Increased SCF/c‐kit by hypoxia promotes autophagy of human placental chorionic plate‐derived mesenchymal stem cells via regulating the phosphorylation of mTOR

Hypoxia triggers physiological and pathological cellular processes, including proliferation, differentiation, and death, in several cell types. Mesenchymal stem cells (MSCs) derived from various tissues have self‐renewal activity and can differentiate towards multiple lineages. Recently, it has been reported that hypoxic conditions tip the balance between survival and death by hypoxia‐induced autophagy, although the underlying mechanism is not clear. The objectives of this study are to compare the effect of hypoxia on the self‐renewal of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and placental chorionic plate‐derived mesenchymal stem cells (CP‐MSCs) and to investigate the regulatory mechanisms of self‐renewal in each MSC type during hypoxia. The expression of self‐renewal markers (e.g., Oct4, Nanog, Sox2) was assessed in both cell lines. PI3K and stem cell factor (SCF) expression gradually increased in CP‐MSCs but were markedly downregulated in BM‐MSCs by hypoxia. The phosphorylation of ERK and mTOR was augmented by hypoxia in CP‐MSCs compared to control. Also, the expression of LC3 II, a component of the autophagosome and the hoof‐shaped autophagosome was detected more rapidly in CP‐MSCs than in BM‐MSCs under hypoxia. Hypoxia induced the expression of SCF in CP‐MSCs and increased SCF/c‐kit pathway promotes the self‐renewal activities of CP‐MSCs via an autocrine/paracrine mechanism that balances cell survival and cell death events by autophagy. These activities occur to a greater extent in CP‐MSCs than in BM‐MSCs through regulating the phosphorylation of mTOR. These findings will provide useful guidelines for better understanding the function of SCF/c‐kit in the self‐renewal and autophagy‐regulated mechanisms that promote of MSC survival. J. Cell. Biochem. 114: 79–88, 2012. © 2012 Wiley Periodicals, Inc.     

Deferoxamine promotes mesenchymal stem cell homing in noise‐induced injured cochlea through PI3K/AKT pathway

Objective

Over 5% of the world's population suffers from disabling hearing loss. Stem cell homing in target tissue is an important aspect of cell‐based therapy, which its augmentation increases cell therapy efficiency. Deferoxamine (DFO) can induce the Akt activation, and phosphorylation status of AKT (p‐AKT) upregulates CXC chemokine receptor‐4 (CXCR4) expression. We examined whether DFO can enhance mesenchymal stem cells (MSCs) homing in noise‐induced damaged cochlea by PI3K/AKT dependent mechanism.

Materials and Methods

Mesenchymal stem cells were treated with DFO. AKT, p‐AKT protein and hypoxia inducible factor 1‐ α (HIF‐1α) and CXCR4 gene and protein expression was evaluated by RT‐ PCR and Western blot analysis. For in vivo assay, rats were assigned to control, sham, noise exposure groups without any treatment or receiving normal, DFO‐treated and DFO +LY294002 (The PI3K inhibitor)‐treated MSCs. Following chronic exposure to 115 dB white noise, MSCs were injected into the rat cochlea through the round window. Number of Hoechst‐ labelled cells was determined in the endolymph after 24 hours.

Results

Deferoxamine increased P‐AKT, HIF‐1α and CXCR4 expression in MSCs compared to non‐treated cells. DFO pre‐conditioning significantly increased the homing ability of MSCs into injured ear compared to normal MSCs. These effects of DFO were blocked by LY294002.

Conclusions

Pre‐conditioning of MSCs by DFO before transplantation can improve stem cell homing in the damaged cochlea through PI3K/AKT pathway activation.

     

Role of hypoxia‐induced fibronectin‐integrin β1 expression in embryonic stem cell proliferation and migration: Involvement of PI3K/Akt and FAK

Cell migration is largely dependent on integrin (IN) binding to the extracellular matrix, and several signaling pathways involved in these processes have been shown to be modified by hypoxia. Therefore, the aim of this study was to determine the influence of hypoxia on fibronectin (FN) and IN β1 expression in mouse embryonic stem cells (mESCs) and their signaling pathways to modulate proliferation. FN and IN β1 expression were significantly increased in hypoxic mESCs by 24 h. Hypoxia also increased cell attachment, which was accompanied by concomitant increases in the binding level of FN and IN β1. Hypoxia‐induced FN expression was mediated by increased phosphatidylinositol 3 kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) phosphorylation, and hypoxia‐inducible factor‐1α (HIF‐1α) expression. Moreover, under hypoxic conditions, focal adhesion kinase (FAK) and Src phosphorylation were increased in a time‐dependent fashion; these increases were blocked by IN β1 antibody. In addition, the hypoxia induced increase of F‐actin distribution and cell migration (activation of matrix metalloproteinase‐2 and ‐9) was inhibited by IN β1 antibody. Indeed, hypoxia increased the level of cell‐cycle regulatory protein and DNA synthesis. In conclusion, hypoxia increases the proliferation and migration of mESCs via FN‐IN β1 production through the PI3K/Akt, mTOR, and HIF‐1α pathways, followed by FAK activation. J. Cell. Physiol. 226: 484–493, 2011. © 2010 Wiley‐Liss, Inc.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

