Sox17 Deficiency Promotes Pulmonary Arterial Hypertension via HGF/c-Met Signaling.

BACKGROUND: In large-scale genomic studies, Sox17, an endothelial-specific transcription factor, has been suggested as a putative causal gene of pulmonary arterial hypertension (PAH); however, its role and molecular mechanisms remain to be elucidated. We investigated the functional impacts and acting mechanisms of impaired Sox17 (SRY-related HMG-box17) pathway in PAH and explored its potential as a therapeutic target. METHODS: In adult mice, Sox17 deletion in pulmonary endothelial cells (ECs) induced PAH under hypoxia with high penetrance and severity, but not under normoxia. RESULTS: Key features of PAH, such as hypermuscularization, EC hyperplasia, and inflammation in lung arterioles, right ventricular hypertrophy, and elevated pulmonary arterial pressure, persisted even after long rest in normoxia. Mechanistically, transcriptomic profiling predicted that the combination of Sox17 deficiency and hypoxia activated c-Met signaling in lung ECs. HGF (hepatocyte grow factor), a ligand of c-Met, was upregulated in Sox17-deficient lung ECs. Pharmacologic inhibition of HGF/c-Met signaling attenuated and reversed the features of PAH in both preventive and therapeutic settings. Similar to findings in animal models, Sox17 levels in lung ECs were repressed in 26.7% of PAH patients (4 of 15), while those were robust in all 14 non-PAH controls. HGF levels in pulmonary arterioles were increased in 86.7% of patients with PAH (13 of 15), but none of the controls showed that pattern. CONCLUSIONS: The downregulation of Sox17 levels in pulmonary arterioles increases the susceptibility to PAH, particularly when exposed to hypoxia. Our findings suggest the reactive upregulation of HGF/c-Met signaling as a novel druggable target for PAH treatment.

Akt1 is involved in renal fibrosis and tubular apoptosis in a murine model of acute kidney injury-to-chronic kidney disease transition.

Maladaptive repair after acute kidney injury (AKI) can predispose patients to chronic kidney disease (CKD). However, the molecular mechanism underlying the AKI-to-CKD transition remains unclear. The Akt signaling pathway has been reported to be involved in the pathological processes of both AKI and CKD. In this study, we investigated the role of Akt1 in a murine model of the AKI-to-CKD transition. Wild-type (WT) and Akt1-/- mice were subjected to unilateral ischemia-reperfusion injury (UIRI), with their kidneys harvested after two days and two, four, and six weeks after UIRI. The dynamic changes in tubulointerstitial fibrosis, markers of tubular epithelial-mesenchymal transition (EMT), and tubular apoptosis were investigated. Akt1 of the three Akt isoforms was activated during the AKI-to-CKD transition. After UIRI, tubulointerstitial fibrosis and tubular EMT were significantly increased in WT mice, but were attenuated in Akt1-/- mice. The expression of the transforming growth factor (TGF)-ß1/Smad was increased in both WT and Akt1-/- mice, but was not different between the two groups. The levels of phosphorylated glycogen synthase kinase (GSK)-3ß, Snail, and ß-catenin in the Akt1-/- mice were lower than those in the WT mice. The number of apoptotic tubular cells and the expression of cleaved caspase-3/Bax were both lower in Akt1-/- mice than in WT mice. Genetic deletion of Akt1 was associated with attenuation of tubulointerstitial fibrosis, tubular EMT, and tubular apoptosis during the AKI-to-CKD transition. These findings were associated with TGF-ß1/Akt1/GSK-3ß/(Snail and ß-catenin) signaling independent of TGF-ß1/Smad signaling. Thus, Akt1 signaling could serve as a potential therapeutic target for inhibiting the AKI-to-CKD transition.

mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation.

mTORC1 plays a key role in autophagy as a negative regulator. The currently known targets of mTORC1 in the autophagy pathway mainly function at early stages of autophagosome formation. Here, we identify that mTORC1 inhibits later stages of autophagy by phosphorylating UVRAG. Under nutrient-enriched conditions, mTORC1 binds and phosphorylates UVRAG. The phosphorylation positively regulates the association of UVRAG with RUBICON, thereby enhancing the antagonizing effect of RUBICON on UVRAG-mediated autophagosome maturation. Upon dephosphorylation, UVRAG is released from RUBICON to interact with the HOPS complex, a component for the late endosome and lysosome fusion machinery, and enhances autophagosome and endosome maturation. Consequently, the dephosphorylation of UVRAG facilitates the lysosomal degradation of epidermal growth factor receptor (EGFR), reduces EGFR signaling, and suppresses cancer cell proliferation and tumor growth. These results demonstrate that mTORC1 engages in late stages of autophagy and endosome maturation, defining a broader range of mTORC1 functions in the membrane-associated processes.

