modulating inflammatory response through TLR4 represents an attractive therapeutic approach to treat ALI. Increasing evidence demonstrates that hyaluronan (HA) can modulate TLR4 activation and has shown early promise as a therapeutic agent in ALI. However, the mechanism associated with HA has not been fully elucidated. In the current study, we sought to determine the effects of HA on lipopolysaccharide (LPS)-induced inflammatory response and gain insights into the mechanism of action in mice with intratracheal instillation of LPS. Our results demonstrate that in contrast to mice challenged with LPS, pretreatment with HA significantly inhibited inflammatory cell recruitment, attenuated lung injury and suppressed the level of cytokine/chemokine in bronchial alveolar lavage fluid (BALF). Investigation of the mechanism responsible for inhibition of LPS activation showed HA treatment significantly inhibited the nuclear translocation of NF-κB p65 and protein expression of myeloid differentiation primary response protein (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) and p38 MAPK, JNK and ERK activation in lung tissue. Furthermore, we compared the protection effect of HA in TLR4-deficient mice with those of genetically matched wild type (WT) mice in an acute model of lung injury. However, in TLR4-deficient mice, HA pretreatment before LPS instillation fail to affect the LPS response. Therefore, our findings suggest that HA pretreatment attenuated LPS-induced ALI and the anti-inflammatory function of HA was partial dependent on TLR4, which shed new light on potential elements that regulate the lung injury response.
During senescence, cells express molecules called senescence-associated secretory phenotype (SASP), including growth factors, proinflammatory cytokines, chemokines, and proteases. The SASP induces a chronic low-grade inflammation adjacent to cells and tissues, leading to degenerative diseases. The anti-inflammatory activity of flavonoids was investigated on SASP expression in senescent fibroblasts. Effects of flavonoids on SASP expression such as IL-1α, IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2 and MMP-3 and signaling molecules were examined in bleomycin-induced senescent BJ cells. In vivo activity of apigenin on SASP suppression was identified in the kidney of aged rats. Among the five naturally-occurring flavonoids initially tested, apigenin and kaempferol strongly inhibited the expression of SASP. These flavonoids inhibited NF-κB p65 activity via the IRAK1/IκBα signaling pathway and expression of IκBζ. Blocking IκBζ expression especially reduced the expression of SASP. A structure-activity relationship study using some synthetic flavones demonstrated that hydroxyl substitutions at C-2',3',4',5 and 7 were important in inhibiting SASP production. Finally, these results were verified by results showing that the oral administration of apigenin significantly reduced elevated levels of SASP and IκBζ mRNA in the kidneys of aged rats. This study is the first to show that certain flavonoids are inhibitors of SASP production, partially related to NF-κB p65 and IκBζ signaling pathway, and may effectively protect or alleviate chronic low-grade inflammation in degenerative diseases such as cardiovascular diseases and late-stage cancer.
Matrix metalloproteinase-13 (MMP-13) plays a critical role in degrading major collagens in human cartilage under some pathological conditions such as osteoarthritis. To establish the therapeutic potential against cartilage degradation, the effects of 12 naturally-occurring triterpenoids and steroids on MMP-13 induction were examined in the human chondrocyte cell line, SW1353. They included coreanoside F1, suavissimoside R1, spicatoside A, 25(S)-ruscogenin, methyl protogracillin, hederagenin, loniceroside A, loniceroside B, loniceroside C, smilaxin A, smilaxin C, and ursolic acid. Among these, only spicatoside A and 25(S)-ruscogenin were found to inhibit MMP-13 expression in IL-1β-treated SW1353 cells at a pharmacologically-relevant concentration of 10 μM. These effects were also supported by the finding that spicatoside A (20 μM) reduced glycosaminoglycan release from IL-1α-treated rabbit joint cartilage culture to some degree. When the cellular mechanisms of action of spicatoside A in MMP-13 inhibition were investigated, the blocking point was not found among the MMP-13 signaling molecules examined such as mitogen-activated protein kinases, activator protein-1, and nuclear transcription factor-κB. Instead, spicatoside A was found to reduce MMP-13 mRNA stability. All of these findings suggest that spicatoside A and 25(S)-ruscogenin have a therapeutic potential for protecting against cartilage breakdown in arthritic disorders.