LPA rescues ER stress‐associated apoptosis in hypoxia and serum deprivation‐stimulated mesenchymal stem cells

Poor viability of transplanted mesenchymal stem cells (MSCs) in the infracted heart has limited their therapeutic efficacy in cardiac repair after myocardial infarction. We previously demonstrated that hypoxia and serum deprivation (hypoxia/SD) induced mitochondria‐dependent apoptosis in MSCs, while lysophosphatidic acid (LPA) could almost completely block this apoptotic process. However, the role of endoplasmic reticulum (ER) stress and its upstream signaling events in hypoxia/SD‐induced MSC apoptosis remain largely unknown. Here we found that hypoxia/SD‐induced MSC apoptosis was associated with ER stress, as shown by the induction of CHOP expression and procaspase‐12 cleavage, while the effects were abrogated by LPA treatment, suggesting ER stress is also a target of LPA. Furthermore, hypoxia/SD induced p38 activation, inhibition of which resulted in decreases of apoptotic cells, procaspase‐12 cleavage and mitochondrial cytochrome c release that function in parallel in MSC apoptosis. Unexpectedly, p38 inhibition enhanced hypoxia/SD‐induced CHOP expression. Interestingly, p38 activation, a common process mediating various biological effects of LPA, was inhibited by LPA in this study, and the regulation of p38 pathway by LPA was dependent on LPA_1/3/Gi/ERK1/2 pathway‐mediated MKP‐1 induction but independent of PI3K/Akt pathway. Collectively, our findings indicate that ER stress is a target of LPA to antagonize hypoxia/SD‐induced MSC apoptosis, and the modulation of mitochondrial and ER stress‐associated apoptotic pathways by LPA is at least partly dependent on LPA_1/3/Gi/ERK/MKP‐1 pathway‐mediated p38 inhibition. This study may provide new anti‐apoptotic targets for elevating the viability of MSCs for therapeutic potential of cardiac repair. J. Cell. Biochem. 111: 811–820, 2010. © 2010 Wiley‐Liss, Inc.     

The transient receptor potential vanilloid‐3 regulates hypoxia‐mediated pulmonary artery smooth muscle cells proliferation via PI3K/AKT signaling pathway

Objectves

Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP channels family of Ca²⁺‐permeant cation channels. In this study, we aim to investigate the role of TRPV3 in pulmonary vascular remodeling and PASMCs proliferation under hypoxia.

Materials and methods

The expression of TRPV3 was evaluated in patients with pulmonary arterial hypertension (PAH) and hypoxic rats, using hematoxylin and eosin (H&E) and immunohistochemistry. In vitro, MTT assay, flow cytometry, Western blotting and immunofluorescence were performed to investigate the effects of TRPV3 on proliferation of PASMCs.

Results

We found that, in vivo, the expression of TRPV3 was increased in patients with PAH and hypoxic rats. Right ventricular hypertrophy measurements and pulmonary pathomorphology data show that the ratio of the heart weight/tibia length (HW/TL), the right ventricle/left ventricle plus septum (RV/LV+S) and the medial width of the pulmonary artery were increased in chronic hypoxic rats. Moreover, the expression of proliferating cell nuclear antigen (PCNA), Cyclin D, Cyclin E and Cyclin A, phospho‐CaMKII (p‐CaMKII) were induced by hypoxia. In vitro, we revealed that hypoxia promoted PASMCs viability, increased the expression of PCNA, Cyclin D, Cyclin E, Cyclin A p‐CaMKII, made more cells from G₀/G₁ phase to G₂/M + S phase, enhanced the microtubule formation, and increased [Ca²⁺]_i, which could be suppressed by Ruthenium Red, an inhibitor of TRPV3, and TRPV3 silencing has similar effects. Furthermore, the up‐regulated expression of PCNA, Cyclin D, Cyclin E and Cyclin A, the increased number of cells in G₂/M and S phase, and the enhanced activation and expression of PI3K and AKT proteins induced by hypoxia and in presence of carvacrol (an agonist of TRPV3), was significantly attenuated by incubation of LY 294002, a specific inhibitor for PI3K/AKT.