Regulation of Epithelial-Mesenchymal Transition of A549 Cells by Prostaglandin D2.

BACKGROUND/AIMS: Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. METHODS: A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-ß1 and TGFß type I receptor (TGFßRI), and protein expression was assessed by immunoblotting and immunofluorescence. RESULTS: Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-ß1 expression, and silencing of TGF-ß1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFßRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. CONCLUSION: Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

Akt1 is involved in tubular apoptosis and inflammatory response during renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (IRI) is one of the major causes of acute kidney injury (AKI). Although Akt is involved in renal IRI, it is unclear as to which Akt isoform plays an important role in renal IRI. In this study, we investigated the role of Akt1 in renal IRI. We subjected the C57BL/6 male mice to unilateral IRI with contralateral nephrectomy. Two days after IRI, IRI-kidneys were harvested. The mice were divided into four groups: wild type (WT) IRI, Akt1-/- IRI, WT sham, and Akt1-/- sham. We found that Akt1, not Akt2 or Akt3, was markedly activated in WT IRI than in WT sham mice. The histologic damage score and serum creatinine level significantly increased in WT IRI mice, the increase being the highest in Akt1-/- IRI mice. The number of TdT-mediated dUTP nick-end labeling (TUNEL)-positive tubular cells and expression of cleaved caspase-3/Bax were higher in Akt1-/- IRI mice than in WT IRI mice. The expression of Bcl-2 was lower in Akt1-/- IRI mice than in WT IRI mice. The expression of tumor necrosis factor-α/interleukin-6/interleukin-1ß and number of F4/80-positive macrophages were markedly higher in Akt1-/- IRI than in WT IRI mice. The expression of phosphorylated nuclear factor-κB p65 was also higher in Akt1-/- IRI mice than in WT IRI mice. Our results show that Akt1 deletion exacerbates kidney damage as it increases tubular apoptosis and inflammatory response during renal IRI. Akt1 could be a potential therapeutic target for developing treatments against IRI-induced AKI.

The effect of desflurane on retinal angiogenesis in a mouse model of oxygen-induced retinopathy.

PURPOSE: Retinopathy of prematurity (ROP) is an ocular disorder that primarily occurs in premature infants and is the most common cause of vision impairment. This study examined the effect of desflurane on angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). METHODS: Mice were randomly allocated to the control (C), ROP control (Rc), or ROP with desflurane exposure (Rd) group. To induce ROP, 7-day-old mice were exposed to 75% oxygen in a chamber for 5 days [postnatal days (P) 7-12], and thereafter returned to room air. Age-matched mice exposed to room air formed the C group. The Rd group was exposed to 8% desflurane for 2 h on P12, P13, and P14 with 40% oxygen. To observe changes in angiogenesis of the retina, mice were sacrificed at P16. RESULTS: The ratio of avascular area/total retinal area was not changed significantly in the Rd group, compared to the Rc group. The expression of endothelial growth factor A (VEGF-A) and hypoxia inducible factor-1α (HIF-1α) in the Rd group and Rc group was not significantly different. CONCLUSIONS: Desflurane does not have a significant influence on retinal angiogenesis via HIF-1α and VEGF-A expression in the OIR mouse model. However, these findings are not directly applicable to premature infants, and it is thus necessary to perform further studies to determine the effect of desflurane on angiogenesis.

7-Oxygenated cholesterol molecules differentially affect the expression of zonula occludens-1 in vascular smooth muscle cells and monocyte/macrophage cells.