Ammonia is considered to be the main neurotoxin responsible for hepatic encephalopathy resulting from liver failure. Liver failure has been reported to alter expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2) at the blood-brain barrier (BBB). The aim of this study was to investigate whether ammonia is involved in abnormalities of expression and activity of P-gp and Mrp2 at the BBB. Hyperammonemic rats were developed by an intraperitoneal injection of ammonium acetate (NH4 Ac, 4.5 mmol/kg). Results showed that Mrp2 function markedly increased in cortex and hippocampus of rats at 6 h following NH4 Ac administration. Significant increase in function of P-gp was observed in hippocampus of rats. Meanwhile, such alterations were in line with the increase in mRNA and protein levels of P-gp and Mrp2. Significant increase in levels of nuclear amount of nuclear factor-κB (NF-κB) p65 was also observed. Primarily cultured rat brain microvessel endothelial cells (rBMECs) were used for in vitro study. Data indicated that 24 h exposure to ammonia significantly increased function and expression of P-gp and Mrp2 in rBMECs, accompanied with activation of NF-κB. Furthermore, such alterations induced by ammonia were reversed by NF-κB inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P-gp and Mrp2 at the BBB via activating NF-κB pathway. Hyperammonemia, a proverbial main factor responsible for neurocognitive disorder and blood-brain barrier (BBB) dysfunction resulting from liver failure, could increase the expression and activity of P-glycoprotein and multidrug resistance-associated protein 2 (Mrp2) at the BBB both in vivo and in vitro. Furthermore, the NF-κB activation stimulated by hyperammonemia may be the potential mechanism underlying such abnormalities induced by hyperammonemia.
Ginsenoside Rb1 is reported to possess anti-fatigue activity, but the mechanisms remain unknown. The aim of this study was to investigate the molecular mechanisms responsible for the anti-fatigue effect of ginsenoside Rb1 on postoperative fatigue syndrome induced by major small intestinal resection (MSIR) in aged rat. Aged rats with MSIR were administrated with ginsenoside Rb1 (15 mg/kg) once a day from 3 days before surgery to the day of sacrifice, or with saline as corresponding controls. Rats without MSIR but going through the same surgery procedure were administrated with saline as blank controls. Anti-fatigue effect was assessed by an open field test; superoxide dismutase, reactive oxygen species and malondialdehyde in skeletal muscle were determined. The mRNA levels of Akt2 and Nrf2 in skeletal muscle were measured by real-time quantitative PCR. The activation of Akt and Nrf2 was examined by western blot and immunohistofluorescence. Our results revealed that ginsenoside Rb1 significantly increased the journey and the rearing frequency, decreased the time of rest in aged rats with MSIR. In addition, ginsenoside Rb1 significantly reduced reactive oxygen species and malondialdehyde release and increased the superoxide dismutase activity of skeletal muscle in aged rats with MSIR. Ginsenoside Rb1 also increased the expression of Akt2 and Nrf2 mRNA, up-regulated Akt phosphorylation and Nrf2 nuclear translocation. These findings indicate that ginsenoside Rb1 has an anti-fatigue effect on postoperative fatigue syndrome in aged rat, and the mechanism possibly involves activation of the PI3K/Akt pathway with subsequent Nrf2 nuclear translocation and induction of antioxidant enzymes.
Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.