Conclusions

These findings suggest that TRPV3 is involved in hypoxia‐induced pulmonary vascular remodeling and promotes proliferation of PASMCs and the effect is, at least in part, mediated via the PI3K/AKT pathway.

     

Up‐regulated basigin‐2 in microglia induced by hypoxia promotes retinal angiogenesis

Retinal microglia cells contribute to vascular angiogenesis and vasculopathy induced by relative hypoxia. However, its concrete molecular mechanisms in shaping retinal angiogenesis have not been elucidated. Basigin, being involved in tumour neovasculogenesis, is explored to exert positive effects on retinal angiogenesis induced by microglia. Therefore, we set out to investigate the expression of basigin using a well‐characterized mouse model of oxygen‐induced retinopathy, which recapitulated hypoxia‐induced aberrant neovessel growth. Our results elucidate that basigin is overexpressed in microglia, which accumulating in retinal angiogenic sprouts. In vitro, conditioned media from microglia BV2 under hypoxia treatment increase migration and tube formation of retinal capillary endothelia cells, compared with media from normoxic condition. The angiogenic capacity of BV2 is inhibited after basigin knockdown by small interfering RNAs. A new molecular mechanism for high angiogenic capacity, whereby microglia cells release basigin via up‐regulation of PI3K‐AKT and IGF‐1 pathway to induce angiogenesis is unveiled. Collectively, our results demonstrate that basigin from hypoxic microglia plays a pivotal pro‐angiogenic role, providing new insights into microglia‐promoting retinal angiogenesis.     

MiR‐29 mediates TGFβ1‐induced extracellular matrix synthesis through activation of PI3K‐AKT pathway in human lung fibroblasts

TGFβ1 is very important in the synthesis and degradation of extracellular matrix, and also in the mediation of human lung fibroblasts proliferation, and miR‐29 plays an important role in this process. To explore the interactions of miR‐29 family members and TGFβ1, the effects of transforming growth factor TGFβ1 on the expression of miR‐29 and whether miR‐29 is involved in pro‐survival signaling pathways mediated by TGFβ1 were examined in human lung fibroblasts. Treatment of the human embryonic lung fibroblast cell line IMR90 with TGFβ1 caused a decrease in expression of miR‐29a/b/c by real‐time PCR analysis. TGFβ1 stimulation increased cell proliferation, colony formation and up‐regulated expression of COL1A1; transfecting with miR‐29a/b/c mimics reverse TGFβ1‐induced phenotype changes in IMR90 cells. Western blot analyses showed that TGFβ1 treatment unchanged total protein expression levels of PI3K or AKT, but the expression levels of p‐PI3K, p‐AKT, and COL1A1 were increased; and miR‐19a/b/c mimics interfering blocked phosphorylation of PI3K or AKT and decreased expression of COL1A1 after TGFβ1 treatment. The results indicate that TGFβ1 beta uses the PI3k‐Akt pathway in these embryonic fibroblasts and miR29 blocks this activation pathway. It indicates a novel biological function of the PI3K‐Akt pathway in IMR90. Elevated expression of miR‐29 may play an important role in the pathogenesis of diseases related to fibrogenic reactions in human lung fibroblasts. J. Cell. Biochem. 114: 1336–1342, 2013. © 2013 Wiley Periodicals, Inc.     

The involvement of histone methylation in osteoblastic differentiation of human periosteum‐derived cells cultured in vitro under hypoxic conditions

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia‐induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum‐derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis‐ and senescence‐associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red‐S‐positive mineralization, and mRNA expression of osteoblast‐related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein‐2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein‐2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.     

Abamectin induces apoptosis and autophagy by inhibiting reactive oxygen species‐mediated PI3K/AKT signaling in MGC803 cells

Abamectin (ABA) is one of the most widely used compounds in agriculture and veterinary medicine. However, the cytotoxicity of ABA in human gastric cells is utterly unknown. In this study, ABA suppressed the proliferation of MGC803 cells by arresting the cell cycle at the G0/G1‐phase. Moreover, ABA induced mitochondrial‐mediated apoptosis by inducing the loss of mitochondrial membrane potential, upregulation of Bax/Bcl‐2, and activation of caspase‐3. ABA significantly improved the LC3‐II/LC3‐I ratio and reduced P62 protein expression in a dose‐dependent manner. Through detection of the reactive oxygen species (ROS) levels, we found ABA induced the accumulation of intracellular ROS and then reduced PI3K/AKT signaling activation related to MGC803 cell apoptosis and autophagy. Our results indicate that ABA exerts cytotoxic effects on human MGC803 cells through apoptosis and autophagy by inhibiting ROS‐mediated PI3K/AKT signaling. Furthermore, ABA may be a potential risk to human gastric health.     

