Hypoxia-inducible Factor-1 (HIF-1) but Not HIF-2 Is Essential for Hypoxic Induction of Collagen Prolyl 4-Hydroxylases in Primary Newborn Mouse Epiphyseal Growth Plate Chondrocytes

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α₂β₂ tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I–III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.     

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.     

Prolyl Hydroxylase 3 (PHD3) Modulates Catabolic Effects of Tumor Necrosis Factor-α (TNF-α) on Cells of the Nucleus Pulposus through Co-activation of Nuclear Factor κB (NF-κB)/p65 Signaling

Recent studies suggest a differential role of prolyl hydroxylase (PHD) isoforms in controlling hypoxia-inducible factor (HIF)-α degradation and activity in nucleus pulposus (NP) cells. However, the regulation and function of PHDs under inflammatory conditions that characterize disc disease are not yet known. Here, we show that in NP cells, TNF-α and IL-1β induce PHD3 expression through NF-κB. Lentiviral delivery of Sh-p65 and Sh-IKKβ confirms that cytokine-mediated PHD3 expression is NF-κB-dependent. It is noteworthy that although both cytokines induce HIF activity, mechanistic studies using Sh-HIF-1α and PHD3 promoter/enhancer constructs harboring well characterized hypoxia response element (HRE) show lack of HIF involvement in cytokine-mediated PHD3 expression. Loss-of-function studies clearly indicate that PHD3 serves as a co-activator of NF-κB signaling activity in NP cells; PHD3 interacts with, and co-localizes with, p65. We observed that when PHD3 is silenced, there is a significant decrease in TNF-α-induced expression of catabolic markers that include ADAMTS5, syndecan4, MMP13, and COX2, and at the same time, there is restoration of aggrecan and collagen type II expression. It is noteworthy that hydroxylase function of PHDs is not required for mediating cytokine-dependent gene expression. These findings show that by enhancing the activity of inflammatory cytokines, PHD3 may serve a critical role in degenerative disc disease.     

Thermal Unfolding Pathway of PHD2 Catalytic Domain in Three Different PHD2 Species: Computational Approaches

Prolyl hydroxylase domain 2 containing protein (PHD2) is a key protein in regulation of angiogenesis and metastasis. In normoxic condition, PHD2 triggers the degradation of hypoxia-inducible factor 1 (HIF-1α) that induces the expression of hypoxia response genes. Therefore the correct function of PHD2 would inhibit angiogenesis and consequent metastasis of tumor cells in normoxic condition. PHD2 mutations were reported in some common cancers. However, high levels of HIF-1α protein were observed even in normoxic metastatic tumors with normal expression of wild type PHD2. PHD2 malfunctions due to protein misfolding may be the underlying reason of metastasis and invasion in such cases. In this study, we scrutinize the unfolding pathways of the PHD2 catalytic domain’s possible species and demonstrate the properties of their unfolding states by computational approaches. Our study introduces the possibility of aggregation disaster for the prominent species of PHD2 during its partial unfolding. This may justify PHD2 inability to regulate HIF-1α level in some normoxic tumor types.     

Intermediary Metabolite Precursor Dimethyl-2-Ketoglutarate Stabilizes Hypoxia-Inducible Factor-1α by Inhibiting Prolyl-4-Hydroxylase PHD2

Hypoxia-inducible factor 1α (HIF-1α), a major mediator of tumor physiology, is activated during tumor progression, and its abundance is correlated with therapeutic resistance in a broad range of solid tumors. The accumulation of HIF-1α is mainly caused by hypoxia or through the mutated succinate dehydrogenase A (SDHA) or fumarate hydratase (FH) expression to inhibit its degradation. However, its activation under normoxic conditions, termed pseudohypoxia, in cells without mutated SDHA or FH is not well documented. Here, we show that dimethyl-2-ketoglutarate (DKG), a cell membrane-permeable precursor of a key metabolic intermediate, α-ketoglutarate (α-KG), known for its ability to rescue glutamine deficiency, transiently stabilized HIF-1α by inhibiting activity of the HIF prolyl hydroxylase domain-containing protein, PHD2. Consequently, prolonged DKG-treatment under normoxia elevated HIF-1α abundance and up-regulated the expression of its downstream target genes, thereby inducing a pseudohypoxic condition. This HIF-1α stabilization phenotype is similar to that from treatment of cells with desferrioxamine (DFO), an iron chelator, or dimethyloxalyglycine (DMOG), an established PHD inhibitor, but was not recapitulated with other α-KG analogues, such as Octyl-2KG, MPTOM001 and MPTOM002. Our study is the first example of an α-KG precursor to increase HIF-1α abundance and activity. We propose that DKG acts as a potent HIF-1α activator, highlighting the potential use of DKG to investigate the contribution of PHD2-HIF-1α pathway to tumor biology.     