Many studies have found a positive association between the progression of hepatocellular carcinoma and DDT exposure. These studies mainly focus on the effect of DDT exposure on cell proliferation and epithelial to mesenchymal transition (EMT) promotion. However, the influence of DDT on cell adhesion of hepatocellular carcinoma remains to be unclear. The aim of our study was to determine the effect of p,p'-DDT on cell adhesion of hepatocellular carcinoma in vitro and in vivo. The data showed that p,p'-DDT, exposing HepG2 cells for 6 days, decreased cell-cell adhesion and elevated cell-matrix adhesion. Strikingly, p,p'-DDT increased reactive oxygen species (ROS) content, and this was accompanied by the activation of JAK/STAT3 pathway. Moreover, ROS inhibitor supplement reversed these effects significantly. However, the addition of ER inhibitor, ICI, had no effect on the p,p'-DDT-induced effects. p,p'-DDT altered the mRNA levels of related adhesion molecules, including inhibition of E-cadherin and promotion of N-cadherin along with CD29. Interestingly, the p,p'-DDT-altered adhesion molecules could be reversed with JAK inhibitor or STAT3 inhibitor. Likewise, p,p'-DDT stimulated the JAK/STAT3 pathway in nude mice, as well as altered the mRNA levels of E-cadherin, N-cadherin, and CD29. Taken together, these results indicate that p,p'-DDT profoundly promotes the adhesion process by decreasing cell-cell adhesion and inducing cell-matrix adhesion via the ROS-mediated JAK/STAT3 pathway. All these events account for the carcinogenic potential of p,p'-DDT in liver.
Dysregulation of signalling pathways by changes of gene expression contributes to hallmarks of cancer. The ubiquitously expressed chaperone protein AAG8 (aging-associated gene 8 protein, encoded by the SIGMAR1 gene) is often found to be overexpressed in various cancers. AAG8 is involved in ER (endoplasmic reticulum)-associated degradation and has been intensively elaborated in neuroscience. However, its rationale in carcinogenesis has rarely been noticed. In this study, we explored the intrinsic oncogenetic roles of AAG8 in cancer cells and found that AAG8 promoted carcinogenesis both in vitro and in vivo. We further characterized AAG8, for the first time to our knowledge, as a STAT3 activator and elucidated that it alternatively activated STAT3 in addition to IL6/JAK pathway. Based on these findings and a drug screening study, we demonstrated that combined inhibition of AAG8 and IL6/JAK signalling synergistically limits cancer cell growth. Taken together, our findings shed light on the fundamental evidences for identification of AAG8 as an oncoprotein and potential target for cancer prevention, as well as highlight the importance of ER proteins in contributing to JAK/STAT signaling and carcinogenesis.
The nuclear factor of activated T cell (NFAT) family of calcium-regulated transcription factors plays a key role in the development and function of the immune system. Calcineurin, a protein phosphatase, activates NFAT by dephosphorylation. The activated NFAT is translocated into the nucleus, where it up-regulates the expression of interleukin 2 (IL-2) and other target genes. Calcineurin inhibitors such as cyclosporine A (CsA) and FK506 are effective immunosuppressant drugs and dramatically increase the success rate of organ transplantation procedures. However, since calcineurin is expressed in most tissues in the body and calcineurin inhibition alters many cellular processes besides immune cell activation, the therapeutic use of calcineurin inhibitors is limited by serious side effects. Thus inhibiting NFAT by other mechanisms such as blocking its binding to DNA could be a more selective and safer approach to target NFAT for therapeutic applications. In peripheral T cells, productive immune responses are dependent upon the cooperative binding of the NFAT/AP-1 transcriptional complex to the promoter regions of genes such as interleukin-2 (IL-2), while NFAT in the absence of AP-1 leads to T cell anergy. Protein transduction domains (PTDs) are able to penetrate cell membranes and can be used to transport exogenous proteins across the cell and nuclear membranes. In this study, we constructed a fusion protein of PTD and a minimum DNA binding domain of human NFAT1 (PTD-ΔNFATminiDBD), which contains two mutations (R466A and T533G) in the AP-1 binding sites. The delivery and functions of this fusion protein in T cells were investigated. The results indicated that PTD-ΔNFATminiDBD could be effectively delivered into T cells and transported into the nucleus. PTD-ΔNFATminiDBD attenuated IL-2 production in T cells and then inhibited T cell proliferation, likely through competing against endogenous NFAT for binding to the IL-2 gene promoter. These results demonstrated that PTD-ΔNFATminiDBD was an effective NFAT inhibitor with a novel mechanism of action and might potentially be used as an immunosuppressant for organ transplantation with higher safety and better tolerance than calcineurin inhibitors.