To investigate the effects of 7-oxygenated cholesterol molecules on the expression of tight junction proteins, we examined the outcomes effects of 7-ketocholesterol (7K), 7α-hydroxycholesterol (7αOHChol) and 7ß-hydroxycholesterol (7ßOHChol) on the expression of the tight-junction protein zonula occludens-1 (ZO-1) using vascular cells. Vascular smooth muscle cells (VSMCs) constitutively express ZO-1, and this expression remained unaffected in the presence of cholesterol. However, the level of ZO-1 protein decreased after exposure to 7K and, to a lesser extent, 7αOHChol and 7ßOHChol. ZO-1 was translocated to the nucleus following treatment with 7K; this translocation was inhibited by z-VAD-fmk, a pan-caspase inhibitor. ZO-1 protein was found to disintegrate in the aorta of ApoE knockout mice fed a high cholesterol diet, whereas it remained intact in the wild-type control. THP-1 monocyte/macrophage cells, which show no expression of ZO-1, were not influenced by treatment with cholesterol, 7K, and 7ßOHChol. However, the treatment of THP-1 cells with 7αOHChol resulted in ZO-1 expression, which largely remained localized on the cytoplasmic membrane. These results indicate the varying effects of 7-oxygenated cholesterol molecules on the expression and localization of ZO-1 depending on cell types, and suggest the contribution of 7-oxygeneted cholesterol molecules to the structural alteration of tight junctions.

The two isoforms of matrix metalloproteinase- 2 have distinct renal spatial and temporal distributions in murine models of types 1 and 2 diabetes mellitus.

BACKGROUND: We recently reported on the enhanced tubular expression of two discrete isoforms of the MMP-2 (full length and N-terminal truncated, FL-MMP-2, NTT-MMP-2) in a murine model and human diabetic kidneys. In the present study, we examined in more detail the temporal and spatial distributions of MMP-2 isoform expression in murine models of Type 1 and Type 2 diabetes mellitus. METHODS: Diabetic models were streptozotocin (STZ)-induced diabetes (Type 1 diabetes mellitus) and db/db mice (Type 2 diabetes mellitus). We quantified the abundance of two isoforms of MMP-2 transcripts by qPCR. A spatial distribution of two isoforms of MMP-2 was analyzed semi-quantitatively according to time after injection of STZ and with increasing age of db/db mice. Furthermore, immunohistochemistry for nitrotyrosine was performed to examine a potential association between oxidative stress and MMP-2 isoform expression. RESULTS: Both isoforms of MMP-2 were upregulated in whole kidneys from STZ and db/db mice. In the case of FL-MMP-2, mRNA levels significantly increased at 12 and 24 weeks in STZ mice, while the isoform expression was significantly increased only at 16 weeks, in the db/db mice. FL-MMP-2 protein levels increased in the cortices and outer medullae of both STZ and db/db mice as a function of the duration of diabetes. For NTT-MMP-2, mRNA levels increased earlier at 4 weeks in STZ mice and at 10 weeks of age in db/db mice. The expression of NTT-MMP-2 also increased, primarily in the cortices of STZ and db/db mice, as a function of the duration of diabetes. Quantitatively, these findings were consistent with the qPCR results in the case of NTT-MMP-2, respectively (STZ 24 weeks, 3.24 ± 3.70 fold; 16 weeks db/db, 4.49 ± 0.55 fold). In addition, nitrotyrosine was expressed primarily in cortex as compared to medulla as a function of the duration of diabetes similar to NTT-MMP-2 expression. CONCLUSIONS: Two isoforms of MMP-2 are highly inducible in two diabetic murine models and become more abundant as a function of time. As the expression patterns were not the same in the two isoforms of MMP-2, it is possible that each isoform has a discrete role in the development of diabetic renal injury.

The effect of sevoflurane on retinal angiogenesis in a mouse model of oxygen-induced retinopathy.

