Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1 alpha in articular chondrocytes: involvement of HIF-1 alpha in the pathogenesis of osteoarthritis

Abstract

Transcription factor hypoxia-inducible factor (HIF)-1 protein accumulates and activates the transcription of genes that are of fundamental importance for oxygen homeostasis - including genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production - under hypoxic conditions. We speculated that HIF-1alpha may have an important role in chondrocyte viability as a cell survival factor during the progression of osteoarthritis (OA). The expression of HIF-1alpha mRNA in human OA cartilage samples was analyzed by real-time PCR. We analyzed whether or not the catabolic factors IL-1beta and H2O2 induce the expression of HIF-1alpha in OA chondrocytes under normoxic and hypoxic conditions (O2 <6%). We investigated the levels of energy generation, cartilage matrix production, and apoptosis induction in HIF-1alpha-deficient chondrocytes under normoxic and hypoxic conditions. In articular cartilages from human OA patients, the expression of HIF-1alpha mRNA was higher in the degenerated regions than in the intact regions. Both IL-1beta and H2O2 accelerated mRNA and protein levels of HIF-1alpha in cultured chondrocytes. Inhibitors for phosphatidylinositol 3-kinase and p38 kinase caused a significant decrease in catabolic-factor-induced HIF-1alpha expression. HIF-1alpha-deficient chondrocytes did not maintain energy generation and cartilage matrix production under both normoxic and hypoxic conditions. Also, HIF-1alpha-deficient chondrocytes showed an acceleration of catabolic stress-induced apoptosis in vitro. Our findings in human OA cartilage show that HIF-1alpha expression in OA cartilage is associated with the progression of articular cartilage degeneration. Catabolic-stresses, IL-1beta, and oxidative stress induce the expression of HIF-1alpha in chondrocytes. Our results suggest an important role of stress-induced HIF-1alpha in the maintenance of chondrocyte viability in OA articular cartilage.

Figure 1

Levels of HIF-1α mRNA in the articular cartilage from patients with osteoarthritis (OA). (a)Representative x-ray film of knee joint and safranin-O staining for hypoxia-inducible factor 1α(HIF-1α) in the degenerated region and intact region of articular cartilage from a 66-year-old woman with OA. Original magnification of histological sections × 200. (b) The mRNA levels of HIF-1α were higher in the degenerated regions than in the intact regions from the same OA sample.

Figure 2

IL-1β and H₂O₂ induce the expression of HIF-1α mRNA in human articular chondrocytes. Under normoxic culture conditions, mRNA levels of hypoxia-inducible factor 1α (HIF-1α) were observed in cultured chondrocytes, whereas HIF-1α protein was undetected in the cells. HIF-1α mRNA was accelerated by IL-1β or H₂O₂ in cultured chondrocytes under hypoxic conditions. Cobalt chloride (CoCl₂), chemical inducer of HIF-1, was used for the positive control. *P < 0.05, **P < 0.01 compared with the control. Cont., control.

Figure 3

Catabolic factors induce the expression of HIF-1α protein in human articular cartilage. (a)Hypoxia-inducible factor 1α (HIF-1α) protein was accelerated by IL-1β or H₂O₂in cultured chondrocytes under hypoxic conditions. (b)Under hypoxic conditions, the inhibitors of PI3K and p38 mitogen-activated protein kinase (MAPK) reduced protein levels of IL-1β-induced HIF-1α expression. Cobalt chloride (CoCl₂), chemical inducer of HIF-1, was used for the positive control. *P < 0.05, **P < 0.01 compared with the control. LY294002: phosphatidylinositol 3-kinase inhibitor; SB203580: p38 mitogen-activated protein kinase inhibitor; PD98059: extracellular signal-regulated kinase inhibitor.

Figure 4

Effect of HIF-1α on ATP production and glycolysis in human articular cartilage. (a)Hypoxia-inducible factor 1α (HIF-1α) depletion by antisense oligonucleotide was assessed by RT-PCR and western blotting analyses. HIF-1α mRNA and protein expressions were reduced in antisense HIF-1α-treated chondrocyte populations. Scrambled oligonucleotide was used as control oligonucleotide. Representative data from four independent experiments are shown. (b)In hypoxia, HIF-1α-deficient chondrocytes showed a significant decrease of free ATP in comparison with control oligonucleotide-treated chondrocytes. Statistical differences were calculated using data from four independent experiments. (c, d) The levels of lactate (c) and glucose transporter-1 (Glu-1) (d) were increased in the scrambled ODN-treated chondrocytes under hypoxic culture conditions compared with normoxic culture condition. In HIF-1α-deficient chondrocytes, both glycolytic activities were reduced under hypoxic conditions. Statistical differences were calculated using data from four independent experiments. ^aP < 0.01, control oligonucleotide hypoxia vs HIF-1α-deficient hypoxia; *P < 0.05, **P < 0.01.

Figure 5

Glycosaminoglycan production and apoptosis induction in HIF-1α-deficient chondrocytes. In the scrambled oligonucleotide-treated groups, the amount of glycosaminoglycan (GAG) produced by cultured chondrocytes was higher under hypoxic conditions than under normoxic conditions. Under hypoxic conditions, GAG levels decreased in chondrocytes deficient in hypoxia-inducible factor 1α (HIF-1α). Statistical differences were calculated using data from four independent experiments. ^aP < 0.01, control oligonucleotide hypoxia vs HIF-1α-antisense hypoxia; *P < 0.05; **P < 0.01.

Figure 6

Apoptosis induction in HIF-1α-deficient chondrocytes. IL-1β was used for apoptosis induction under hypoxic or normoxic conditions in chondrocytes lacking hypoxia-inducible factor 1α (HIF-1α), treated with oligonucleotide, and cultured without oligonucleotide, and also not exposed to the oligonucleotide or HIF-1α antisense nucleotides. IL-1β-induced apoptosis was significantly increased in HIF-1α-deficient chondrocytes compared with chondrocytes treated with scrambled oligonucleotide under both normoxic and hypoxic culture conditions. Statistical differences were calculated using data from four independent experiments. **P < 0.01, control oligonucleotide group vs HIF-1α-antisense group.