Knockdown Of HIF-1α Expression Research Articles

HIF-1α Regulates Osteogenesis of Periosteum-Derived Stem Cells Under Hypoxia Conditions via Modulating POSTN Expression.

Periosteum is indispensable in bone repair and is an important source of skeletal stem cells (SSCs) for endogenous bone regeneration. However, there are only a few studies about SSCs in periosteum. The craniomaxillofacial bone regeneration is done under the hypoxia microenvironment, in which HIF-1α plays an important role. The effect of HIF-1α on periosteum-derived stem cells (PDSCs) and the mechanisms of PDSCs activation under hypoxia conditions are unknown. In this study, the calvarial bone defect was established, with the periosteum removed or retained. Results show that the bone regeneration was severely impaired in the periosteum removed group. Moreover, pluripotent PDSCs isolated from the periosteum were positive for mesenchymal stem cell (MSC) markers. To determine the role of HIF-1α, the expression of HIF-1α was knocked down in vivo and in vitro, impairing the bone regeneration or osteogenesis of PDSCs. Furthermore, the knockdown of HIF-1α expression also reduced periostin (POSTN) expression, and recombinant POSTN addition partly rescued the osteogenic inhibition. Finally, to explore the mechanism under POSTN activation, the phosphorylation level of the PI3K/AKT pathway was assessed in transfected PDSCs. The phosphorylation level of PI3K and AKT was enhanced with HIF-1α overexpression and inhibited with HIF-1α knockdown, and the addition of PI3K activator or AKT activator could partly rescue POSTN expression. In conclusion, as a potential target to promote bone repair under the hypoxia microenvironment, HIF-1α can regulate the osteogenic differentiation of PDSCs via the PI3K/AKT/POSTN pathway, which lay a solid foundation for periosteum-based craniomaxillofacial bone regeneration.

MiR-126 regulates angiogenesis in myocardial ischemia by targeting HIF-1α

Promoting angiogenesis by targeting various angiogenic regulators has emerged as a new treatment strategy for myocardial ischemia (MI). MicroRNA-126 (miR-126) has been identified as the main regulator of compensatory angiogenesis; however, its role in MI is unclear. A rat MI model and an EA. hy926 endothelial cell hypoxia model were constructed and it was found that miR-126 was highly expressed in both models. The knockdown of HIF-1α expression in EA. hy926 cells in turn downregulated VEGF and CD34 expression and consequently inhibited angiogenesis. MiR-126 inhibitor inhibited EA. hy926 cell migration and tube formation as well as downregulated VEGF and CD34 expression, and these were reversed by transfection of miR-126 mimics. Rescue tests using miR-126 and HIF-1α demonstrated that miR-126-mediated regulation of angiogenesis was dependent on HIF-1α. In summary, miR-126 regulates the occurrence and progression of angiogenesis during MI via HIF-1α and may be a potential new therapeutic target.

Abstract 149: Endothelial-specific Overexpression of Metallothionein Prevents Diabetes Mellitus-induced Impairment in Ischemia Angiogenesis via Preservation of Hif-1á/sdf-1 in Endothelial Progenitor Cells

