Dehydrocostuslactone suppresses angiogenesis in vitro and in vivo through inhibition of Akt/GSK-3β and mTOR signaling pathways

The traditional Chinese medicine component dehydrocostuslactone (DHC) isolated from Saussurea costus (Falc.) Lipschitz, has been shown to have anti-cancer activity. Angiogenesis is an essential process in the growth and progression of cancer. In this study, we demonstrated, for the first time, the anti-angiogenic mechanism of action of DHC to be via the induction of cell cycle progression at the G0/G1 phase due to abrogation of the Akt/glycogen synthase kinase-3β (GSK-3β)/cyclin D1 and mTOR signaling pathway. First, we demonstrated that DHC has an anti-angiogenic effect in the matrigel-plug nude mice model and an inhibitory effect on human umbilical vein endothelial cell (HUVEC) proliferation and capillary-like tube formation in vitro. DHC caused G0/G1 cell cycle arrest, which was associated with the down-regulation of cyclin D1 expression, leading to the suppression of retinoblastoma protein phosphorylation and subsequent inhibition of cyclin A and cdk2 expression. With respect to the molecular mechanisms underlying the DHC-induced cyclin D1 down-regulation, this study demonstrated that DHC significantly inhibits Akt expression, resulting in the suppression of GSK-3β phosphorylation and mTOR expression. These effects are capable of regulating cyclin D1 degradation, but they were significantly reversed by constitutively active myristoylated (myr)-Akt. Furthermore, the abrogation of tube formation induced by DHC was also reversed by overexpression of Akt. And the co-treatment with LiCl and DHC significantly reversed the growth inhibition induced by DHC. Taken together, our study has identified Akt/GSK-3β and mTOR as important targets of DHC and has thus highlighted its potential application in angiogenesis-related diseases, such as cancer.     

Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O⁻₂ signaling

To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2(-/-) mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 ± 0.04 to 0.62 ± 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ± 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.     

Poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer:DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10(3)-fold and 1.5±0.7×10(3)-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p?=?0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.     

Wnt/β-catenin pathway promotes acute lung injury induced by LPS through driving the Th17 response in mice

T helper cell 17 (Th17), one type of CD4⁺ T cell, plays an important role in regulating the acute lung injury (ALI) inflammatory response. Recent studies showed that Wnt/β-catenin pathway could modulate the differentiation and the function of CD4⁺ T cell. However, whether Wnt/β-catenin could regulate the differentiation and function of Th17 in the development and progress of ALI induced by lipopolysaccharide (LPS) is still unknown. To test this, we used dickkopf1 (Dkk-1) to block the Wnt/β-catenin pathway and LiCl to activate the Wnt/β-catenin pathway by instillation to the murine model of ALI. Our results revealed that activation of Wnt/β-catenin pathway significantly aggravated the LPS-induced lung inflammation. Meanwhile, we observed that activation of Wnt/β-catenin pathway promoted Th17 response by analyzing CD4⁺ T cells and the related cytokines secretions. Enhanced Th17 response was responsible for the further neutrophils infiltration and pro-inflammatory cytokines production. In addition, activation of Wnt/β-catenin pathway resulted in induced expression of retinoic acid related orphan receptor-γt (RORγt) via histone acetyltransferase p300. These data suggested that Wnt/β-catenin pathway might be a potential target to treat the LPS-induced inflammation in ALI.     

17β-estradiol modulates NGF and BDNF expression through ERβ mediated ERK signaling in cortical astrocytes

17β-estradiol is known to exert neurotrophic and neuroprotective effects through classical estrogen receptors [ERs], ERα and ERβ, on a variety of cell types either by genomic or non-genomic actions. The actions of estradiol on glial cells are important to maintain metabolic functions of the nervous system. Astrocytes are considered to be active participants in brain activity because of their ability to release growth factors, including neurotrophins. Present in vitro studies show that 17β-estradiol modulates NGF and BDNF expression in time-dependent manner and ERK acts as secondary messenger for estradiol’s action. 17β-estradiol is involved in survival of cortical astrocytes. In conclusion, this study indicates vital role of ERβ mediated ERK signalling for regulation of NGF and BDNF expression along with cell viability of cortical astrocytes which further confirms the role of ERs, particularly ERβ in glial cells’ functions and viability.     

