| ObjectiveWith the improvement of people’s living standards and changes of lifestyle, the incidence of obesity dramatically increased, which has developed into one of the serious diseases threatening public health. As a global disease, obesity has been paid more and more attention. Obesity is closely related to the pathogenesis of many diseases, increases the risk of type II diabetes, cardiovascular diseases and various cancers and other diseases. Excessive accumulation of energy in bodies can disrupt adipose tissue homeostasis and energy balance, thus leading to obesity, insulin resistance and the occurrence of the above dieases. Adipose tissues play important roles in regulating energy balance. Therefore, it has become the key step to maintain adipose tissue homeostasis and balance energy for the control of obesity and obesity-related diseases.Two different types of adipose tissues, white adipose tissues (WAT) and brown adipose tissues (BAT) reside in mammals. WAT is responsible for fat storage and energy balance. Excessive energy causes adipocyte hypertrophy and pathological expansion of WAT especially visceral WAT, which contributes to WAT inflammation, insulin resistance and subsequent metabolic syndromes. In contrast, BAT is specialized to burn fat for great heat, playing an important role in maintaining the body temperature and energy balance. In response to cold or excess feeding, BAT-specific uncoupling protein 1 (UCP1) mediates uncoupled respiration and dissipate energy, which plays a natural resistance to obesity. Recently, a newly identified type of brown-like fat cells, termed beige or brite adipocytes were found within certain WAT depots upon appropriate stimulation. Beige adipocytes also expresses UCP1 and gene expression pattern that is distinct from either white or classical brown adipocytes, but it also can burn fat for thermogenesis through uncoupled respiration, produces beneficial effects against obesity, diabetes and metabolic diseases.Adipose-derived stem cells (ADSCs), as a kind of mesenchymal stem cells (MSCs), has the unique characteristics of stem cells, referred to sternness, including two aspects that are self-renewal capacity and multipotency. ADSCs are keystone of tissue homeostasis by balancing self-renewal and differentiation divisions. ADSCs are the main source of adipocytes in adipose tissues, which play essential roles in energy balance and nutrient homeostasis. Since the differentiation of ADSCs into white or beige adipocytes may determine the development of obesity or not, genes or proteins that control the differentiation destination of ADSCs would become promising targets for obesity treatment.Programmed cell death 4 (Pdcd4) is initially found to upregulate during apoptosis. So far, Pdcd4 has been well recognized as a tumor suppressor and regulates gene expression through influencing translation and transcription. Recently, emerging evidences have demonstrated the involvement of Pdcd4 in inflammatory or metabolic diseases. Our previous study have shown that in response to high-fat diet, Pdcd4 promotes the pathological expansion of epididymal WAT and the development of obesity partially through inhibiting a modulator of lipid homeostasis, LXR-a. Considering the important roles of ADSCs in tissue homeostasis, we raised the hypothesis that Pdcd4 might promote diet-induced obesity by regulating ADSC function. In order to clarify the effects of Pdcd4 on ADSCs, we have performed the following study. ADSCs from WT and Pdcd4-/- mice were isolated and cultured in vitro, then were tested for their differences on sternness, proliferation and adipogenic differentiation, underlying mechanisms were further explored, and related findings were verified by in vivo animal experiments.MethodsⅠ. The effects of Pdcd4 deficiency on stem cell-related makers of ADSCs1. Isolation and culture of ADSCsEpididymal fat pads from WT and Pdcd4-/- mice were minced and digested with collagenase for 45 minutes at 37℃. After a