Among the mammalian matrix metalloproteinases (MMPs), MMP-1, -3 and -13 are collagenases. Particularly, MMP-13 is important for the degradation of major collagens in cartilage under certain pathological conditions such as osteoarthritis. To establish a potential therapeutic strategy for cartilage degradation disorders, the effects of 11 ginseng saponins (ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rg5, Rk1 and F4) on MMP-13 induction were examined in a human chondrocyte cell line, SW1353. Among these, several saponins including ginsenoside Rc, Rd, Rf, Rg3 and F4 were found to inhibit MMP-13 expression in IL-1β-treated SW1353 cells at non-cytotoxic concentrations (1-50 μM). The most prominent inhibitors were ginsenosides F4 and Rg3. Ginsenoside F4 inhibited MMP-13 expression 33.5% (P<0.05), 57.9% (P<0.01) and 90.0% (P<0.01) at 10, 30 and 50 μM, respectively. Significantly, ginsenoside F4 was found to strongly inhibit activation of p38 mitogen-activated protein kinase in signal transduction pathways (86.6 and 100.0% inhibition at 30 and 50 μM, P<0.01). The MMP-13 inhibitory effect was also supported by the finding that ginsenosides F4 and Rg3 reduced glycosaminoglycan release from IL-1α-treated rabbit joint cartilage culture to some degree. Taken together, these results indicate that several ginsenosides inhibit MMP-13 expression in IL-1β-treated chondrocytes. Ginsenoside F4 and Rg3 blocked cartilage breakdown in rabbit cartilage tissue culture. Thus, it is suggested that certain ginsenosides have therapeutic potential for preventing cartilage collagen matrix breakdown in diseased tissues such as those found in patients with arthritic disorders.
(-)-Epigallocatechin-3‑gallate (EGCG), the predominant constituent of green tea, has been demonstrated to be neuroprotective against stroke in rats. However, the precise mechanism of EGCG responsible for neuroprotective activity remains unclear and no established treatment for decreasing the resulting neurological damage of stroke exists. The present study was designed to investigate the neuroprotective mechanism of EGCG on transient focal cerebral ischemia in rats. EGCG, when applied immediately following ischemia, significantly decreased the expression of endoplasmic reticulum stress (ERS)‑related markers, [glucose‑regulated protein 78 (GRP78), C/EBP‑homologous protein (CHOP) and caspase‑12] and apoptosis 24 h following reperfusion. EGCG treatment also significantly reduced infarct volumes and increased neurological scores which was correlated with elevated levels of TRPC6 and phosphorylation of cAMP/Ca2+ response element‑binding protein (p‑CREB) activity, and decreased calpain‑specific aII‑spectrin breakdown product (SBDP145) activity. When mitogen‑activated protein kinase kinase (MEK) activity was specifically inhibited, the neuroprotective effect of EGCG was attenuated and a correlated decrease in CREB activity was observed. In conclusion, the results clearly demonstrated that intracerebroventricular injection of EGCG immediately following ischemia, inhibits ERS and improves the neurological status of rats that have undergone middle cerebral artery occlusion via the inhibition of calpain‑mediated TRPC6 proteolysis and the subsequent activation of CREB via the MEK/extracellular signal-regulated kinases (ERK) pathway.
Lipoxins (LXs) and their analogues are known to display potent anti-inflammatory actions. Previously, we reported that lipoxin A4 (LXA4) possessed powerful anti-inflammatory properties in acute pancreatitis in rats and that it may ameliorate the concomitant acute lung injury by reducing cytokine generation and inhibiting neutrophil activation. Considering that the vascular endothelium plays an important role during adherence, migration and activation of leukocytes, the present study was designed to investigate the effects of LXA4 on the inflammatory response induced by tumor necrosis factor α (TNF-α) in human pulmonary microvascular endothelial cells (HPMECs) and explore the potential mechanisms involved in these processes. We found that LXA4 markedly down-regulated the expression of monocyte chemotactic protein-1 (MCP-1), E-selectin, and interleukin-6 (IL-6) mRNA, as well as intercellular adhesion molecule-1 (ICAM-1) in TNF-α-exposed HPMECs. Moreover, LXA4 inhibited the phosphorylation and nuclear translocation of nuclear factor-κB/p65 (NF-κB/p65) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in HPMECs following TNF-α stimulation. Heme oxygenase-1 (HO-1), a cytoprotective enzyme, was up-regulated by LXA4 in both non- and TNF-α-stimulated HPMECs. In conclusion, the protective effects of LXA4 to ALI may be executed through inhibition inflammation pathways of NF-κB and p38 MAPK and up-regulation of cytoprotective HO-1.
