The Tyrosine Phosphatase SHP-1 Regulates Hypoxia Inducible Factor-1α (HIF-1α) Protein Levels in Endothelial Cells under Hypoxia

Introduction

The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS) formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.

Results

SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn) increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin). SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS) further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A) resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132) returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS) formation, as measured by oxidation of H₂-DCF and DHE fluorescence.

Conclusions

SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.     

Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair

Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10⁻⁶ M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes including Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 was downregulated following Icariin treatment for 14 days. In a differentiation assay using bone marrow mesenchymal stem cells (MSCs) carrying HIF-1α floxed allele, the promotive effect of Icariin on chondrogenic differentiation is largely decreased following Cre recombinase-mediated deletion of HIF-1α in MSCs as indicated by Alcian blue staining for proteoglycan synthesis. In an alginate hydrogel 3D culture system, Icariin increases Safranin O positive (SO+) cartilage area. This phenotype is accompanied by upregulation of HIF-1α, increased proliferating cell nuclear antigen positive (PCNA+) cell numbers, SOX9+ chondrogenic cell numbers, and Col2 expression in the newly formed cartilage. Coincide with the micromass culture, Icariin treatment upregulates mRNA levels of Sox9, Col2α1, aggrecan and Col10α1 in the 3D cultures. We then generated alginate hydrogel 3D complexes incorporated with Icariin. The 3D complexes were transplanted in a mouse osteochondral defect model. ICRS II histological scoring at 6 and 12 weeks post-transplantation shows that 3D complexes incorporated with Icariin significantly enhance articular cartilage repair with higher scores particularly in selected parameters including SO+ cartilage area, subchondral bone and overall assessment than that of the controls. The results suggest that Icariin may inhibit PHD activity likely through competition for cellular iron ions and therefore it may serve as an HIF-1α activator to promote articular cartilage repair through regulating chondrocyte proliferation, differentiation and integration with subchondral bone formation.     

Time-Dependent Stabilization of Hypoxia Inducible Factor-1α by Different Intracellular Sources of Reactive Oxygen Species

Intratumoral hypoxia is a major obstacle in the development of effective cancer chemotherapy, decreasing the efficacy of anti-neoplastic drugs in several solid tumours. The hypoxic environment, through its master regulator hypoxia inducible factor-1 (HIF-1), is able to maintain an anti-apoptotic potential through activation of critical genes associated with drug resistance. Besides affecting metabolism and motility of tumour cells, hypoxia also paradoxically increases production of reactive oxygen species (ROS), which contribute to stabilize HIF-1 through a redox-mediated inhibition of its proteolysis. Here we reported that 1% O₂ hypoxia increases the resistance of human metastatic melanoma cells to conventional chemotherapy with etoposide, and that the increase in chemoresistance strongly depends on ROS delivery due to hypoxia. We reported a biphasic redox-dependent role of HIF-1, involving mitochondrial complex III and NADPH oxidase as oxidants sources, synergising in enhancing survival to chemotherapy. The feed-forward loop engaged by hypoxia involves first an HIF-1-dependent vascular endothelial growth factor-A (VEGF-A) autocrine production and, in the later phase, activation of NADPH oxidase from VEGF/VEGFR2 interaction, finally leading to a further redox-dependent long lasting stabilization of HIF-1. We therefore identified a redox-dependent circuitry linking hypoxia-driven ROS to VEGF-A secretion and to enhanced melanoma cell survival to etoposide chemotherapy.     

Expression of HIF-1α and Its Target Genes in the Nanorana parkeri Heart:Implications for High Altitude Adaptation

Hypoxia-inducible factor 1 alpha(HIF-1α) and its target genes vascular endothelial growth factor(VEGF) and transferrins(TF) play an important role in native endothermic animals' adaptation to the high altitude environments. For ectothermic animals – especially frogs – it remains undetermined whether HIF-1α and its target genes(VEGF and TF) play an important role in high altitude adaptation, too. In this study, we compared the gene sequences and expression of HIF-1α and its target genes(VEGF and TF) between three Nanorana parkeri populations from different altitudes(3008 m a.s.l., 3440 m a.s.l. and 4312 m a.s.l.). We observed that the c DNA sequences of HIF-1A exhibited high sequence similarity(99.38%) among the three altitudinally separated populations; but with increasing altitude, the expression of HIF-1A and its target genes(VEGF and TF) increased significantly. These results indicate that HIF-1α plays an important role in N. parkeri adaptation to the high altitude, similar to its role in endothermic animals.     

Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma

Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.     