Overexpression of heat shock protein 70 enhanced mesenchymal stem cell treatment efficacy in phosgene‐induced acute lung injury

In our previous study, we have confirmed that in phosgene‐induced acute lung injury (ALI) rats, mesenchymal stem cells (MSCs) can treat the disease. Moreover, heat shock protein 70 (Hsp70) can be used as a protective protein, and Hsp70 upregulated drastically when exposed to stressful conditions. We aimed to assess that MSCs overexpressed Hsp70 could enhance the capacity of MSCs and have a good therapeutic effect on phosgene‐induced ALI. We transduced MSCs with Hsp70 and then we tested the function of the transduced MSCs. Sprague Dawley rats inhaled phosgene in a closed container for 5 minutes. The transduced MSCs and MSCs were administered via the trachea immediately. Rats in each group were killed at 6, 24, and 48 hours after exposure. Compared to MSCs, MSCs overexpressed Hsp70 enhanced MSCs viability, antiapoptotic ability, and migration ability, and these effects disappeared when using the phosphatidylinositol 3?kinase/protein kinase B (PI3K/AKT) pathway inhibitor. Furthermore, the results of pathological alterations improved. The lung wet‐to‐dry ratio declined. The lung injury index total protein content and total cells in bronchoalveolar lavage fluid (BALF) also declined. The level of tumor necrosis factor α declined and the level of interleukin‐10 improved in BALF and serum. MSCs overexpressed Hsp70 can enhance the capacity and efficacy of MSCs in the treatment of phosgene‐induced ALI and may be mediated through the PI3k/AKT signaling pathway. This article introduces a new approach to stem cell therapy for improving the efficacy of phosgene‐induced ALI.     

Bone marrow‐derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion‐induced lung injury

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM‐MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion‐induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM‐MSCs. Seventy mice were pre‐treated with BM‐MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro‐vascular endothelial cells (HPMVECs) were pre‐conditioned with BM‐MSCs by oxygen‐glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI‐treated mice, administration of BM‐MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD‐treated HPMVECs, co‐culture with BM‐MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM‐MSCs decreased the level of PI3K class I and p‐Akt while the expression of PI3K class III was increased. Finally, BM‐MSCs‐induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM‐MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM‐MSCs and will help to develop new cell‐based therapeutic strategies in lung injury.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

Linc01014 regulates gefitinib resistance in oesophagus cancer via EGFR‐PI3K‐AKT‐mTOR signalling pathway

This study aimed to explore the underlying mechanism of linc01014 in oesophagus cancer gefitinib resistance. Gefitinib‐resistant oesophagus squamous cell carcinoma (ESCC gefitinibR) cell lines were constructed by using different gefitinib treatment in FLO‐1, KYAE‐1, TE‐8 and TE‐5 cell lines and confirmed by MTS50 and proliferation assays. Expression of linc01014 was overexpressed/silenced in FLO‐1 cells followed by gefitinib treatment, and then, the apoptosis‐associated markers Bax and Bcl‐2, and PI3KCA in PI3K signalling pathway were determined using Western blotting. MST50 and morphology analyses showed that ESCC gefitinibR cell lines presented obvious gefitinib resistance than their parental ESCC cell lines. ESCC gefitinibR cell lines showed significantly higher proliferation abilities than their parental ESCC cell lines after treating with gefitinib. Overexpression of linc01014 significantly inhibited the apoptosis of FLO‐1 cells induced by gefitinib and silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib. Silencing linc01014 could significantly increase the gefitinib chemotherapy sensitivity of oesophagus cancer via PI3K‐AKT‐mTOR signalling pathway.     