BACKGROUND: Sevoflurane is commonly used in general anesthesia for premature neonates. The main mechanism of retinopathy of prematurity (ROP) is increased levels of vascular endothelial growth factor (VEGF). For the investigation of sevoflurane's effect on angiogenesis, the angiogenesis and VEGF expression in the retina were measured after administering sevoflurane in an oxygen-induced retinopathy mice model. MATERIALS AND METHODS: The mice were divided into the normoxic group (Nc and Ns group; n = 6) and the ROP group (C, Rc, and Rs group; n = 6). Rc group were exposed to 75% oxygen for 5 days beginning on postnatal day (P) 7, and then returned to room air. Age-matched mice in the C group were exposed to room air. To observe angiogenesis of the retina, the mice were sacrificed on P16. The Rs group was exposed to 2 vol% sevoflurane for 2 h on P12, P13, and P14 with 40% oxygen. RESULTS: The angiogenic area and the spreading distance of vessels on P4 were statistically decreased in the Ns group, compared to the Nc group. The avascular area on P16 was significantly increased and the expression of VEGF was suppressed in the Rs group compared to the Rc group. CONCLUSIONS: Sevoflurane can inhibit retinal angiogenesis via suppressing VEGF expression in an OIR mice model with exposure to relative hypoxia. Nevertheless, it is still difficult to apply the results of this study immediately to humans because of the heterogeneity of responses to sevoflurane.

Transcript Levels of Androgen Receptor Variant 7 and Ubiquitin-Conjugating Enzyme 2C in Hormone Sensitive Prostate Cancer and Castration-Resistant Prostate Cancer.

PURPOSE: This study is designed to identify the androgen receptor variant 7 (AR-V7) status, clinical significance of AR-V7 in hormone sensitive prostate cancer (HSPC). Then, we evaluated AR-V7 and changes of its target gene, ubiquitin-conjugating enzyme E2C (UBE2C) which is an anaphase-promoting complex/cyclosome (APC/C)-specific ubiquitin-conjugating enzyme, in castration-resistant prostate cancer (CRPC) in serial tumor biopsies from patients receiving androgen deprivation therapy. METHODS: We used RT-PCR and Q-PCR assay to evaluate AR-V7, androgen receptor full length (AR-FL), and UBE2C in tumor biopsies from patients with HSPC and CRPC. We examined associations between mRNA expression of AR-V7 and clinicopathologic factors. Furthermore, to identify other potential genes involved in the development of CRPC, RNA sequencing was conducted, using paired prostate cancer (PCa) tissues obtained immediately prior to treatment and at the time of therapeutic resistance. RESULTS: A total of 13 HSPC patients and three CRPC patients were enrolled. Neither a high Gleason score (score of 8 and 9) nor a high risk of PCa (a high risk of locally advanced PCa according to NCCN guidelines) was correlated with mRNA expression of AR-V7 in HSPC (P = 0.153 and P = 0.215). The mRNA expression of AR-FL, but not AR-V7, was significantly associated with the mRNA expression of UBE2C level in HSPC (P = 0.007). However, increased expression of AR-V7, not AR-FL, paralleled increased expression of UBE2C in the CRPC specimens (P = 0.03). AR-V7 expression status before ADT was likely related to shorter CRPC development in patients treating ADT. The result of the RNA-sequencing analysis using serial samples from the same patient before and after castration demonstrated an increased level of the PI3K regulatory subunit 1 (P = 0.018). CONCLUSION: Our study revealed the role of UBE2C as a marker of the androgen signaling pathway in PCa. Differential gene expression analysis using serial samples from the same patient before and after castration revealed potential genes and pathways involved in development of CRPC. Further studies are needed to determine whether these genes and pathways are potential therapeutic target for CRPC. Prostate 77:60-71, 2017. © 2016 Wiley Periodicals, Inc.

5-Lipoxygenase in monocytes emerges as a therapeutic target for intimal hyperplasia in a murine wire-injured femoral artery.