filtration, stromal vascular fraction (SVF) was obtained and cultured in culture medium. The attached ADSCs were passaged at about 90% confluence. The 3rd to 5th passages of the cells were used in the experiments.2. The detection of stem cell surface makersFlow cytometry were used to detect the expression of positive markers CD 105, CD90 and negative markers CD11b, CD11c on WT and Pdcd4-/- ADSCs.3. The effects of Pdcd4 deficiency on stemness makers of ADSCsTotal RNA was extracted from WT and Pdcd4-/- ADSCs and SVF, and was reversely transcripted into cDNA. The sternness markers Nanog, Oct4 and Sox2 were detected by Real-time PCR.Ⅱ. The effects of Pdcd4 deficiency on self-renewal of ADSCs1. The effects of Pdcd4 deficiency on proliferation of ADSCsIn vitro study:First, the cell vitality of WT and Pdcd4-/- ADSCs at 12 h and 24 h after inoculating into the plates were detected by CCK-8 experiment, and the ability of colony formation was detected by clone formation assay. Then, the effects of Pdcd4 deficiency on proliferation and cell cycle were tested by the EdU incorporation assay and PI staining.In vivo study:the cell dividing efficiency in epididymal fat pads was assayed by intraperitoneal injection of EdU into normal diet (ND)-fed WT and Pdcd4-/- mice, respectively. After 24 h, EdU signal was detected in epididymal fat tissues from WT and Pdcd4-/- mice.2. The effects of Pdcd4 deficiency on signaling pathway during proliferation of ADSCsFirst, the proteins were extracted from WT and Pdcd4-/- ADSCs, and the expression of p-AKT and cell cycle protein cyclinDl were detected by Western-blot assay. To confirm the contribution of AKT signaling pathway during proliferation of ADSCs, inhibitor MK2206 was used to inhibit AKT activation, then the expression of p-AKT and cyclinD1 as well as the cell proliferation were detected though Western-blot or EdU incorporation assay.Ⅲ. The effects of Pdcd4 deficiency on adipogenic differentiation of ADSCs1. The effects of Pdcd4 deficiency on adipogenic differentiation of ADSCs in vitroWT and Pdcd4-/- ADSCs were performed classical white adipogenic differentiation using adipogenic differentiation medium, respectively. The mRNA level of C/EBPa, PPAR-y, adiponectin and aP2 of differentiating ADSCs at 0,4,8 and 12 day post induction were detected by Real-time PCR. At 18 day post induction, the morphology of lipid droplets were observed by Oil Red O staining, and the optical density value was measured after eluting Oil Red O. Immunocytochemistry and Real-time PCR were used to detect the expression of UCP1 at 12 day post induction.2. The effects of Pdcd4 deficiency on differentiation of ADSCs in HFD mice(1) The establishment of diet-induced obesity modelsWT and Pdcd4-/- mice used in experiments at the age of 8 weeks were given a high-fat diet (HFD, containing 15% fat and 15% cholesterol) feeding or a normal diet (ND) feeding for 24 weeks.(2) The detection of beige adipocyte markersAfter 24 weeks of HFD feeding, the protein and total RNA of WT and Pdcd4-/-mice were extracted, the expression of UCP1 were detected by Western-blot assay and Real-time PCR. Besides, WT and Pdcd4-/- WAT were used to detect the expression of UCP1 by immunofluorescence staining.3. The effects of Pdcd4 deficiency on the levels of lactate produced by ADSCsThe supernatants from ADSC cultures before and after 12 day’s adipogenic induction were collected, respectively, and then were used to detect the levels of lactate produced by ADSCs.ResultsI. Pdcd4 deficiency increases the expression levels of stem cell-related makers on ADSCs1. Pdcd4 deficiency increases the expression levels of surface makers on ADSCsADSCs from WT and Pdcd4-/- mice were isolated and expanded in vitro, stem cell-related phenotypes were detected. No morphological difference was observed between WT and Pdcd4-/- ADSCs. Stem cell-related surface markers CD 105 and CD90 were positively expressed on both WT and Pdcd4-/- ADSCs, together with negative or rare expression of CD11b and CD11c. Differently, Pdcd4-/- ADSCs displayed relatively higher levels of CD 105 and CD90 as compared to WT ADSCs.2. Pdcd4 deficiency enhances the stemness makers of ADSCsWe detected the mRNA levels of sternness markers Nanog, Oct4 and Sox2 on ADSCs. Significantly higher levels of Nanog and Oct4 mRNA, but not Sox2 mRNA were detected on Pdcd4-/- ADSCs as compared to WT ADSCs. Interestingly, all the detected sternness markers including Nanog, Oct4, and Sox2 showed higher levels in Pdcd4-/- SVF than in WT SVF.