Diabetes mellitus associated dysfunction of endothelial progenitor cells (EPCs) may contribute to dysregulation of endothelial regeneration. Whether oxidative protection of EPCs by an antioxidant, such as the protein metallothionein (MT) can have positive angiogenic effects and by what mechanisms remain unclear. Endothelial-specific MT overexpression (JTMT) mice were made diabetic by high fat diet followed by streptozocin administration (HFD/STZ). Diabetic hind limb ischemia (HLI) was established by femoral artery ligation. Bone marrow mononuclear cells (MNCs) collected from JTMT mice were transplanted into db/db mice with HLI. Blood reperfusion was monitored. High glucose and hypoxia conditions in culture were adopted to mimic diabetic ischemia. Compared with wild-type (WT) littermates, JTMT mice were resistant to diabetes-induced impairment in ischemia angiogenesis and blood reperfusion in HFD/STZ diabetes. Similarly, transplantation of MT overexpressing MNCs showed better therapeutic effect on db/db mice with HLI than transplantation of WT MNCs. These changes were accompanied by increased mobilization and infiltration of EPCs in JTMT mice, and enhanced incorporation of EPCs into capillaries in JTMT MNCs transplanted db/db mice. Furthermore, EPCs from JTMT mice exhibited augmented cell survival, tube formation, and migration capacities under diabetic ischemia-like conditions. Mechanistically, the expression of hypoxia-inducible factor 1α (HIF-1α) and the secretion of stromal cell-derived factor (SDF-1) in blood and expression in ischemic tissues were upregulated in JTMT and JTMT-MNC transplanted db/db mice, and in cultured JTMT EPCs, which were accompanied by marked amelioration of oxidative stress. However, MT-mediated elevation of SDF-1 and improvements of function in EPCs were all abrogated by siRNA knockdown of HIF-1α expression without effect on the anti-oxidative capacity of MT. Endothelial-specific MT elevation is sufficient to protect against diabetes mellitus-induced impairment of ischemia angiogenesis by promoting EPC function. The benefits of MT are predominantly mediated by upregulation of the HIF-1α/SDF-1 pathway.

MEK2 Negatively Regulates Lipopolysaccharide-Mediated IL-1β Production through HIF-1α Expression.

LPS-activated macrophages require metabolic reprogramming and glucose uptake mediated by hypoxia-inducible factor (HIF)-1 α and glucose transporter 1 (Glut1) expression for proinflammatory cytokine production, especially IL-1β. This process is tightly regulated through activation of MAPK kinases, including the MEK/ERK pathway as well as several transcription factors including HIF-1α. Although MAPK kinase (MEK) 2 deficiency had no significant effect on NO, TNF-α, or IL-12 production in response to LPS challenge, MEK2-deficient murine bone marrow-derived macrophages (BMDMs) exhibited lower IL-10 production. Importantly, MEK2-deficient BMDMs exhibited a preserved ERK1/2 phosphorylation, higher HIF-1α and Glut1 levels, and substantially increased IL-1β as well as IL-6 production in response to LPS stimulation. Knockdown of HIF-1α expression via short interference RNA decreased the level of HIF-1α expression in MEK2-deficient BMDMs and decreased IL-1β production in response to LPS treatment. Furthermore, we performed gain of function experiments by overexpressing MEK2 protein in RAW264.7 cells. LPS stimulation of MEK2 overexpressed in RAW264.7 cells led to a marked decreased IL-1β production. Finally, we investigated the role of Mek1 and Mek2 double and triple mutation on ERK phosphorylation, HIF-1α expression, and IL-1β production. We found that MEK2 is the major kinase, which inversely proportionally regulates HIF-1α and IL-1β expression independent of ERK activation. Our findings demonstrate a novel regulatory function for MEK2 in response to TLR4 activation in IL-1β production through modulating HIF-1α expression.

Rutaecarpine Suppresses Proliferation and Promotes Apoptosis of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia Possibly Through HIF-1α-Dependent Pathways.

The aim of this study is to investigate the potential roles of Rutaecarpine (Rut) in hypoxia-induced human pulmonary artery smooth muscle cells (HPASMCs) model. HPASMCs were cultured with or without hypoxia followed by Rut administration. Cytotoxicity and cell proliferation were assessed by CCK-8 and Cell counting method. Flow cytometry was used for the measurement of cell apoptosis rates. The mRNA expression of hypoxia-induced factor (HIF)-1α and protein levels of HIF-1α, p53, p21, erythropoietin, and vascular endothelial growth factor were determined by quantitative real-time polymerase chain reaction and Western blot, respectively. Rut inhibited the proliferation of HPASMCs with IC50 value of 43.5 μmol·L. Hypoxia significantly increased proliferation and decreased apoptosis in HPASMCs, whereas Rut rescued this phenomenon at the appropriate concentration. Meanwhile, Rut effectively decreased the protein and mRNA expressions of HIF-1α. Knockdown of HIF-1α expression by small interfering RNA (siRNA) significantly enhanced the proapoptotic effect rather than antiproliferation effect of Rut in HPASMCs. Moreover, Rut simultaneously reduced proliferating cell nuclear antigen protein expression, whereas increased p53 and p21 protein levels. However, no significant difference was observed in the protein levels of vascular endothelial growth factor and erythropoietin. Our results demonstrated that Rut exerted protective effects on HPASMCs against hypoxia partly through the HIF-1α-dependent signaling pathway.