Neuroprotectin D1 (NPD1) may serve an endogenous neuroprotective role in brain ischemic injury, yet the underlying mechanism involved is poorly understood. In the present study, we aimed to investigate whether intracerebroventricular (ICV) injection of NPD1 is neuroprotective against transient focal cerebral ischemia. We also sought to verify the neuroprotective mechanisms of NPD1. Rats subjected to 2 h ischemia followed by reperfusion were treated with NPD1 at 2 h after reperfusion. PD98059 was administered 20 min prior to surgery. Western blot analysis was performed to detect the protein levels of calpain-specific aII-spectrin breakdown products of 145 kDa (SBDP145), transient receptor potential canonical (subtype) 6 (TRPC6) and phosphorylation of cAMP/Ca2+-response element binding protein (p-CREB) at 12, 24 and 48 h after reperfusion. The immunoreactivity of p-CREB and TRPC6 was measured by quantum dot‑based immunofluorescence analysis. Infarct volume and neurological scoring were evaluated at 48 h after reperfusion. NPD1, when applied at 2 h after reperfusion, significantly reduced infarct volumes and increased neurological scores at 48 h after reperfusion, accompanied by elevated TRPC6 and p-CREB activity, and decreased SBDP145 activity. When mitogen‑activated protein kinase kinase (MEK) activity was specifically inhibited, the neuroprotective effect of NPD1 was attenuated and correlated with decreased CREB activity. Our results clearly showed that ICV injection of NPD1 at 2 h after reperfusion improves the neurological status of middle cerebral artery occlusion (MCAO) rats through the inhibition of calpain‑mediated TRPC6 proteolysis and the subsequent activation of CREB via the Ras/MEK/ERK pathway.
Chemotherapy resistance represents a major problem for the treatment of patients with lung carcinomas. Parthenolide (PN), a naturally occurring small molecule found in herb feverfew, has been used in clinical treatment. Although its importance in treating the chemotherapy resistance has been shown, the pharmacological benefits of PN for lung cancer with multidrug resistance are underappreciated. Using human lung epithelial carcinoma A549 and A549 derived DOX-resistant A549/DOX cell lines, we found that PN enhanced the apoptotic cytotoxicity of DOX in A549/DOX cells. PN inhibited P-glycoprotein (P-gp) up-regulation and promoted the intracellular accumulation of DOX in A549/DOX cells. PN also exhibited inhibitory effect on NF-κB activation in A549/DOX cells, suggesting that inhibition of NF-κB was involved in attenuating P-gp expression by PN. Moreover, we found that PN could also effectively inhibit the HSP70 up-regulation in A549/DOX cells. Further studies revealed a positive correlation between HSP70 and P-gp expression. Overexpression of HSP70 upregulated P-gp expression independently of NF-κB activation in A549 cells, and knockdown of HSP70 caused a reduced expression of P-gp in A549/DOX cells. RT-PCR experiments showed that HSP70 modulated the P-gp expression mainly at transcription level. Taken together, PN can reverse DOX resistance through suppressing P-gp expression by mechanisms involving attenuation of NF-κB activation and HSP70 up-regulation. Our results not only provide insight into potential use of PN in reversing P-gp mediated MDR to facilitate lung cancer chemotherapy, but also highlight a potential role of HSP70 in the development of drug resistance.