β-Catenin mediates cyclic strain-stimulated cardiomyogenesis in mouse embryonic stem cells through ROS-dependent and integrin-mediated PI3K/Akt pathways

Wnt/β-catenin signaling regulates various cellular events involved in the proliferation and differentiation and these events are affected sensitively by applying to mechanical stimuli. However, the mechanisms by which mechanical force stimulates cardiomyogenesis are not extensively explored. In this study we investigated the cellular mechanisms by which β-catenin signaling regulates cardiac differentiation of strain-subjected embryonic stem (ES) cells. The application of cells to cyclic strain increased beating cardiomyocyte foci with the attendant increases of Cx 43 and Nkx 2.5 proteins. Anti-oxidants such as vitamin C or N-acetyl cysteine (NAC) blocked the strain-mediated increases of Cx 43, Nkx 2.5, and α5/β1 integrins. These anti-oxidants also suppressed the activation of phosphoinositide 3-kinase (PI3K) and Akt in cyclic strain-subjected cells. Western blot analysis revealed that PI3K is a critical downstream effector of β1 integrin signaling and mediates Cx 43 and Nkx 2.5 expression in cyclic strain-applied ES cells. Cyclic strain increased the expression of β-catenin and stimulated its nuclear translocation from the cytosol, which was prevented by anti-oxidant treatment. In addition, the application to cyclic strain increased mRNA expression of β-catenin target genes, Axin2 and c-myc, as well as the phosphorylation of glycogen synthase kinase-3β. Furthermore, the blockage of β-catenin by its specific siRNA transfection diminished the cellular levels of Cx 43 and Nkx 2.5 proteins and the number of beating cardiomyocyte foci. Collectively, these results suggest that β-catenin-mediated signaling is required for cyclic strain-stimulated cardiomyogenesis through ROS-dependent and integrin-mediated PI3K-Akt signaling cascades.     

Arctigenin prevents the progression of osteoarthritis by targeting PI3K/Akt/NF-κB axis: In vitro and in vivo studies

Osteoarthritis (OA), which is principally featured by progressive joint metabolic imbalance and subsequent degeneration of articular cartilage, is a common chronic joint disease. Arctigenin (ATG), a dietary phyto-oestrogen, has been described to have potent anti-inflammatory effects. Nevertheless, its protective effects on OA have not been clearly established. The target of our following study is to evaluate the protective effects of ATG on IL-1β–induced human OA chondrocytes and mouse OA model. Our results revealed that the ATG pre-treatment effectively decreases the level of pro-inflammatory mediators, such as prostaglandin E2 (PGE2), nitrous oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-6 and tumour necrosis factor alpha (TNF-α) in IL-1β–induced human chondrocytes. In addition, ATG protects against the degradation of extracellular matrix (ECM) under the stimulation of IL-1β and the possible mechanism might be connected with the inactivation of phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor-kappa B (NF-κB) axis. Furthermore, a powerful binding capacity between ATG and PI3K was also uncovered in our molecular docking research. Meanwhile, ATG may act as a protector on the mouse OA model. Collectively, all these findings suggest that ATG could be utilized as a promising therapeutic agent for the treatment of OA.     

Maltol prevents the progression of osteoarthritis by targeting PI3K/Akt/NF-κB pathway: In vitro and in vivo studies

Osteoarthritis (OA), a prevalent degenerative arthritis disease, principle characterized by the destruction of cartilage and associated with the inflammatory response. Maltol, a product formed during the processing of red ginseng (Panax ginseng, CA Meyer), has been reported to have the potential effect of anti-inflammatory. However, its specific mechanisms are not demonstrated. We investigated the protective effect of maltol in the progression of OA both in vitro and in vivo experiments. Human chondrocytes were pre-treated with maltol (0, 20, 40, 60 μM, 24 hours) and incubated with IL-1β (10 ng/mL, 24 hours) in vitro. Expression of PGE2, TNF-α and NO was measured by the ELISA and Griess reaction. The expression of iNOs, COX-2, aggrecan, ADAMTS-5, MMP-13, IκB-α, p65, P-AKT, AKT, PI3K and P-PI3K was analysed by Western blotting. The expression of collagen II and p65-active protein was detected by immunofluorescence. Moreover, the serious level of OA was evaluated by histological analysis in vivo. We identified that maltol could suppress the IL-1β-stimulated generation of PGE2 and NO. Besides, maltol not only suppressed the production of COX-2, iNOs, TNF-α, IL-6, ADAMTS-5, MMP-13, but also attenuated the degradation of collagen II and aggrecan. Furthermore, maltol remarkably suppressed the phosphorylation of PI3K/AKT and NF-κB induced by IL-1β in human OA chondrocytes. Moreover, maltol could block the cartilage destroy in OA mice in vivo. To date, all data indicate maltol is a potential therapeutic agent by inhibiting inflammatory response via the regulation of NF-κB signalling for OA.     