Investigation of the Regulation of Roundabout4 by Hypoxia-Inducible Factor-1α in Microvascular Endothelial Cells

We determined if hypoxia-inducible factor-1α (HIF-1α) and Roundabout4 (Robo4) colocalized in fibrovascular membranes (FVM) from patients with proliferative diabetic retinopathy (PDR), and investigated the regulation of HIF-1α on Robo4 in microvascular endothelial cells under normoxic and hypoxic conditions in vitro. Immunofluorescence and confocal laser scanning microscopy were done to analyze the colocalization of HIF-1α and Robo4 in the FVM. Expression of HIF-1α was knocked down by small interfering RNA (siRNA) technology to study its effects on Robo4 expression of human retinal endothelial cells (HREC) and human dermal microvascular endothelial cells (HDMEC) under normoxic and/or hypoxic conditions. Full-length human HIF-1α gene was transfected into HREC and HDMEC using GFP lentivirus vectors to overexpress HIF-1α under normoxic conditions. The HIF-1α and Robo4 mRNA and protein expressions were quantified by real-time PCR and Western blot. A cell proliferation, migration assay, and flow cytometry were used to analyze the effect of HIF-1α regulation on Robo4 in HREC under hypoxic conditions. Colocalization of HIF-1α and Robo4 in vessels of FVM was confirmed by immunofluorescence staining. Knockdown of HIF-1α expression by siRNA in the HREC and HDMEC inhibited Robo4 expression in mRNA and protein level, while overexpressed HIF-1α increased Robo4 mRNA and protein expression. Silencing HIF-1α in endothelial cells under hypoxic conditions inhibited cell invasion and proliferation, which showed that HIF-1α and Robo4 overexpression due to hypoxic conditions correlated with HREC migration and proliferation. Both HIF-1α and Robo4 may have a vital role during the formation of FVM. The increased or decreased expression of Robo4 by stimulation or knockdown of HIF-1α suggesting that Robo4 is positively regulated by HIF-1α under normoxic and hypoxic conditions in microvascular endothelial cells in vitro. The HIF-1α gene promotes HREC invasion and proliferation by transcriptionally upregulating Robo4 under hypoxic conditions.

Loss of HIF-1α impairs GLUT4 translocation and glucose uptake by the skeletal muscle cells

Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. The molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1α in the homeostatic response to various cellular stimuli, including insulin under normoxic conditions. Thus we hypothesized HIF-1α is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12 myocytes in which HIF-1α is knocked down by short-hairpin RNA and examined the intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1α expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the protein kinase B substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMP-activated protein kinase activation were abrogated as well. In addition, expression of the constitutively active mutant of HIF-1α (CA-HIF-1α) or upregulation of endogenous HIF-1α in C2C12 cells shows AS160 phosphorylation comparable to the insulin-stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1α. Taken together, HIF-1α is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in, e.g., type 2 diabetes.