Effect of Proline Analogues on Activity of Human Prolyl Hydroxylase and the Regulation of HIF Signal Transduction Pathway

Hypoxia inducible factor 1 (HIF-1) plays a pivotal role in cellular responses to hypoxia. Prolyl hydroxylase 3 (PHD3) degrades HIF-1α under normoxic conditions through the hydroxylation of HIF-1α for proteolysis. Inhibiting PHD3 activity is crucial for up-regulating HIF-1α, thereby acting as a potential target for treating hypoxia-related diseases. In this study, two proline analogues (PA1 and PA2) were screened as PHD3 inhibitors with apparent EC₅₀ values of 1.53 and 3.17 µM respectively, indicating good inhibition potency. Nine proteins, significantly regulated by PA1, were identified using 2-DE coupled with MALDI-TOF/TOF MS. Pyruvate kinase isozymes M1/M2 (PKM) and alpha-enolase 1 (ENO1), which are key modulators of glycolysis, are directly regulated by HIF-1α. Moreover, VEGF, a signal protein stimulating angiogenesis, was strongly promoted by PA1. Our findings suggest that PA1 stabilized HIF-1α as well as up-regulated glycolysis and angiogenesis proteins. Herein, for the first time, we systematically studied proline analogue PA1 as a PHD3 inhibitor, which provides innovative evidence for the treatment of HIF-related diseases.     

Human meniscus cells express hypoxia inducible factor-1α and increased SOX9 in response to low oxygen tension in cell aggregate culture

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)α(I) in response to 5% oxygen, and this hypoxia-induced expression of P4Hα(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1α inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1α gene and downstream target genes (namely, those encoding P4Hα(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation.     

Hypoxia promotes redifferentiation and suppresses markers of hypertrophy and degeneration in both healthy and osteoarthritic chondrocytes

Introduction

Hypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation.

Methods

Monolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured.

Results

In hypoxia, both healthy and OA chondrocytes had higher human collagen type II, α1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, α1 gene (COL10A1), and human collagen type I, α1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1α and HIF-2α had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time.

Conclusions

Hypoxic culture of human chondrocytes has long been acknowledged to result in increased matrix accumulation, but still little is known of its effects on catabolism. We show herein that the increased expression of matrix proteins, combined with decreased expression of numerous degradative enzymes by hypoxia, minimizes but does not abolish differences between redifferentiated healthy and OA chondrocytes. Hypoxia-induced HIF expression is associated with hypertrophic marker and degradative enzyme downregulation and increased measures of redifferentiation in both healthy and OA chondrocytes. Therefore, though HIFs may be involved in the pathogenesis of OA, conditions that promote HIF expression in vitro promote matrix accumulation and decrease degradation and hypertrophy, even in cells from OA joints.     

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.     

Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells

Background

The endothelin B receptor (ET_BR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.

Principal Findings

Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET_BR, enhance the expression and activity of HIF-1α and HIF-2α that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET_BR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET_BR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α, ET_BR expression is associated with low PHD2 levels. In melanoma xenografts, ET_BR blockade by ET_BR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels.

Conclusions

In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET_BR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.     

Prolyl-4-hydroxylase Domain Protein 2 Controls NF-κB/p65 Transactivation and Enhances the Catabolic Effects of Inflammatory Cytokines on Cells of the Nucleus Pulposus

Prolyl-4-hydroxylase (PHD) proteins are key in sensing tissue hypoxia. In nucleus pulposus (NP) cells, our previous work demonstrated that PHD isoforms have a differential contribution in controlling hypoxia-inducible factor (HIF)-α degradation and activity. Recently we have shown that a regulatory relationship exists between PHD3 and inflammatory cytokines in NP cells. With respect to PHD2, the most abundant PHD isoform in NP cells, very little is known concerning its function and regulation under inflammatory conditions that characterize intervertebral disc degeneration. Here, we show that PHD2 is a potent regulator of the catabolic activities of TNF-α; silencing of PHD2 significantly decreased TNF-α-induced expression of catabolic markers including SDC4, MMP-3, MMP-13, and ADAMTS5, as well as several inflammatory cytokines and chemokines, while partially restoring aggrecan and collagen II expression. Use of NF-κB reporters with ShPHD2, SiHIF-1α, as well as p65^−/−, PHD2^−/−, and PHD3^−/− cells, shows that PHD2 serves as a co-activator of NF-κB/p65 signaling in HIF-1-independent fashion. Immunoprecipitation of endogenous and exogenously expressed tagged proteins, as well as fluorescence microscopy, indicates that following TNF-α treatment, PHD2 interacts and co-localizes with p65. Conversely, loss of function experiments using lentivirally delivered Sh-p65, Sh-IKKβ, and NF-κB inhibitor confirmed that cytokine-dependent PHD2 expression in NP cells requires NF-κB signaling. These findings clearly demonstrate that PHD2 forms a regulatory circuit with TNF-α via NF-κB and thereby plays an important role in enhancing activity of this cytokine. We propose that during disc degeneration PHD2 may offer a therapeutic target to mitigate the deleterious actions of TNF-α, a key proinflammatory cytokine.