Given the importance of leukotrienes in vascular inflammation induced by local tissue injury, this study investigated the role for 5-lipoxygenase (5-LO) in monocytes in the development of intimal hyperplasia. As a mechanistic study, the importance of monocyte 5-LO in monocyte-macrophage differentiation with subsequent infiltration in neointima was evaluated. In a mouse model of wire-injured femoral artery, intimal hyperplasia started as early as 2wks after injury, and luminal area and blood flow were reduced due to increased neointima formation. Time-dependent increases in macrophage infiltration were observed in neointima and showed a positive relationship with neointima volume. In 5-LO-deficient (KO) mice or wild-type (WT) mice treated with an inhibitor of 5-LO activating protein (MK886, 1 and 10mg/kg), intimal hyperplasia and macrophage infiltration into neointima were reduced, but monocyte adhesion to injured luminal surface was not inhibited, which suggested 5-LO participates in monocyte-macrophage differentiation. In an in vitro study, monocyte-macrophage differentiation was found to be increased by high mobility group box 1 protein (HMGB1), but this effect was attenuated in cells isolated from 5-LO-KO mice. Furthermore, macrophage infiltration and intimal hyperplasia were more prominent in 5-LO-KO mice transplanted with monocytes from WT mice than in 5-LO-KO mice transplanted with monocytes from 5-LO-KO mice. Taken together, it was suggested that 5-LO in monocytes played a pivotal role in monocyte-macrophage differentiation and subsequent infiltration of macrophage in neointima, leading to vascular remodeling after vascular injury.

Fibroblast Growth Factor 12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. APPROACH AND RESULTS: Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK (mitogen-activated protein kinase) pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. CONCLUSIONS: In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis.

Regulation of vascular smooth muscle phenotype by cross-regulation of krüppel-like factors.

Regulation of vascular smooth muscle cell (VSMC) phenotype plays an essential role in many cardiovascular diseases. In the present study, we provide evidence that krüppel-like factor 8 (KLF8) is essential for tumor necrosis factor α (TNFα)-induced phenotypic conversion of VSMC obtained from thoracic aorta from 4-week-old SD rats. Stimulation of the contractile phenotype of VSMCs with TNFα significantly reduced the VSMC marker gene expression and KLF8. The gene expression of KLF8 was blocked by TNFα stimulation in an ERK-dependent manner. The promoter region of KLF8 contained putative Sp1, KLF4, and NFκB binding sites. Myocardin significantly enhanced the promoter activity of KLF4 and KLF8. The ectopic expression of KLF4 strongly enhanced the promoter activity of KLF8. Moreover, silencing of Akt1 significantly attenuated the promoter activity of KLF8; conversely, the overexpression of Akt1 significantly enhanced the promoter activity of KLF8. The promoter activity of SMA, SM22α, and KLF8 was significantly elevated in the contractile phenotype of VSMCs. The ectopic expression of KLF8 markedly enhanced the expression of SMA and SM22α concomitant with morphological changes. The overexpression of KLF8 stimulated the promoter activity of SMA. Stimulation of VSMCs with TNFα enhanced the expression of KLF5, and the promoter activity of KLF5 was markedly suppressed by KLF8 ectopic expression. Finally, the overexpression of KLF5 suppressed the promoter activity of SMA and SM22α, thereby reduced the contractility in response to the stimulation of angiotensin II. These results suggest that cross-regulation of KLF family of transcription factors plays an essential role in the VSMC phenotype.

CTCF-mediated Chromatin Loop for the Posterior Hoxc Gene Expression in MEF Cells.

Modulation of chromatin structure has been proposed as a molecular mechanism underlying the spatiotemporal collinear expression of Hox genes during development. CCCTC-binding factor (CTCF)-mediated chromatin organization is now recognized as a crucial epigenetic mechanism for transcriptional regulation. Thus, we examined whether CTCF-mediated chromosomal conformation is involved in Hoxc gene expression by comparing wild-type mouse embryonic fibroblast (MEF) cells expressing anterior Hoxc genes with Akt1 null MEFs expressing anterior as well as posterior Hoxc genes. We found that CTCF binding between Hoxc11 and -c12 is important for CTCF-mediated chromosomal loop formation and concomitant posterior Hoxc gene expression. Hypomethylation at this site increased CTCF binding and recapitulated the chromosomal conformation and posterior Hoxc gene expression patterns observed in Akt1 null MEFs. From this work we found that CTCF at the C12|11 does not function as a barrier/boundary, instead let the posterior Hoxc genes switch their interaction from inactive centromeric to active telomeric genomic niche, and concomitant posterior Hoxc gene expression. Although it is not clear whether CTCF affects Hoxc gene expression solely through its looping activity, CTCF-mediated chromatin structural modulation could be an another tier of Hox gene regulation during development. © 2016 IUBMB Life, 68(6):436-444, 2016.

Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma.

