Hif-αs modulation of Sox9-dependent extracellular matrix production: insight into intervertebral disc cell activity under hypoxia

Abstract

Introduction: Intervertebral disc (IVD) degeneration leads to loss of mechanical function of motion segment and is attributed to deregulated extracellular matrix components in the nucleus pulposus (NP). The NP is rich in type II collagen (encoded by COL2A1) and aggrecan (encoded by AGC1), which are known to be regulated by the master chondrogenic transcription factor, Sox9 (1). In situ studies of human degenerated NP illustrated a reduction of COL2A1 and absence of AGC1 expression, whereas SOX9 expression level remained high (2). This indicates that there may be other factors in the IVD that can regulate the Sox9-dependent matrix expression. Recent studies in aged rabbit IVD suggest that the abnormal NP cells may be contributed by a migration of the Sox9-expressing prechondrocytic cells from the annulus fibrosus (AF) adjacent to the endplate (EP) (3, 4). Among the hypoxia inducible factor-alpha subunits (Hif-α), Hif-1α and Hif-2α (EPAS1) have been illustrated as important transcription factors in maintaining disc cell and matrix homeostasis, particularly in the hypoxic NP region (5, 6, 7).Furthermore, hypoxia promotes Notch signaling protein expression for disc cell maintenance in degenerated NP (8). We hypothesized that Hif-αs may regulate the Sox9-dependent transcription of the extracellular matrix genes in response to IVD degeneration. Firstly, we aimed at identification of Sox9 and Hif-αs co-expression in cells of non-degenerated and degenerated IVD. Secondly, we tested the modulatory effects of Hif-1α/Hif-2α on the expression of Col2a1 and Agc1 using Sox9-expressing mouse prechondrocytic cells as a model. Materials and methods: Intervertebral discs were harvested from wild-type C57BL/6N mice at 3 and 6 month-old. Disc degeneration was induced by an insertion of 25G needle into contralateral AF of 4-month old Lewis rat and discs were harvested post 2 to 4 weeks of operation. Lumbar IVD of scoliosis patients and degenerative disc disease patient were collected. All animal and human works were approved by local ethical committee. Comparative immunohistochemistry staining of Hif-1α, Hif-2α and Sox9 were studied in parrafilm sections of IVD. Luciferase-based promoter assays based on specific Col2a1 or Agc1 cis-acting elements via the over-expression for each of the HIF-α subunits and/or for Sox9 in ATDC5 cells were performed. Results: Relative co-expression patterns of Hif-1α, Hif-2α and Sox9 were detected in AF, NP and EP of the mouse, rat and human IVD. Intracellular expression of Hif-1α, Hif-2α and Sox9 were confirmed by immunofluorescence. Luciferase-based promoter assay showed that Sox9 is a dominant transcription factor in the activation of transcription of Col2a1 and Agc1 in mouse chondroprogenitor cells. Overt additive transcriptional up-regulation of Col2a1 and Agc1 was observed with a co-expression of HIF-1α and Sox9. Strikingly, Hif-2α inhibited the Sox9–dependent transcriptional up-regulation of Col2a1 and Agc1. Conclusion: Our findings indicate the presence of Hif-αs/Sox9 expressing cells in IVD. Additionally, our data suggest that Hif-αs can modulate Sox9-mediated transcriptional regulation of Col2a1 and Agc1 in prechondrocytic cells, implicating a possible role of dynamic Hif-α-Sox9 interaction in the IVD degeneration process. Further investigations of the orchestration mechanisms of Hif-αs/Sox9 in the IVD may provide insights in potential strategies to alleviate disc degeneration.