17β-Estradiol promotes cell proliferation in rat osteoarthritis model chondrocytes via PI3K/Akt pathway

Osteoarthritis (OA) is the most common cause of musculoskeletal pain and disability. The importance of chondrocytes in the pathogenesis of OA is unequivocal. 17β-estradiol (E2) has a potential protective effect against OA. However, the mechanism of E2 in OA chondrocytes remains unclear. In this study, we investigated the regulative effect of E2 on cell growth and the relationship between E2 and the PI3K/Akt pathway in rat OA model chondrocytes (pretreated with interleukin-1β). We found that E2 induced chondrocyte proliferation, and increased the expression level of Akt simultaneously, especially the expression level of P-Akt. Furthermore, the inhibition of P-Akt could block chondrocyte proliferation induced by E2. These results suggest that PI3K/Akt activation induced by E2 may be an important factor in the mechanism of E2 in cell proliferation in rat OA model chondrocytes, and help further understanding the role of E2 in OA progression.     

The cardioprotective effect of dihydromyricetin prevents ischemia–reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways

Reperfusion therapy is widely used to treat acute myocardial infarction (AMI). However, further injury to the heart induced by rapidly initiating reperfusion is often encountered in clinical practice. A lack of pharmacological strategies in clinics limits the prognosis of patients with myocardial ischemia-reperfusion injury (MIRI). Dihydromyricetin (DMY) is one of the most abundant components in vine tea, commonly known as the tender stems and leaves of Ampelopsis grossedentata. The aim of this study was to evaluate the cardioprotection of DMY against myocardial ischemia-reperfusion (I/R) injury and to further investigate the underlying mechanism. An I/R injury was induced by left anterior descending coronary artery occlusion in adult male rats in vivo and a hypoxia–reoxygenation (H/R) injury in H9c2 cardiomyocytes in vitro. We found that DMY pretreatment provided significant protection against I/R-induced injury, including enhanced antioxidant capacity and inhibited apoptosis in vivo and in vitro. This effect correlated with the activation of the PI3K/Akt and HIF-1α signaling pathways. Conversely, blocking Akt activation with the PI3K inhibitor LY294002 effectively suppressed the protective effects of DMY against I/R-induced injury. In addition, the PI3K inhibitor partially blocked the effects of DMY on the upregulation of Bcl-2, Bcl-xl, procaspase-3, -8, and -9 protein expression and the downregulation of HIF-1α, Bnip3, Bax, Cyt-c, cleaved caspase-3, -8, and -9 protein expression. Collectively, these results showed that DMY decreased the apoptosis and necrosis by I/R treatment, and PI3K/Akt and HIF-1α plays a crucial role in protection during this process. These observations indicate that DMY has the potential to exert cardioprotective effects against I/R injury and the results might be important for the clinical efficacy of AMI treatment.     

Resolvin D1 attenuates inflammation in lipopolysaccharide-induced acute lung injury through a process involving the PPARγ/NF-κB pathway

Background

Docosahexaenoic acid (DHA) and DHA-derived lipid mediators have recently been shown to possess anti-inflammatory and pro-resolving properties. In fact, DHA can down-regulate lipolysaccharide (LPS)-induced activation of NF-κB via a PPARγ-dependent pathway. We sought to investigate the effects of the novel DHA-derived mediator resolvin D1 (RvD1) on LPS-induced acute lung injury and to determine whether these effects occur via a PPARγ-dependent pathway.

Methods

BALB/c mice aged 6–8 weeks were randomly divided into seven groups: two control groups receiving saline or RvD1 (600 ng) without LPS; a control group receiving LPS only; an experimental group receiving RvD1 (300 ng) or RvD1 (600 ng), followed by LPS; a group receiving the PPARγ antagonist GW9662; and a group receiving GW9662, then RvD1 (600 ng) and finally LPS. LPS (50 μM) and saline were administered intratracheally. RvD1 was injected intravenously 24 h and 30 min before LPS, while GW9662 was injected intravenously 30 min before RvD1. Mice were killed at 6, 12, and 24 h. Samples of bronchoalveolar lavage fluid (BALF) were analyzed for cell counts and cytokine analysis. Lung tissues were collected for histology, Western blotting and electrophoretic mobility shift assays (EMSAs).