Hypoxia inducible factor‐1α is involved in the neurodegeneration induced by isoflurane in the brain of neonatal rats

More and more data show isoflurane, a commonly used volatile anesthetic has dual effects on neuron fate. However, the underlying mechanisms that can explain the apparent paradox are poorly understood. Hypoxia inducible factor (HIF)-1α, a transcription factor, has been found regulating both prosurvival and prodeath pathways in the CNS. Previously, we found that isoflurane can activate HIF-1α under normoxic conditions in vitro and HIF-1α has been found to be involved in the pre-conditioning effect of isoflurane in various organs. Here, we investigated whether HIF-1α is a contributing factor in the neurodegenration in rodent primary cultured neurons and in developing rat brain. Isoflurane dose-dependently induced apoptotic neurodegeneration in neonatal rats as assessed by S100β, cleaved caspase 3 and poly-(ADP-ribose) polymerase (PARP), respectively. Notably, isoflurane up-regulates HIF-1α protein levels in vivo and in vitro during induction of neurodegeneration. Likewise, isoflurane resulted in a significant elevation of cytosonic calcium levels in neuron cultures. Furthermore, knockdown of HIF-1α expression in cultured neurons attenuated isoflurane-induced neurotoxicity. Finally, Morris water maze (MWM) test showed neonatal exposure to isoflurane impaired juvenile learning and memory ability in rats. These findings indicate that HIF-1α is involved in the neurodegeneration induced by isoflurane in the brain of neonatal rats, suggesting HIF-1α may be a candidate for the dual effects of isoflurane on neuron fate.

Hypoxia Induces Osteogenesis-Related Activities and Expression of Core Binding Factor .ALPHA.1 in Mesenchymal Stem Cells

Mesenchymal stem sells (MSCs) have received much attention in the field of bone tissue engineering due to their biological capability to differentiate into osteogenic lineage cells. Hypoxia-inducible factor 1alpha (HIF-1α) plays an important role in the MSC-related bone regeneration during hypoxia, while core binding factor alpha 1 (Cbfα1) is a transcription regulator that is involved in the chondrocyte differentiation and ossification. In the present study, we investigated the effects of hypoxia on biological capability of MSCs. MSCs were isolated from adult rabbit bone marrow, and were cultured in vitro under normoxia (air with 5% CO(2)) or hypoxia (5% CO(2) and 95% N(2)). The proliferation of MSCs, alkaline phosphatase (ALP) activity, and production of collagens type I and type III (Col I/III) were examined. The expression levels of HIF-1α and Cbfα1 were measured by real-time PCR and western blot analyses. We found that hypoxia significantly induced the proliferation of MSCs and increased ALP activity and the production of Col I/III. Moreover, hypoxia increased the expression of Cbfα1 mRNA after 12 h, whereas the expression of HIF-1α mRNA was increased after 1 h of hypoxia. Knockdown of HIF-1α expression with a small interfering RNA significantly increased the expression levels of Cbfα1 protein either under the normoxia or hypoxia condition. Our results indicate that hypoxia enhances MSCs to differentiate into osteogenic lineage cells and suggest that Cbfα1 may be negatively regulated by HIF-1α.

Expression and function of hypoxia inducible factor-1 alpha in human melanoma under non-hypoxic conditions

BackgroundHypoxia inducible factor-1 alpha (HIF-1α) protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP), vertical growth phase (VGP) and metastatic (MET) melanomas.ResultsHIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part of the oxygen-dependent regulation domain in WM1366 and WM9 melanoma cells. Over-expression of HIF-1α and its splice variant in the RGP cell line SbCl2 resulted in a small increase in soft agar colony formation and a large increase in matrigel invasion relative to control transfected cells. Knockdown of HIF-1α expression by siRNA in the MET WM9 melanoma cell line resulted in a large decrease in both soft agar colony formation and matrigel invasion relative to cells treated with non-specific siRNA. There is a high level of ERK1/2 phosphorylation in WM9 cells, indicating an activated Ras-Raf-MEK-ERK1/2 MAPK pathway. Treatment of WM9 cells with 30 μM U0126 MEK inhibitor, decreased ERK1/2 phosphorylation and resulted in a decrease in HIF-1α expression. However, a 24 h treatment with 10 μM U0126 totally eliminated Erk1/2 phosphorylation, but did not change HIF-1alpha levels. Furthermore, siRNA knockdown of MEK siRNA did not change HIF-1alpha levels.ConclusionWe speculate that metabolic products of U0126 decrease HIF-1alpha expression through "off target" effects. Overall our data suggest that increased HIF-1α expression under normoxic conditions contributes to some of the malignant phenotypes exhibited by human melanoma cells. The expanded role of HIF-1α in melanoma biology increases its importance as a therapeutic target.