Ⅱ. Pdcd4 deficiency increases the self-renewal of ADSCs1. Pdcd4 deficiency increases the proliferation of ADSCs through promoting S-phase entryThe result from CCK-8 and colony formation assay showed that Pdcd4 deficiency increases the proliferation of ADSCs. EdU staining showed that the EdU-positive cells entering S phase obviously increased in Pdcd4-/- ADSCs compared with those in WT ADSCs. Cell cycle analysis further substantiated that Pdcd4 deficiency promotes the G1/S phase transition, as suggested by higher percentages of cells at the S phase and lower percentages of cells at G1 phase in Pdcd4-/- ADSCs than those in WT ADSCs.In vivo EdU incorporation assay showed that obviously higher percentages of EdU-positive cells were observed in Pdcd4-/- WAT than in WT WAT, thus indicating that Pdcd4 deficiency could improve the sternness of ADSCs in mice. Collectively, these data suggest that Pdcd4 deficiency increases the proliferation of ADSCs by promoting S-phase entry.2. AKT activation is responsible for the S-phase elevation caused by Pdcd4 deficiency in ADSCsNext, we detected the activation status of AKT in WT and Pdcd4-/- ADSCs. Compared with WT ADSCs, Pdcd4-/- ADSCs had a significant upregulation in AKT phosphorylation. Notably, enhanced AKT activation in Pdcd4-/- ADSCs was accompanied by a marked increase in the expression of cyclinDl. In WT ADSCs, blockade of AKT signaling significantly inhibited cell proliferation, but had no significant influence on the decline of cyclinDl.On the contrary, the expression of cyclinDl decreased substantially in Pdcd4-/- ADSCs, and very few detectable EdU-positive cells was consistently observed. These data suggest that AKT activation is responsible for the S-phase elevation caused by Pdcd4 deficiency in ADSCs.Ⅲ. Pdcd4 deficiency drives the transdifferentiation of ADSCs into beige adipocytes1. Pdcd4 deficiency drives the transdifferentiation of ADSCs into beige adipocytes in vitroThe mRNA levels of white adipocyte markers including C/EBPa, adiponectin, PPAR-a, and αP2 during the adipogenic differentiation at 0,4,8,12 day post induction were detected. As shown in the results, all mentioned genes underwent a rapid up-regulation in both WT and Pdcd4-/- ADSCs. Whereas, the expression levels in Pdcd4-/- ADSCs were obviously lower at 12 day post induction. Relatively lower levels of lipids were detected in Pdcd4-/- adipocytes as measured by Oil Red O staining. Of note, the expression of UCP1 was remarkably higher in Pdcd4-/- adipocytes at 12 day post induction, which were confirmed by immunocytochemistry. These data revealed that Pdcd4 deficiency might drive the transdifferentiation of ADSCs from white adipocyte into UCP1-expressing beige adipocytes.2. Pdcd4 deficiency drives the formation of beige adipocytes in WAT from HFD-fed miceWe detected the protein and mRNA levels of UCP1 in epididymal WT and Pdcd4-/- WAT, little amounts of UCP1 were detected in ND-fed WT and Pdcd4"7" WAT. In response to HFD, Pdcd4-/- WAT displayed an obvious elevation in UCP1 expression, while WAT from WT mice showed no significant change, these data were consistent with the results of immunohistochemistry, demonstrating that Pdcd4 deficiency in mice promotes beige adipocyte differentiation in WAT upon HFD feeding.3. Pdcd4 deficiency increases the levels of lactate produced by ADSCsA remarkable elevation of lactate was observed in Pdcd4-/- ADSCs compared with WT ADSCs. Even in differentiating Pdcd4-/- ADSCs at 12 day