DNA damage activates p53 and its downstream target genes, which further leads to apoptosis or survival either by the cell cycle arrest or by DNA repair. In many tumors, the heat shock protein 27 (Hsp27) is expressed at high levels to provide protection against anticancer drugs. However, the roles of Hsp27 in p53-mediated cellular responses to DNA damage are controversial. Here, we investigated the interplay between the phosphorylation status of Hsp27 and p53 in kidney 293A (HEK293A) cells and found that over-expressing phosphorylated Hsp27 mimics (Hsp27-3D) activated p53/p21 in an ATM-dependent manner. In addition, incubation with doxorubicin (Dox), an anticancer drug, induced Hsp27 phosphorylation in human adenocarcinoma cells (MCF-7). In contrast, inhibition of Hsp27 phosphorylation retarded both p53 induction and p21 accumulation, and led to cell apoptosis. Furthermore, phosphorylated Hsp27 increased p53 nuclear importing and its downstream target gene expression such as p21 and MDM2, while de-phosphorylated Hsp27 impeded this procession. Taken together, our data suggest that Hsp27, in its phosphorylated or de-phosphorylated status, plays different roles in regulating p53 pathway and cell survival.
Adiponectin secreted from adipose tissues plays a role in the regulation of energy homeostasis, food intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin significantly inhibited GNRH secretion from GT1-7 hypothalamic GNRH neuron cells. In this study, we further investigated the effect of adiponectin on hypothalamic KISS1 gene transcription, which is the upstream signal of GNRH. We found that globular adiponectin (gAd) or AICAR, an artificial AMPK activator, decreased KISS1 mRNA transcription and promoter activity. Conversely, inhibition of AMPK by Compound C or AMPKα1-SiRNA augmented KISS1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the translocation of specificity protein-1 (SP1) from cytoplasm to nucleus; however, Compound C and AMPKα1-siRNA played an inverse role. Our experiments in vivo demonstrated that the expression of Kiss1 mRNA was stimulated twofold in the Compound C-treated rats and decreased about 60-70% in gAd- or AICAR-treated rats compared with control group. The numbers of kisspeptin immunopositive neurons in the arcuate nucleus region of Sprague Dawley rats mimicked the same trend seen in Kiss1 mRNA levels in animal groups with different treatments. In conclusion, our results provide the first evidence that adiponectin reduces Kiss1 gene transcription in GT1-7 cells through activation of AMPK and subsequently decreased translocation of SP1.
INTRODUCTION:
Carbon monoxide (CO) released from CORM-2 has anti-inflammatory function, but the critical molecule mediating the inflammation inhibition has not been elucidated. Previous studies indicate that CORM-2 can activate Nrf2, a key transcription factor regulating host defense against oxidative stress and inflammation-related disorders. In this study we use Nrf2 knockout mice to determine the role of Nrf2 in mediating the CO anti-inflammatory action.
METHODS:
We compared CORM-2's inhibiting effect on pro-inflammatory cytokine expressions (TNF-α, IL-1β and IL-6 and iNOS) in primary peritoneal macrophages, mouse liver and brain tissues from Nrf2(+/+) and Nrf2(-/-) mice. We further assayed the inflammatory cell infiltration in both liver and brain tissues of the Nrf2(+/+) and Nrf2(-/-) mice. Finally, we examined CORM's influence on mouse mortality in a mouse sepsis model.
RESULTS:
Our results showed that CORM-2 dramatically inhibited the expression of pro-inflammatory cytokines in Nrf2(+/+) mice, but not in Nrf2(-/-) mice. Furthermore CORM-2 substantially decreased LPS-induced mouse mortality of Nrf2(+/+) mice, but not of Nrf2(-/-) mice.
CONCLUSION:
We conclude that Nrf2 is indispensable for CORM-2 inhibition of LPS-induced inflammation.
BACKGROUND:
The present study was designed to prepare and find the optimum active preparation or fraction from Korea Red Ginseng inhibiting matrix metalloproteinase-13 (MMP-13) expression, because MMP-13 is a pivotal enzyme to degrade the collagen matrix of the joint cartilage.
METHODS:
From total red ginseng ethanol extract, n-BuOH fraction (total ginsenoside-enriched fraction), ginsenoside diol-type-enriched fraction (GDF), and ginsenoside triol-type-enriched fraction (GTF) were prepared, and ginsenoside diol type-/F4-enriched fraction (GDF/F4) was obtained from Panax ginseng leaf extract.