Protosappanin‐A and oleanolic acid protect injured podocytes from apoptosis through inhibition of AKT‐mTOR signaling

Protosappanin‐A (PrA) and oleanolic acid (OA), which are important effective ingredients isolated from Caesalpinia sappan L., exhibit therapeutic potential in multiple diseases. This study focused on exploring the mechanisms of PrA and OA function in podocyte injury. An in vitro model of podocyte injury was induced by the sC5b‐9 complex and assays such as cell viability, apoptosis, immunofluorescence, quantitative real‐time polymerase chain reaction, and western blot were performed to further investigate the effects and mechanisms of PrA and OA in podocyte injury. The models of podocyte injury were verified to be successful as seen through significantly decreased levels of nephrin, podocin, and CD2AP and increased level of desmin. The sC5b‐9‐induced podocyte apoptosis was inhibited in injured podocytes treated with PrA and OA, accompanied by increased protein levels of nephrin, podocin, CD2AP, and Bcl2 and decreased levels of desmin and Bax. The p‐AKT/p‐mTOR levels were also reduced by treatment of PrA and OA while AKT/mTOR was unaltered. Further, the effects of PrA and OA on injured podocytes were similar to that of LY294002 (a PI3K‐AKT inhibitor). PrA and OA were also seen to inhibit podocyte apoptosis and p‐AKT/p‐mTOR levels induced by IGF‐1 (a PI3K‐AKT activator). Our data demonstrate that PrA and OA can protect podocytes from injury or apoptosis, which may occur through inhibition of the abnormal activation of AKT‐mTOR signaling.     

Circular RNA YAP1 acts as the sponge of microRNA‐21‐5p to secure HK‐2 cells from ischaemia/reperfusion‐induced injury

Circular RNA YAP1 (circYAP1) was reported to participate in progression of gastric cancer. However, the role of circYAP1 in acute kidney injury (AKI) remains obscure. We attempted to examine the effects of circYAP1 on ischaemia/reperfusion‐stimulated renal injury. AKI model was established by treating HK‐2 cells in ischaemia/reperfusion (I/R) environment. CircYAP1 expression in blood of AKI patients and I/R‐treated HK‐2 cells was evaluated via RT‐qPCR. CCK‐8, flow cytometry, ELISA and ROS assay were executed to test the impact of circYAP1 on cell viability, apoptosis, inflammatory cytokines and ROS generation. Bioinformatic analysis was executed to explore miRNA targets. The relativity between circYAP1 and miR‐21‐5p was verified by RT‐qPCR and luciferase assay. The functions of miR‐21‐5p in I/R‐triggered injury were reassessed. PI3K/AKT/mTOR pathway was detected by Western blot. Down‐regulated circYAP1 was observed in AKI blood samples and I/R‐treated HK‐2 cells. CircYAP1 overexpression expedited cell growth and weakened secretion of inflammatory factors and ROS generation in I/R‐disposed cells. Besides, we found circYAP1 could sponge to miR‐21‐5p. Interestingly, miR‐21‐5p overexpression overturned the repressive effects of circYAP1 on cell injury. Moreover, PI3K/AKT/mTOR pathway was activated by circYAP1 via inhibiting miR‐21‐5p. We demonstrated that circYAP1 activated PI3K/AKT/mTOR pathway and secured HK‐2 cells from I/R injury via sponging miR‐21‐5p.     

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL‐induced osteoclastogenesis

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL‐induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F‐actin ring formation and tartrate‐resistant acid phosphatase (TRAP) staining in dose‐dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK‐related trigger RANKL by phosphorylation JNK/ERK/p38‐MAPK, IκBα/p65‐NF‐κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K‐AKT‐NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL‐induced RANK‐TRAF6 association and RANKL‐related gene and protein markers such as NFATc1, Cathepsin K, MMP‐9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.     

Akt‐ and Erk‐mediated regulation of proliferation and differentiation during PDGFRβ‐induced MSC self‐renewal

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self‐renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet‐derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF‐BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl‐2′‐deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT‐PCR, and by long‐term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR‐β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E‐BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ‐induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self‐renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self‐renewal strategies.     

PKR activity is required for acute leukemic cell maintenance and growth: A role for PKR‐mediated phosphatase activity to regulate GSK‐3 phosphorylation

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p‐T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant‐negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time‐dependent augmentation of AKT S473 and GSK‐3α S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK‐3α was not dependent on PI3K activity. PKR inhibition augmented levels of p‐S473 AKT and p‐S21/9 GSK‐3α/β in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK‐3α or β phosphorylation in the presence of the AKT inhibitor, A443654. Pre‐treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2α, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2α phosphorylation. The effects of PKR inhibition on AKT and GSK‐3 phosphorylation were found to be, in part, PP2A‐dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK‐3. J. Cell. Physiol. 221: 232–241, 2009. © 2009 Wiley‐Liss, Inc