Hypoxia Inducible Factor-2α Regulates the Development of Retinal Astrocytic Network by Maintaining Adequate Supply of Astrocyte Progenitors

Here we investigate the role of hypoxia inducible factor (HIF)-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26^CreERT2, Tie2^Cre, and GFAP^Cre. Global Hif-2α disruption by Rosa26^CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2^Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP^Cre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP⁺ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2⁺ and PDGFRα⁺ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other''s development, mediated at least in part by HIF-2α.     

Deletion of Hypoxia-Inducible Factor-1α in Adipocytes Enhances Glucagon-Like Peptide-1 Secretion and Reduces Adipose Tissue Inflammation

It is known that obese adipose tissues are hypoxic and express hypoxia-inducible factor (HIF)-1α. Although some studies have shown that the expression of HIF-1α in adipocytes induces glucose intolerance, the mechanisms are still not clear. In this study, we examined its effects on the development of type 2 diabetes by using adipocyte-specific HIF-1α knockout (ahKO) mice. ahKO mice showed improved glucose tolerance compared with wild type (WT) mice. Macrophage infiltration and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor α (TNFα) were decreased in the epididymal adipose tissues of high fat diet induced obese ahKO mice. The results indicated that the obesity-induced adipose tissue inflammation was suppressed in ahKO mice. In addition, in the ahKO mice, serum insulin levels were increased under the free-feeding but not the fasting condition, indicating that postprandial insulin secretion was enhanced. Serum glucagon-like peptide-1 (GLP-1) levels were also increased in the ahKO mice. Interestingly, adiponectin, whose serum levels were increased in the obese ahKO mice compared with the obese WT mice, stimulated GLP-1 secretion from cultured intestinal L cells. Therefore, insulin secretion may have been enhanced through the adiponectin-GLP-1 pathway in the ahKO mice. Our results suggest that the deletion of HIF-1α in adipocytes improves glucose tolerance by enhancing insulin secretion through the GLP-1 pathway and by reducing macrophage infiltration and inflammation in adipose tissue.     

Hypoxia-inducible Factor-1α (HIF-1α) Protein Diminishes Sodium Glucose Transport 1 (SGLT1) and SGLT2 Protein Expression in Renal Epithelial Tubular Cells (LLC-PK1) under Hypoxia

In this work, we demonstrated the regulation of glucose transporters by hypoxia inducible factor-1α (HIF-1α) activation in renal epithelial cells. LLC-PK₁ monolayers were incubated for 1, 3, 6, or 12 h with 0% or 5% O₂ or 300 μm cobalt (CoCl₂). We evaluated the effects of hypoxia on the mRNA and protein expression of HIF-1α and of the glucose transporters SGLT1, SGLT2, and GLUT1. The data showed an increase in HIF-1α mRNA and protein expression under the three evaluated conditions (p < 0.05 versus t = 0). An increase in GLUT1 mRNA (12 h) and protein expression (at 3, 6, and 12 h) was observed (p < 0.05 versus t = 0). SGLT1 and SGLT2 mRNA and protein expression decreased under the three evaluated conditions (p < 0.05 versus t = 0). In conclusion, our results suggest a clear decrease in the expression of the glucose transporters SGLT1 and SGLT2 under hypoxic conditions which implies a possible correlation with increased expression of HIF-1α.     

Xanthine Oxidase Mediates Hypoxia-Inducible Factor-2α Degradation by Intermittent Hypoxia

Sleep-disordered breathing with recurrent apnea produces chronic intermittent hypoxia (IH). We previously reported that IH leads to down-regulation of HIF-2α protein via a calpain-dependent signaling pathway resulting in oxidative stress. In the present study, we delineated the signaling pathways associated with calpain-dependent HIF-2α degradation in cell cultures and rats subjected to chronic IH. Reactive oxygen species (ROS) scavengers prevented HIF-2α degradation by IH and ROS mimetic decreased HIF-2α protein levels in rat pheochromocytoma PC12 cell cultures, suggesting that ROS mediate IH-induced HIF-2α degradation. IH activated xanthine oxidase (XO) by increased proteolytic conversion of xanthine dehydrogenase to XO. ROS generated by XO activated calpains, which contributed to HIF-2α degradation by IH. Calpain-induced HIF-2α degradation involves C-terminus but not the N-terminus of the HIF-2α protein. Pharmacological blockade as well as genetic knock down of XO prevented IH induced calpain activation and HIF-2α degradation in PC12 cells. Systemic administration of allopurinol to rats prevented IH-induced hypertension, oxidative stress and XO activation in adrenal medulla. These results demonstrate that ROS generated by XO activation mediates IH-induced HIF-2α degradation via activation of calpains.