Results

At all three time points, groups receiving either dose of RvD1 followed by LPS had significantly lower total leukocyte counts and levels of TNF-α and IL-6 levels in BALF than did the group given only LPS. RvD1 markedly attenuated LPS-induced lung inflammation at 24 h, based on hematoxylin-eosin staining of histology sections. RvD1 activated PPARγ and suppressed IκBα degradation and NF-κB p65 nuclear translocation, based on Western blots and EMSAs. The PPARγ inhibitor GW9662 partially reversed RvD1-induced suppression of IκBα degradation and p65 nuclear translocation.

Conclusions

These results suggest that RvD1 may attenuate lung inflammation of LPS-induced acute lung injury by suppressing NF-κB activation through a mechanism partly dependent on PPARγ activation.     

Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages

Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to activate innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking, and here we show in macrophages that Rab31-GTP is additionally and specifically recruited to early-stage phagosomes. At phagocytic cups, Rab31 is first recruited during the phosphoinositide transition from PI(4,5)P₂ to PI(3,4,5)P₃, and it persists on PI(3)P-enriched phagosomes. During early phagocytosis, we find that Rab31 recruits the signaling adaptor APPL2. siRNA depletion of either Rab31 or APPL2 reduces FcγR-mediated phagocytosis. Mechanistically, this corresponds with a delay in the transition to PI(3,4,5)P₃ and phagocytic cup closure. APPL2 depletion also reduced PI3K/Akt signaling and enhanced p38 signaling from FcγR. We thus conclude that Rab31/APPL2 is required for key roles in phagocytosis and prosurvival responses of macrophages. Of interest, in terms of localization and function, this Rab31/APPL2 complex is distinct from the Rab5/APPL1 complex, which is also involved in phagocytosis and signaling.     

MicroRNA-181a suppresses norethisterone-promoted tumorigenesis of breast epithelial MCF10A cells through the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway

BackgroundResearch suggests that hormone replacement therapy may increase the risk of breast cancer, and progestins such as norethisterone (NET) play a key role in this phenomenon. We have demonstrated that microRNA-181a (miR-181a) suppresses NET-promoted breast cancer cell survival. Nonetheless, the effects of NET and miR-181a on the tumorigenesis of human breast epithelial cells have not yet been elaborated.MethodsAssays of cell viability, proliferation, migration, apoptosis, and colony formation were performed to investigate the pro-tumorigenesis effect of NET and the effects of miR-181a on human breast epithelial MCF10A cells. The expressions of cell-proliferation-related genes and apoptotic factors were analyzed by quantitative RT-PCR and Western blot in MCF10A cells treated with NET and miR-181a.ResultsNET significantly increased MCF10A cell viability, proliferation, migration, and colony formation, but reduced cellular apoptosis. In addition, NET increased the expression of progesterone receptor membrane component 1 (PGRMC1), EGFR, B-cell lymphoma 2, cyclin D1, and proliferating cell nuclear antigen, but decreased the expression of pro-apoptosis factors, such as Bax, caspase-7, and caspase-9. Overexpression of miR-181a strongly inhibited the effects of NET on MCF10A cells and abrogated NET-stimulated PGRMC1, EGFR, and mTOR expression.ConclusionsActivation of the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway is the primary mechanism underlying the pro-tumorigenesis effects of NET on human breast epithelial MCF10A cells. Additionally, miR-181a can suppress the effects of NET on these cells. These data suggest a therapeutic potential for miR-181a in reducing or preventing the risk of breast cancer in hormone replacement therapy using NET.     