Role of HIF-1α in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells

Angiotensin-converting enzyme (ACE) enhances the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which contribute to the pathogenesis of hypoxic pulmonary hypertension (HPH). Previous reports have demonstrated that hypoxia upregulates ACE expression, but the underlying mechanism is unknown. Here, we found that ACE is persistently upregulated in PASMCs on the transcriptional level during hypoxia. Hypoxia-inducible factor 1alpha (HIF-1alpha), a key transcription factor activated during hypoxia, was able to upregulate ACE protein expression under normoxia, whereas knockdown of HIF-1alpha expression in PASMCs inhibited hypoxia-induced ACE upregulation. Furthermore, HIF-1alpha can bind and transactivate the ACE promoter directly. Therefore, we report that ACE is a novel target of HIF-1alpha. Recently, a homolog of ACE, ACE2, was reported to counterbalance the function of ACE. In contrast to ACE, we found that ACE2 mRNA and protein levels increased during the early stages of hypoxia and decreased to near-baseline levels at the later stages after HIF-1alpha accumulation. Thus HIF-1alpha inhibited ACE2 expression, and the accumulated ANG II catalyzed by ACE is a key mediator in the downregulation of ACE2 by HIF-1alpha. Moreover, a reduction of ACE2 expression in PASMCs by RNA interference was accompanied by significantly enhanced proliferation and migration during hypoxia. We conclude that ACE is directly regulated by HIF-1alpha, whereas ACE2 is regulated in a bidirectional way during hypoxia and may play a protective role during the development of HPH. In sum, these findings contribute to the understanding of the pathogenesis of HPH.

Hypoxia and hypoxia-inducible factor-1α promote growth factor-induced proliferation of human vascular smooth muscle cells

Hypoxia is thought to be a stimulus for the excessive proliferation of vascular smooth muscle cells (VSMC) that contributes to pulmonary hypertension, but the mechanisms involved are unknown. Here we tested whether hypoxia-inducible factor 1-alpha (HIF-1alpha), a master regulator of the transcriptional response to hypoxia, is involved in the enhanced mitogen-induced proliferative responses of hypoxic VSMC. Exposure to moderate hypoxia (5% O(2)) enhanced the proliferative responses of human pulmonary artery SMC (HPASMC) to mitogens including platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF), compared with those in normoxia (20% O(2)). Moderate hypoxia elicited increased cellular HIF-1alpha levels, shown by Western blot analysis, and also enhanced PDGF-, FGF-2-, and EGF-induced expression of HIF-1alpha. Knockdown of HIF-1alpha or HIF-1beta levels in HPASMC with specific small interfering RNAs inhibited FGF-2-stimulated proliferation of HPASMC incubated in either 5% or 20% O(2) but failed to inhibit the comitogenic effect of hypoxia. Knockdown of HIF-1alpha similarly inhibited PDGF-stimulated proliferation, whereas HIF-2alpha knockdown had no effect on HPASMC proliferation. Knockdown of HIF-1alpha expression also inhibited growth factor-induced expression of cyclin A. We conclude that HIF-1alpha promotes proliferative responses of human VSMC to FGF-2, PDGF, and EGF by mechanisms that may involve HIF-1-dependent expression of cyclin A, but HIF is apparently not crucial to the enhancement of FGF-2-, PDGF-, and EGF-induced proliferation of VSMC that occurs during hypoxia.