UNLABELLED: Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling up-regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL-6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL-6, with a concomitant decrease of hypoxia-inducible factor 1 alpha (HIF-1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF-κB) p65 subunit to positively regulate the transcription of HIF-1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF-1α and CD133 expression were not observed in Toll-like receptor 4/IL-6 double-knockout mice. Long-term silencing of CD133 by a lentiviral-based approach inhibited cancer cell-cycle progression and suppressed in vivo tumorigenicity by down-regulating expression of cytokinesis-related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF-1α proteins. CONCLUSION: IL-6/STAT3 signaling induces expression of CD133 through functional cooperation with NF-κB and HIF-1α during liver carcinogenesis. Targeting STAT3-mediated CD133 up-regulation may represent a novel, effective treatment by eradicating the liver tumor microenvironment.

Probucol inhibits LPS-induced microglia activation and ameliorates brain ischemic injury in normal and hyperlipidemic mice.

AIM: Increasing evidence suggests that probucol, a lipid-lowering agent with anti-oxidant activities, may be useful for the treatment of ischemic stroke with hyperlipidemia via reduction in cholesterol and neuroinflammation. In this study we examined whether probucol could protect against brain ischemic injury via anti-neuroinflammatory action in normal and hyperlipidemic mice. METHODS: Primary mouse microglia and murine BV2 microglia were exposed to lipopolysaccharide (LPS) for 3 h, and the release NO, PGE2, IL-1ß and IL-6, as well as the changes in NF-κB, MAPK and AP-1 signaling pathways were assessed. ApoE KO mice were fed a high-fat diet containing 0.004%, 0.02%, 0.1% (wt/wt) probucol for 10 weeks, whereas normal C57BL/6J mice received probucol (3, 10, 30 mg·kg(-1)·d(-1), po) for 4 d. Then all the mice were subjected to focal cerebral ischemia through middle cerebral artery occlusion (MCAO). The neurological deficits were scored 24 h after the surgery, and then brains were removed for measuring the cerebral infarct size and the production of pro-inflammatory mediators. RESULTS: In LPS-treated BV2 cells and primary microglial cells, pretreatment with probucol (1, 5, 10 µmol/L) dose-dependently inhibited the release of NO, PGE2, IL-1ß and IL-6, which occurred at the transcription levels. Furthermore, the inhibitory actions of probucol were associated with the downregulation of the NF-κB, MAPK and AP-1 signaling pathways. In the normal mice with MCAO, pre-administration of probucol dose-dependently decreased the infarct volume and improved neurological function. These effects were accompanied by the decreased production of pro-inflammatory mediators (iNOS, COX-2, IL-1, IL-6). In ApoE KO mice fed a high-fat diet, pre-administration of 0.1% probucol significantly reduced the infarct volume, improved the neurological deficits following MCAO, and decreased the total- and LDL-cholesterol levels. CONCLUSION: Probucol inhibits LPS-induced microglia activation and ameliorates cerebral ischemic injury in normal and hyperlipidemic mice via its anti-neuroinflammatory actions, suggesting that probucol has potential for the treatment of patients with or at risk for ischemic stroke and hyperlipidemia.

Akt3 knockdown induces mitochondrial dysfunction in human cancer cells.

Akt/PKB plays a pivotal role in cell proliferation and survival. However, the isotype-specific roles of Akt in mitochondrial function have not been fully addressed. In this study, we explored the role of Akt in mitochondrial function after stable knockdown of the Akt isoforms in EJ human bladder cancer cells. We found that the mitochondrial mass was significantly increased in the Akt1- and Akt3-knockdown cells, and this increase was accompanied by an increase in TFAM and NRF1. Akt2 knockdown did not cause a similar effect. Interestingly, Akt3 knockdown also led to severe structural defects in the mitochondria, an increase in doxorubicin-induced senescence, and impairment of cell proliferation in galactose medium. Consistent with these observations, the mitochondrial oxygen consumption rate was significantly reduced in the Akt3-knockdown cells. An Akt3 deficiency-induced decrease in mitochondrial respiration was also observed in A549 lung cancer cells. Collectively, these results suggest that the Akt isoforms play distinct roles in mitochondrial function and that Akt3 is critical for proper mitochondrial respiration in human cancer cells.