post-induction, more lactate were also detected, which consisted with the relatively higher levels of UCP1 compared with those in WT ADSCs, revealed lactate as a pivotal contributor to the occurrence of beige adipocytes.Conclusion1. Pdcd4 deficiency enhances the stemness of ADSCs in mice.2. AKT activation is responsible for the S-phase elevation caused by Pdcd4 deficiency in ADSCs.3. Pdcd4 deficiency drives the transdifferentiation of ADSCs into beige adipocytes.ObjectiveStress granules (SGs) are translationally silent cytoplasmic ribonucleoprotein complexes that assemble during various types of cellular stresses such as heat shock, viral infection, oxidative and endoplasmic reticulum stresses. In response to environmental emergency, cells can trigger a sudden translational arrest, leading to rapid polysome disassembly and subsequent assembly of heterogeneous mRNAs and translation factors. Typical SG markers include TIA-1 (T-cell-restricted intracellular antigen-1, TIA-1),FXR1 (Fragile X mental retardation-relate protein 1, FXR1), eIF4A(Eukaryotic-initiating factors 4A)and eIF4B are often as a symbol of formation. So far, SGs have been implicated in various diseases including viral infection, inflammatory diseases, cancer, and multiple neurodegenerative diseases.As a translation inhibition factor, Pdcd4 can bind with eIF4A through its MA3 structure domain and suppress the helicase activity, thus inhibiting cap-dependent protein translation. Pdcd4 can inhibit the growth of tumor cells, thus inhibiting neoplastic transformation and tumor progression. Recently, we have demonstrated the involvement of Pdcd4 in obesity, adipose inflammation, and atherosclerosis. Pdcd4 deficiency brings an obvious alleviation in high-fat diet (HFD)-induced adipose ER stress and hepatic oxidative stress, suggesting that Pdcd4 is involved in the process of HFD- induced ER stress and oxidative stress. However, the precise roles of Pdcd4 during these stress processes remain largely unknown, the possible link between Pdcd4 and SGs is yet to be determined.Oxidative stress is an imbalance state of oxidation and anti-oxidation in the bodies. Oxidized low density lipoprotein (ox-LDL) is a typical biomarker for obesity and related metabolic synrome, which contributes to the enhanced oxidative stress in macrophages and some other cells through promoting and augmenting the generation of reactive oxygen species. In the present study, we used ox-LDL or HFD as stress signals to test the roles of Pdcd4 in SG formation, and then explored the possible mechanisms.Methods1. SG formation in macrophages and liver tissues from HFD-fed miceDiet-induced obesity model were established in WT and Pdcd4-/- mice, the formation of TIA-1+SGs in the peritoneal macrophage and liver tissues were detected by the immunofluorescence.2. Role of Pdcd4 in SG formationMacrophages from WT or Pdcd4-/- mice were treated with or without ox-LDL, TIA-1+SGs were detected by the immunofluorescence, the cells containing SGs was compared between WT and Pdcd4-/- macrophages.3. The location of Pdcd4 in SGsWT macrophages or Hela cells transfected with pEGFP-C1-Pdcd4 plasmids were stimulated with ox-LDL, the expression and location of Pdcd4 and SG makers (TIA-1, FXR1 and eIF4A) were detected by immunofluorescence.4. The involvement of Pdcd4 in SGs through the RNA-binding regionIt has been recognized that two important regions, referred to as RBM1 and RBM2 within Pdcd4. Based on pEGFP-Cl-Pdcd4 (Pdcd4-WT), we constructed different truncated plasmids Pdcd4-Dl, Pdcd4-D2 and Pdcd4-D1+2, which were depleted of RBM1, RBM2, both RBM1 and RBM2, respectively. After transfection into HepG2 cells, the formation of TIA-1+SGs in response to ox-LDL were detected by the immunofluorescence staining, the SGs number were statistically analyzed.5. The regulation of Pdcd4 on SG formation1) Hela cells were transfected with pEGFP-C1 or pEGFP-C1-Pdcd4 plasmids respectively. After stimulation with ox-LDL, the expression of p-AKT and p-eIF2a were