MIF Plays a Key Role in Regulating Tissue-Specific Chondro-Osteogenic Differentiation Fate of Human Cartilage Endplate Stem Cells under Hypoxia

摘要

class="head no_bottom_margin" id="sec1title">IntroductionDegenerative disc disease (DDD) is considered to be the most important cause of low back pain (LBP), which is one of the most common reasons for activity limitation (). Many pathological factors account for DDD, such as cell senescence, inflammatory factors, and extracellular matrix degradation (, , ). A declining metabolic exchange due to the accumulation of waste products and nutrition insufficiency appears to be the most important of these mechanisms (). The intervertebral disc (IVD) is the largest avascular organ in the human body, and the metabolic exchange is predominantly reliant on the diffusion effect across cartilage endplate (CEP) in the mature IVD (). The CEP is a thin horizontal layer of hyaline cartilage that separates the IVD from the vertebral body. Blood vessels in the adjacent vertebral bones do not reach the inner component of the discs but end at the interface between IVD and the vertebrae (). Because it is the most important metabolic exchange channel, many studies assert that the degeneration of CEP may initiate DDD ().Chondrification characteristics are considered to be critical in the physiological function of CEP. Changes in the cartilaginous biochemical content of CEP are closely related to IVD degeneration. The matrix of CEP is primarily composed of collagen type II, which is not only necessary for cartilage to resist compressive forces but is also instrumental in the transport properties of CEP. In addition, CEP ossification reduces the solute transport, which finally leads to DDD (). However, the mechanisms responsible for the loss of chondrification and the onset of ossification remain unclear.Recent studies by our group demonstrated the presence of stem cells in the CEP. These cartilage endplate stem cells (CESCs) exhibited superior capacity for chondrogenic and osteogenic differentiation to those of bone marrow mesenchymal stem cells (BM-MSCs) (). This differentiation property attracted our attention because it is likely that CESCs play an important role in the restoration and regeneration of CEP, and the direction of chondrogenesis and osteogenesis in CESCs may be responsible for CEP chondrification and ossification.As an avascular tissue, IVD remains in a hypoxic microenvironment (), and the oxygen tension within CEP is as low as 1% (). Hypoxia greatly affects the chondrogenesis and osteogenesis of MSCs (), which indicates that physiological hypoxia may regulate the chondro-osteogenic differentiation of CESCs to maintain a balance of chondrification and ossification in CEP.The hypoxia-inducible factor-1α subunit/macrophage migration inhibitory factor (HIF1A/MIF) pathway is one of the most important signaling pathways in response to hypoxia. HIF1A is a key cellular regulator in responding to hypoxia; MIF, which has been recognized as a downstream target of HIF1A, acts as a regulator of innate immunity and can regulate many biological activities (, ). MIF is highly involved in cartilage metabolism. MIF knockdown in zebrafish embryos can lead to undeveloped jaw cartilage (href="#bib19" rid="bib19" class=" bibr popnode">Ito et al., 2008). Moreover, MIF was observed to be involved in the degenerative process of CEP (href="#bib56" rid="bib56" class=" bibr popnode">Xiong et al., 2014). In mouse neural progenitor cells, MIF promoted survival and maintenance by upregulating the expression of SOX6, which is also a member of the SOX family and is co-expressed with SOX9 in all chondroprogenitors, indicating that MIF may initiate chondrogenesis in progenitor cells (href="#bib34" rid="bib34" class=" bibr popnode">Ohta et al., 2013). In addition, MIF was also involved in bone metabolism (href="#bib35" rid="bib35" class=" bibr popnode">Onodera et al., 2002). Transgenic mice overexpressing MIF exhibited osteoporosis, implying that the overexpression of MIF may lead to poor osteogenesis capability (href="#bib36" rid="bib36" class=" bibr popnode">Onodera et al., 2006). Obviously, the cartilage and bone metabolism was closely related to the chondrogenic and osteogenic differentiation of stem cells; however, the impact of the HIF1A/MIF pathway upon chondro-osteogenic differentiation is rarely reported. The transcription factor SOX9 is the master regulator of chondrogenesis and is expressed in pre-chondrocytes and differentiated chondrocytes during skeletal development (href="#bib16" rid="bib16" class=" bibr popnode">Healy et al., 1996). The transcription factor RUNX2 acts as the master regulator of skeletogenesis; its expression is necessary for osteoblast differentiation and maturation (href="#bib10" rid="bib10" class=" bibr popnode">Ducy et al., 1997). In this study, we investigated how the HIF1A/MIF pathway regulated SOX9 and RUNX2, which resulted in a change in the chondro-osteogenic differentiation fate of CESCs.