HIF-1α regulates configuration and maintenance of articular cartilage through induction of anabolic factors and suppression of catabolic factors

Abstract

Purpose: Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in hypoxic conditions for cell survival in various tissues. In chondrocytes, previous studies showed that HIF-1α was necessary for chondrocyte survival in endochondral ossification and for interzone formation in joint development; however, the underlying molecular mechanisms remain unclear. Moreover, physiological and pathological functions of HIF-1α in articular cartilage are yet to be revealed. Here we have investigated HIF-1α functions and the underlying mechanisms of the configuration and maintenance of articular cartilage by HIF-1α. Methods: We generated tissue-specific knockout mice of HIF-1α by mating Sox9-Cre knock-in mice (Sox9-Cre) or tamoxifen-inducible Col2a1-Cre transgenic mice (Col2a1-CreERT2), with mice homozygous for a floxed Hif1a allele (Hif1afl/fl). For analyses of cartilage configuration, we examined Sox9-Cre;Hif1afl/fl mice at E15.5, E18.5 and P1. To analyze articular cartilage maintenance and osteoarthritis (OA) progression after maturation, we injected tamoxifen into 7-week-old Col2a1-CreERT2;Hif1afl/fl mice, and created a surgical OA model by inducing instability in the knee joints one week later. OA severity was quantified by the OARSI histopathology grade 8 weeks after surgery. For histological analyses of these knockout mice, we performed Safranin O staining, TUNEL staining, and immunofluorescence. Quantitative gene expression in limb cartilages of Sox9-Cre;Hif1afl/fl and the control Hif1afl/fl neonates was determined based on real-time RT-PCR analyses using samples obtained directly from their limb cartilages. To assess the catabolic ability of HIF-1α, we measured the concentration of aggrecan released from the 3-week-old mouse femoral heads cultured with or without CoCl2, a potent enhancer of HIF-1α protein stability, utilizing dimethylmethylene blue dye-binding assay. Mouse primary articular chondrocytes were used for further in vitro functional analyses. Results: Sox9-Cre;Hif1afl/fl embryos and neonates showed dwarfism with severe limb shortening throughout the embryonic stages and died in the perinatal period. This phenotype was consistent with previous reports using Prx1-Cre and Col2a1-Cre mice. Notably, the knee joints of Sox9-Cre;Hif1afl/fl embryos showed a widespread epiphysis with a massive cavity continuing from the joint surface forming a trumpet shape, in which the columnar architecture was completely abolished and the cell numbers were vastly reduced(Fig.1). Safranin O staining showed a decrease in proteoglycan content in comparison with the control Hif1afl/fl embryo. TUNEL staining accentuated ectopic apoptosis broadly across the limb cartilage, and immunofluorescence showed enhanced expression of Mmp13 surrounding the defective area. When we created the experimental OA model one week after the tamoxifen injection into Col2a1-CreERT2;Hif1afl/fl mice, the OA development in the knee joints was markedly accelerated as compared to the control Hif1afl/fl joints. Apoptosis and Mmp13 expression were upregulated by the conditional knockout of HIF-1α after maturation in articular cartilage, as well as in the limb cartilage. To reveal altered gene expression by HIF-1α deficiency, we obtained RNA samples directly from the limb cartilages of Sox9-Cre;Hif1afl/fl and the control Hif1afl/f embryo. Real-time RT-PCR using these samples revealed increases of catabolic factors including Mmp13 and Mmp9, and decreases of anabolic factors including Col2a1 and Sox9 by the conditional knockout of HIF-1α. When we deleted HIF-1α in primary articular chondrocytes from Hif1afl/fl mice by adenoviral vector expressing Cre recombinase, expressions of the catabolic and the anabolic genes were changed in ways similar to those in the in vivo analyses, under both normoxic and hypoxic (1% O2 concentration) conditions. Similar results were also obtained under both O2 conditions by HIF-1α silencing in primary articular chondrocytes from WT mice using siRNA. Furthermore, in the organ culture of mouse femoral heads, stabilizing HIF-1α protein by CoCl2 treatment markedly decreased aggrecan release into the medium. Conclusions: HIF-1α regulates the configuration and maintenance of articular cartilage through induction of anabolic factors and suppression of catabolic factors. Elucidation of the molecular network related to HIF-1α may lead to cartilage regeneration and OA treatment.