RESULTS:
The n-BuOH fraction, GDF, and GDF/F4 clearly inhibited MMP-13 expression compared to interleukin-1β-treated SW1353 cells (human chondrosarcoma), whereas the total extract and ginsenoside diol-type-enriched fraction did not. In particular, GDF/F4, the most effective inhibitor, blocked the activation of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun-activated protein kinase (JNK), and signal transducer and activator of transcription-1/2 (STAT-1/2) among the signal transcription pathways involved. Further, GDF/F4 also inhibited the glycosaminoglycan release from interleukin-1α-treated rabbit cartilage culture (30.6% inhibition at 30 μg/mL).
CONCLUSION:
Some preparations from Korean Red Ginseng and ginseng leaves, particularly GDF/F4, may possess the protective activity against cartilage degradation in joint disorders, and may have potential as new therapeutic agents.
OBJECTIVE:
Investigate the therapeutic effect of regional arterial infusion (RAI) with Aspirin-Triggered Lipoxin A4 (ATL) in experimental severe acute pancreatitis (SAP) in rats.
MATERIALS AND METHODS:
SAP was induced by injection of 5% sodium taurocholate into the pancreatic duct. Rats with SAP were treated with ATL (the ATL group) or physiological saline (the SAP group) infused via the left gastric artery 30 min after injection of sodium taurocholate. The sham group was subjected to the same surgical procedure, though without induction of SAP. Serum levels of amylase, phospholipase A2 (PLA2), interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured at 12 and 24 h after induction of SAP. Ascitic fluid, the pancreatic index (wet weight ratio) and myeloperoxidase (MPO) levels in the pancreas were determined and histopathological findings were evaluated. The expression of intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), NF-κB p65, and heme oxygenase-1 (HO-1) in the pancreas were estimated by immunofluorescence and western blot, respectively.
RESULTS:
ATL rats had lower serum levels of TNF-α, IL-1β, and IL-6 (P<0.01), PLA2 (P<0.05), and amylase levels (P<0.05) studied as compared with the SAP group. The pancreatic index in the ATL group decreased only at 24 h as compared with the SAP group (P<0.05). The histopathological findings and MPO levels in the pancreas significantly decreased in the ATL group as compared to the SAP group (P<0.05 and P<0.01, respectively). Immunofluorescence and western blot showed that ATL attenuated the expression of NF-κB p65, ICAM-1 and PECAM-1 in the pancreas, and increased the expression of HO-1 in SAP animals.
CONCLUSIONS:
We demonstrated that RAI with ATL attenuated the severity of experimental SAP, maybe achieved by improving the expression of HO-1, and down-regulating the NF-κB signaling pathway, with decreased expression of ICAM-1 and PECAM-1 and reduced generation of pro-inflammatory cytokines.
OBJECTIVE:
The protective effect of sodium tanshinone IIA sulfonate (STS) pretreatment against experimental myocardial ischemia/reperfusion (I/R) has been demonstrated previously, however its therapeutic effects and mechanism of action still remain unclear. The objective of this study was to investigate the therapeutic time window and potential mechanism of STS action on myocardial I/R injury in a rat model of myocardial I/R.
METHODS:
Rats received 30 min ischemia by complete ligation of the left ascending coronary artery, and then were reperfused for 24 h. STS (8 mg/kg) was administered intravenously 15 min before and at 0, 0.5, 1, 2, 4, 6 h after reperfusion. The infarct size and several consequences of myocardial I/R including myocardial zymogram, antioxidant status, cardiac function and microstructure disorder were evaluated 24 h after reperfusion. Furthermore, the effect of STS on heme oxygenase-1 (HO-1) protein expression and nuclear factor-κB (NF-κB) activation were also evaluated.
RESULTS:
In the present study, the time point of optimal cardioprotective effect of STS was within 2 h after reperfusion, with declining effect at 4 h and no effect at 6 h after the onset of reperfusion. In addition, STS-mediated cytoprotection against oxidative stress and inflammatory responses was correlated with an increased HO-1 activity..