detected by western-blot assay.2) The macrophages from WT or Pdcd4-/- mice were treated with ox-LDL, the expression of p-AKT and p-eIF2a were detected by western-blot assay.3) The macrophages from Pdcd4-/- mice were treated with MK2206 and then stimulated with ox-LDL, the expression of p-AKT and p-eIF2a were detected by western-blot assay.4) The macrophages were isolated from HFD WT or Pdcd4-/- mice the expression of p-AKT and p-eIF2a were detected by western-blot assay.Results1. Pdcd4 deficiency reduces the SG formation in macrophages and liver tissues from HFD-fed miceWe detected the formation of SGs in macrophages and liver tissues using TIA-1 as a SG marker. Obvious TIA-1+SGs were observed in macrophages from WT obese mice, whereas much less TIA-1+ SGs were detected in those from Pdcd4-/- lean mice, similar change was observed in liver sections. These data indicate the involvement of SGs in HFD-induced stress responses, and importantly, reveal Pdcd4 as a key contributor to SG formation induced by HFD stimulus.2. Pdcd4 deficiency reduces the formation of TIA-1+ SGs in macrophages in response to ox-LDLIn response to ox-LDL, the TIA-1+ SGs in WT macrophages obviously increased as suggested by significantly elevated percentage of cells containing TIA-1+ SGs as compared to those in non-treated macrophages. Whereas Pdcd4-/- macrophages showed reduced percentage of cells containing TIA-1+ SGs, and the SGs were only observed in a few of Pdcd4-/- macrophages. These results support that Pdcd4 deficiency causes a resistance to SG formation in response to ox-LDL, thus revealing the key role of Pdcd4 in ox-LDL-induced SGs.3. Pdcd4 is co-localized with specific SG markers in ox-LDL-stimulated macrophagesIn ox-LDL-stimulated macrophages, all the detected SG markers including TIA-1, FXR1 and eIF4A displayed apparent co-localization with Pdcd4, suggesting that Pdcd4 is an important component of ox-LDL-induced SGs.4. Ectopic Pdcd4 is co-localized with specific SG markers in ox-LDL-stimulated HeLa cellsIn ox-LDL-stimulated HeLa cells which were transfected with pEGFP-Cl-Pdcd4 plasmids, the SG markers including TIA-1, FXR1 and eIF4A also displayed co-localization with Pdcd4. These data are partially in consistent with the observed finding in macrophages, thereby suggesting that ox-LDL treatment also drives exogenous Pdcd4 into SG assembly.5. Pdcd4 participates in the formation of SGs through its RNA-binding regionAfter transfection into HepG2 cells, lots of apparent GFP+ SGs were detected in Pdcd4-WT-overexpressed cells in response to ox-LDL. In contrast, in cells expressing Pdcd4-D2 or Pdcd4-D1+2, the formation of GFP+ TLA-1+ SGs remarkably decreased, which were hardly detected. For cells expressing Pdcd4-D1, there was no significant decrease in the number of GFP+ TIA-1+ SGs. These data indicate that Pdcd4 participates in the assembly of ox-LDL-induced SGs mainly through its RNA-binding region RBM2.6. Pdcd4 regulates ox-LDL-stimulated SGs through AKT-eIF2a axisIn response to ox-LDL, Pdcd4-/- macrophages displayed a significant elevation in AKT phosphorylation, and a decline in eIF2a phosphorylation as ompared to WT macrophages, suggesting that the reduction of p-eIF2a in Pdcd4-/- macrophages might be caused by the activation of AKT. After blocking AKT phosphorylation using MK2206, the expression of p-eIF2a reversed to a relatively higher level, accompanied by an obvious increase of SG formation. Results from WT and Pdcd4-/- mice fed on HFD further confirmed the regulatory role of AKT-eIF2a axis in Pdcd4-involved SGs. These findings indicate that Pdcd4 promotes the formation of ox-LDL or HFD-induced SGs dependent on eIF2a phosphorylation, which is regulated through AKT-eIF2a axis at least partially.Conclusion1. Ox-LDL or HFD can induce the formation of SGs.2. Pdcd4 participates in the formation of SGs through its RNA-binding region.3. Pdcd4 regulates ox-LDL-stimulated SGs through AKT-eIF2a axis. |
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