Lnk is an important modulator of insulin-like growth factor-1/Akt/peroxisome proliferator-activated receptor-gamma axis during adipogenesis of mesenchymal stem cells.

Adipogenic differentiation of mesenchymal stem cells (MSCs) is critical for metabolic homeostasis and nutrient signaling during development. However, limited information is available on the pivotal modulators of adipogenic differentiation of MSCs. Adaptor protein Lnk (Src homology 2B3 [SH2B3]), which belongs to a family of SH2-containing proteins, modulates the bioactivities of different stem cells, including hematopoietic stem cells and endothelial progenitor cells. In this study, we investigated whether an interaction between insulin-like growth factor-1 receptor (IGF-1R) and Lnk regulated IGF-1-induced adipogenic differentiation of MSCs. We found that wild-type MSCs showed greater adipogenic differentiation potential than Lnk (-/-) MSCs. An ex vivo adipogenic differentiation assay showed that Lnk (-/-) MSCs had decreased adipogenic differentiation potential compared with wild-type MSCs. Interestingly, we found that Lnk formed a complex with IGF-1R and that IGF-1 induced the dissociation of this complex. In addition, we observed that IGF-1-induced increase in the phosphorylation of Akt and mammalian target of rapamycin was triggered by the dissociation of the IGF-1R-Lnk complex. Expression levels of a pivotal transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ) and its adipogenic target genes (LPL and FABP4) significantly decreased in Lnk (-/-) MSCs. These results suggested that Lnk adaptor protein regulated the adipogenesis of MSCs through the IGF-1/Akt/PPAR-γ pathway.

Regulation of retinal angiogenesis by endothelial nitric oxide synthase signaling pathway.

Angiogenesis plays an essential role in embryo development, tissue repair, inflammatory diseases, and tumor growth. In the present study, we showed that endothelial nitric oxide synthase (eNOS) regulates retinal angiogenesis. Mice that lack eNOS showed growth retardation, and retinal vessel development was significantly delayed. In addition, the number of tip cells and filopodia length were significantly reduced in mice lacking eNOS. Retinal endothelial cell proliferation was significantly blocked in mice lacking eNOS, and EMG-2-induced endothelial cell sprouting was significantly reduced in aortic vessels isolated from eNOS-deficient mice. Finally, pericyte recruitment to endothelial cells and vascular smooth muscle cell coverage to blood vessels were attenuated in mice lacking eNOS. Taken together, we suggest that the endothelial cell function and blood vessel maturation are regulated by eNOS during retinal angiogenesis.

Mechanical stretch enhances the expression and activity of osteopontin and MMP-2 via the Akt1/AP-1 pathways in VSMC.

Hemodynamic forces causing mechanical stretch (MS) of vascular smooth muscle cells (VSMC) play an important role in vascular remodeling, but the underlying mechanism involved is not fully understood. Thus, this study investigated whether osteopontin (OPN) expression in VSMC was induced by MS, and identified the intracellular signaling pathways involved in OPN production. The plasma level of OPN and its expression in aortic tissue were increased in various animal models of hypertension including spontaneous hypertensive rats and hypertensive mice induced by angiotensin II or L-NAME. When aortic VSMC was stimulated with MS, OPN production was increased, which was markedly attenuated in VSMC treated with PI3K/Akt inhibitor as well as in Akt1-depleted cells, but not in Akt2-depleted cells, suggesting a pivotal role of Akt1 isoform in OPN expression in VSMC. In the promoter assay, MS increased OPN promoter activity, which was attenuated when the region harboring AP-1 binding sites was mutated. The MS-enhanced promoter activity and OPN expression were also decreased in cells treated with AP-1 siRNA or inhibitor. Moreover, MS-induced MMP-2 production was attenuated in cells treated with OPN siRNA or anti-OPN antibody as well as in OPN-deficient VSMC cultured from aorta of OPN deficient mice. In in vivo experiments, the expressions of OPN and MMP-2 were increased in the aortic tissues from hypertensive mice, but these increases were markedly attenuated in OPN-deficient mice with hypertension. In conclusion, these results suggested that OPN expression in the hypertensive vasculature was increased via signaling pathways that involve Akt1/AP-1, leading to vascular remodeling by increasing the production of MMP-2.