CONCLUSIONS:
STS could ameliorate cardiac dysfunction and variation of myocardial zymogram, up-regulate antioxidant systems. Moreover, modulation of HO-1 was involved in STS induced cardioprotection..
Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
BACKGROUND:
Mitochondrial dysfunction has been reported to be one of the contributing factors of sepsis-associated encephalopathy (SAE). Mitochondrial biogenesis controls mitochondrial homeostasis and responds to changes in cellular energy demand. In addition, it is enhanced or decreased due to mitochondrial dysfunction during SAE. The aim of this study was to explore the changes of mitochondrial biogenesis of astrocytes under septic conditions.
METHODS:
Lipopolysaccharide (LPS; 50 ng/ml) and interferon-γ (IFN-γ; 200 U/ml) were incubated with astrocytes to model the effects of a septic insult on astrocytes in vitro. The mitochondrial ultrastructure and volume density were evaluated by transmission electron microscopy. Intracellular adenosine triphosphate (ATP) levels were detected by the firefly luciferase system. The expression of protein markers of mitochondrial biogenesis and the binding ability of mitochondrial transcription factor A (TFAM) were determined by western blot and electrophoretic mobility shift assays, respectively. The mitochondrial DNA (mtDNA) content was detected by real-time polymerase chain reaction.
RESULTS:
The number of mildly damaged mitochondria was found to be significantly greater after treatment for 6 hours, as compared with at 0 hour (P < 0.05). The mitochondrial volume density was significantly elevated at 24 hours, as compared with at 0 hour (P < 0.05). The ATP levels at 6 hours, 12 hours, and 24 hours were significantly greater than those at 0 hour (P < 0.05). The protein markers of mitochondrial biogenesis were significantly increased at 6 hours and 12 hours, as compared with at 0 hour (P < 0.05). The TFAM binding activity was not significantly changed among the four time points analyzed. The mtDNA contents were significantly increased at 12 hours and 24 hours, as compared with at 0 hour (P < 0.05).
CONCLUSIONS:
Under septic conditions, mitochondrial biogenesis of astrocytes increased to meet the high-energy demand and to promote mitochondrial recovery. Furthermore, the TFAM-DNA binding ability was not sensitive to sepsis-induced injury.
BACKGROUND:
Homeobox A10 (HOXA10), a key transcription factor, plays a critical role in endometrial receptivity by regulating the expression of downstream target genes, such as β3-integrin (ITGB3), but little is understood about the mechanisms of the posttranslational modification of HOXA10 during embryo implantation.
OBJECTIVE:
The aim of this study was to assess the effect of HOXA10 acetylation by p300/CREB-binding protein-associated factor (PCAF) in the embryo implantation process.
METHODS:
The association of HOXA10 with PCAF was detected by coimmunoprecipitation, Western blotting, and confocal immunofluorescent assays. A luciferase reporter assay, Western blotting, quantitative real-time PCR, and chromatin immunoprecipitation techniques were used to determine the effect of PCAF on HOXA10 protein stability and the HOXA10-mediated regulation of ITGB3 expression. HOXA10-PCAF association on embryo implantation was evaluated using a BeWo spheroid attachment assay. PCAF expression in the eutopic endometrium of women with endometriosis and fertile controls was measured by Western blotting technique.
RESULTS:
PCAF was identified as an HOXA10-interacting protein and inhibited HOXA10-mediated ITGB3 transcription via acetylating HOXA10 at K338 and K339. Overexpressing or knocking down PCAF in Ishikawa cells showed that PCAF not only down-regulated HOXA10-mediated ITGB3 protein expression but also diminished HOXA10-mediated embryo adhesiveness by acetylating HOXA10 (P < .05). Furthermore, we found aberrantly high PCAF expression in the eutopic endometrium of women with a diagnosis of endometriosis compared with the fertile controls (P < .05).
CONCLUSIONS:
These observations demonstrate that 1) HOXA10 associates with and is acetylated by PCAF at lysines K338 and K339 in Ishikawa cells and 2) HOXA10-PCAF association impairs embryo implantation by inhibiting ITGB3 protein expression in endometrial epithelial cells.
