Original articleInteraction of SOX5 with SOX9 promotes warfarin-induced aortic valve interstitial cell calcification by repressing transcriptional activation of LRP6
Graphical abstract
Introduction
Calcific aortic valve disease has become the most common type of valvular disease in developed countries, representing an increasing health burden in the aged population [1]. Even though knowledge, experience and diagnostic techniques for addressing this disease are growing, the only treatment for severe CAVD is surgical or transcatheter aortic valve replacement with a prosthetic valve [2]. Calcification is well-known to play a critical role during the deterioration of valve leaflet structure and function. The formation of calcific nodes is no longer considered a passive consequence of degeneration but an active complex process involving cellular and molecular pathways. Since the exact mechanism underlying the initiation and progression of valvular calcification has not been determined [3,4], there is no preventive approach or pharmacological treatment available to hinder or reverse the progression of CAVD. The early stages of CAVD are associated with endothelial cell dysfunction and inflammation. During the progression of CAVD, the pro-osteogenic factor-mediated phenotypic switching of aortic valve interstitial cells (AVICs) into an osteoblast-like phenotype is thought to be the fundamental step for AVIC calcification. Many osteoblastic pathways, including receptor activator of nuclear factor kappa B (RANK)/receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG), Wnt/β-catenin, and bone morphogenetic proteins (BMPs), are reported to be involved in calcification [5]. BMP2 is a key protein in phenotypic switching of VICs and thus in the development of aortic valve calcification. The downregulation of matrix Gla-protein (MGP) as an endogenous inhibitor of BMP2 signaling is thought to be an important mechanism of aortic valve calcification [6,7]. MGP is a vitamin K-dependent protein. Vitamin K antagonists, such as warfarin, inhibit the recycling of vitamin K, thereby inducing inactive vitamin K-dependent proteins. Warfarin is important for thromboprophylaxis in the presence of atrial fibrillation or a mechanical prosthetic heart valve. Both animal and human studies have implicated warfarin use in increasing vascular and aortic valve calcification [8,9].
SOX (SRY-related HMG-box) family proteins are a conserved group of transcriptional regulators defined by the presence of a highly conserved high mobility group (HMG) domain that mediates DNA binding. SOX proteins are divided into eight groups from A-H. SOX9 belonging to the SOX E group has been reported as an important regulator involved in CAVD. Reduced nuclear SOX9 promotes early onset CAVD in mice and calcification in cultured porcine aortic valve interstitial cells (pAVICs) [10,11]. Partner factors are usually required for the functions of SOX proteins, and SOX9 can form a homodimer to mediate its transcriptional activity in collagen gene expression during chondrocyte development. Although SOX9 often forms dimers, SOX9 dimers further require SOXD protein (SOX5/6) dimers that bind to nearby DNA sites for their activity in many stages of chondrocyte development [12]. SOX5 divided into long SOX5 and short SOX5 isoform, but only long-SOX5 isoform is structurally and functionally equivalent to SOX6. Most research focuses on long SOX5, but for simplicity most authors have referred to it as SOX5 [13]. SOX5 has been reported to play an important role in chondrogenesis, the development of the nervous system and human diseases [[14], [15], [16], [17]].To the best of our knowledge, whether SOX5 is involved in AVIC calcification has not been studied to date.
Low-density lipoprotein receptor-related protein 6 (LRP6) belongs to the low-density lipoprotein receptors (LDLRs) family and accumulating evidence points to the critical role of LRP6 in cardiovascular health. Moreover, presenting the well-appreciated roles in canonical signaling regulating lipid metabolism, blood pressure, cardiac development, cardiac valve disease, LRP6 also inhibits non-canonical Wnt signals that accelerate arterial smooth muscle cell proliferation and vascular calcification [18]. Thus, a deep understanding of LRP6 in other calcification model will contribute to the clinical therapy and new drug development for cardiovascular calcification.
Because of the close association of SOX5 and SOX9, we hypothesized that SOX5 is involved in AVIC calcification. In this study, we first investigated the role of SOX5 in warfarin-induced porcine AVIC (pAVIC) calcification. We found that both knockdown and overexpression of SOX5 attenuated warfarin-induced pAVIC calcification. Warfarin treatment increased the binding of SOX5 to SOX9 and promoted the nuclear export of SOX9. Knockdown of SOX5 decreased the total level of SOX9 expression but preserved its transcriptional regulation of LRP6. Overexpression of SOX5 led to the multimeric complexes formation of SOX5 and reduced its binding to SOX9, resulted in similar effect with SOX5 knockdown. Our results demonstrated the special role of SOX5 in warfarin-induced pAVIC calcification by interfering with SOX9 function.
Section snippets
Human aortic valve tissue samples
Calcific aortic valves were obtained from patients with rheumatic heart valve disease and chronic atrial fibrillation who received warfarin therapy before surgical valve replacement. Control aortic valves were obtained from patients who received heart transplants, and the valve leaflets were inspected by gross examinations and microscopic examinations of hematoxylin and eosin-stained cryosections to confirm the absence of calcification. This study was approved by the Ethics Committee of the
SOX5 expression in human and mouse calcific and noncalcific aortic valves
SOX5 and SOX6 have a high degree of sequence identity to each other and are likely to have redundant functions in chondrocytes [23]. We first analyzed SOX5 and SOX6 expression in pAVICs and hAVICs. In contrast to the redundant expression of SOX6 observed in chondrocytes [24,25], quantitative PCR (qPCR) showed that SOX6 mRNA is absent in AVICs (P < 0.05; Fig. 1A–C). We then detect mRNA expression levels of SOX5 and SOX6 after warfarin stimulation in pAVICs, and found that after 4 days of
Discussion
The major findings of this study include the following: (1) The expression level of SOX5 by AVICs is decreased in human calcific aortic valve tissues. (2) Both overexpression and knockdown of SOX5 decreased AVIC calcification induced by warfarin. (3) Overexpression of SOX5 leads to multimeric complex formation of SOX5 and resulted in decreased binding of SOX5 to SOX9 after warfarin treatment. Knockdown of SOX5 decreased the binding of SOX5 to SOX9 as well. (4) SOX9 without SOX5 can stay in
Author contributions
Conceptualization, W.S. and X.K., Methodology, M.Q., Y.L., J.L., and J.G.; Investigation, M.Q., Y.L., J.L. and Y.J.; Writing–Original Draft, Y.L., M.Q and W.S.; Writing–Review & Editing, M.Q., Y.L. and W.S.; Funding Acquisition, W.S. and X.K.; Resources, X.K., W.S. and Y.S.; Supervision, W.S. and X.K..
Disclosures
None.
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (No.81570247, No.81627802, No.81900442), the National Key R&D Program of China (2019YFA0210100), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX19_1154) and the Six Talent Peaks project in Jiangsu Province (No. 2015-WSN-29). Dr. Wei Sun is an Assistant Fellow at the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, and Dr. Xiangqing Kong is a
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These authors contribute equally to this work.