The RCAN1.4-calcineurin/NFAT signaling pathway is essential for hypoxic adaption of intervertebral discs

Bao Huang,Junhui Liu,Yue Huang,Fengdong Zhao,Zhi Shan,Jian Chen,Xiaoan Wei,Yongqing He,Shengwen Li

doi:10.1038/s12276-020-0441-x

Abstract

Calcipressin-1, also known as regulator of calcineurin 1 (RCAN1), can specifically bind calcineurin at or near the calcineurin A catalytic domain and downregulate calcineurin activity. However, whether RCAN1 affects the hypoxic intervertebral disc (IVD) phenotype through the calcineurin/NFAT signaling pathway remains unclear. First, we confirmed the characteristics of the degenerative nucleus pulposus (NP) by H&E, safranin O/fast green and Alcian blue staining, and detected increased RCAN1 levels in the degenerative NP by immunohistochemistry. Then, we demonstrated that the protein level of RCAN1.4 was higher than that of RCAN1.1 and progressively elevated from the control group to the Pfirrmann grade V group. In vitro, both hypoxia (1% O2) and overexpression of HIF-1α reduced the protein level of RCAN1.4 in rat NP cells in a dose- and time-dependent manner. We further found that miRNA-124, through a nondegradative pathway (without the proteasome or lysosome), suppressed the expression of RCAN1.4. As expected, calcineurin in NP cells was activated and primarily promoted nuclear translocation of NFATc1 under hypoxia or RCAN1.4 siRNA transfection. Furthermore, SOX9, type II collagen and MMP13 were elevated under hypoxia, RCAN1.4 siRNA transfection or NFATc1 overexpression. Using chromatin immunoprecipitation (ChIP) and a luciferase reporter assay (with mutation), we clarified that NFATc1 increasingly bound the SOX9 promotor region (bp −367~−357). Interaction of HIF-1α and NFATc1 promoted MMP13 transcription. Finally, we found that FK506 reversed hypoxia-induced activation of the calcineurin/NFAT signaling pathway in NP cells and an ex vivo model. Together, these findings show that the RCAN1.4-calcineurin/NFAT signaling pathway has a vital role in the hypoxic phenotype of NP cells. RCAN1.4 might be a therapeutic target for degenerative disc diseases.

Highlights

Intervertebral disc degeneration (IVDD) is a leading cause of low back pain (LBP), one of the most common clinical complaints[1,2]
The regulator of calcineurin 1 (RCAN1) protein level was visibly increased in the IDD group by immunohistochemistry (Fig. 1c)
We first demonstrated that RCAN1.4 is positively associated with disc degeneration and that hypoxiainduced downregulation of RCAN1.4 activated the calcineurin/NFAT signaling pathway to facilitate SOX9 and MMP13 expression

Summary

Introduction

Intervertebral disc degeneration (IVDD) is a leading cause of low back pain (LBP), one of the most common clinical complaints[1,2]. Intervertebral discs (IVDs) include the central hydrated chondrocyte-like nucleus pulposus (NP), which is inferiorly and superiorly bounded by the cartilaginous endplate (CEP) and laterally surrounded by the concentric lamellae of the annulus fibrosus (AF). IVDs are a complex tissue that accommodates high biomechanical forces and permits a range of motions between adjacent vertebrae[3]. The high proteoglycan content in the central water-binding NP is an important component of IVDs, which absorb stress and maintain the structure and function of the spine[4]. The protein level of HIF-1α in the degenerative disc is generally reduced[10,11,12]

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Results

Conclusion