Inhibition of PI3K/Akt/NF-κB signaling with leonurine for ameliorating the progression of osteoarthritis: In vitro and in vivo studies

Osteoarthritis (OA) is characterized as the degeneration and destruction of articular cartilage. In recent decades, leonurine (LN), the main active component in medical and edible dual purpose plant Herba Leonuri, has been shown associated with potent anti-inflammatory effects in several diseases. In the current study, we examined the protective effects of LN in the inhibition of OA development as well as its underlying mechanism both in vitro and in vivo experiments. In vitro, interleukin-1 beta (IL-1β) induced over-production of prostaglandin E2, nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6 and tumor necrosis factor alpha were all inhibited significantly by the pretreatment of LN at a dose-dependent manner (5, 10, and 20 µM). Moreover, the expression of thrombospondin motifs 5 (ADAMTS5) and metalloproteinase 13 (MMP13) was downregulated by LN. All these changes led to the IL-1β induced degradation of extracellular matrix. Mechanistically, the LN suppressed IL-1β induced activation of the PI3K/Akt/NF-κB signaling pathway cascades. Meanwhile, it was also demonstrated in our molecular docking studies that LN had strong binding abilities to PI3K. In addition, LN was observed exerting protective effects in a surgical induced model of OA. To sum up, this study indicated LN could be applied as a promising therapeutic agent in the treatment of OA.     

Combination of valproic acid and ATRA restores RARβ2 expression and induces differentiation in cervical cancer through the PI3K/Akt pathway

Epigenetic silencing of the tumor suppressor gene, RARβ2, through histone deacetylation has been established as an important process of cervical carcinogenesis. This pivotal role has led to the suggestion that a combination of retinoids selective for RARβ2 with histone deacetylase (HDAC) inhibitors may have therapeutic potential. Valproic acid (VPA), a HDAC inhibitor, has a critical role in the regulation of gene expression through histone acetylation and causes transformed cells to undergo growth arrest, differentiation, and apoptosis. Therefore, we hypothesized that the combination of VPA and ATRA could restore RARβ2 expression, thus resulting in enhanced anti-neoplastic activity in cervical cancer. Here, we show that VPA combined with ATRA led to hyperacetylation of histone H3 and a significant alteration of gene expression in cervical cancer cells, including RARβ2 gene expression, which was upregulated 50- to 90-fold. The combination therapy effectively inhibited the growth of cervical cancer cells more than the single agent treatment both in vitro and in vivo. The additive effects were associated with a significant upregulation of p21(CIP1) and p53 as well as a pronounced decrease in p-Stat3. Furthermore, the combined treatment led to cell cycle arrest predominantly at the G1 phase, and it preferentially induced cell differentiation rather than apoptosis in cervical cancer cells. The differentiation program was determined by the presence of E-cadherinmediated adhesion and activation of the PI3K/Akt pathway. Taken together, these results provide new insight into the mechanisms of enhanced antitumor activity of the HDAC inhibitor and ATRA regimen, thus offering a new therapeutic strategy for cervical cancer patients.     

Mechanotransduction activates α₅β₁ integrin and PI3K/Akt signaling pathways in mandibular osteoblasts

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and β-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α(5)β(1) integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α(5)β(1) integrin to upregulate expression of the proteins β-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α(5)β(1) integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α(5)β(1) integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.     

Integrin alpha x stimulates cancer angiogenesis through PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression in blood vessel endothelial cells

Integrin alpha x (ITGAX), a member of the integrin family, usually serves as a receptor of the extracellular matrix. Recently, accumulating evidence suggests that ITGAX may be involved in angiogenesis in dendritic cells. Herein, we report a direct role of ITGAX in angiogenesis during tumor development. Overexpression of ITGAX in human umbilical vein endothelial cells (HUVECs) enhanced their proliferation, migration, and tube formation and promoted xenograft ovarian tumor angiogenesis and growth. Further study showed that overexpression of ITGAX activated the PI3k/Akt pathway, leading to the enhanced expression of c-Myc, vascular endothelial growth factor-A (VEGF-A), and VEGF receptor 2 (VEGFR2), whereas, the treatment of cells with PI3K inhibitor diminished these effects. Besides, c-Myc was observed to bind to the VEGF-A promoter. By Co-Immunoprecipitation (Co-IP) assay, we manifested the interaction between ITGAX and VEGFR2 or the phosphorylated VEGFR2. Immunostaining of human ovarian cancer specimens suggested that endothelial cells of micro–blood vessels displayed strong expression of VEGF-A, c-Myc, VEGFR2, and the PI3K signaling molecules. Also, overexpression of ITGAX in HUVECs could stimulate the spheroid formation of ovarian cancer cells. Our study uncovered that ITGAX stimulates angiogenesis through the PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression during cancer development.