Selective regulation of RANKL/RANK/OPG pathway by heparan sulfate through the binding with estrogen receptor β in MC3T3-E1 cells
Introduction
The homeostasis between osteoblastic bone formation and osteoclastic bone resorption is critical for bone metabolism during the continuous reconstruction of human bone tissue. The altered balance can cause various metabolic bone diseases, among which osteoporosis is the most typical one [1,2]. As a prevalent metabolic bone disease, osteoporosis is characterized by reduced bone mass and increased bone fragility, leading to infliction upon more than one billion people around the world [3].
RANKL/RANK/OPG signaling pathway is implicated in the regulation of bone metabolism and closely related to the biological function of osteoclasts. An in-depth understanding of RANKL/RANK/OPG pathway is important for the exploration of the etiology and pathogenesis of various metabolic bone diseases, including osteoporosis. Numerous studies have been performed to investigate this from different perspectives. It has been reported that RANKL/RANK/OPG pathway mediated transcellular signal transduction between osteoblasts and osteoclasts is the key mechanism underlying the regulation of bone formation and bone resorption [4], while the maintained optimal RANKL/OPG ratio is essential to regulate bone remodeling [5].
Generally, the RANKL/RANK/OPG signal pathway is initiated by the binding between RANK receptors on the surface of osteoclast precursor cells and RANKL released by osteoblasts, then the downstream signaling cascade can be activated upon the participation of tumor necrosis factor receptor-related factors (TRAFs). RANK can bind to TRAF2, TRAF5 and TRAF6, among which TRAF6 is closely related to the generation of osteoclasts, probably through the nuclear factor kappa-B (NF-κB) pathway, c-Jun N-terminal kinases (JNK) pathway, protein kinase B (PKB) pathway, calcineurin/nuclear factor of activated T cells (CN/NFATcl) pathway, etc. Through such multi-pathway signaling network, RANKL eventually functions to facilitate the differentiation and maturation of osteoclasts [[6], [7], [8], [9]]. In contrast, OPG is a decoy receptor for osteoblast differentiation which competes with RANKL and inhibits the interaction between RANKL and RANK, thereby stymieing osteoblast-induced differentiation and fusion of osteoclast precursor, meanwhile, regulating the differentiation, proliferation and apoptosis of osteoclasts [10].
HS is a linear anionic polysaccharide with repeating sulfated disaccharide units, which is covalently linked to the residues of serine and glycine in the protein core structure [11], constituting the HSPGs (heparan sulfate proteoglycans) on cell surfaces and extracellular basement membranes. Previous studies have validated the remarkable osteogenesis effect of HS, but the mechanism is perplexing since the biological roles of various signaling molecules at each stage of bone healing vary enormously [[12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]]. The long chain of HS can bind to growth factors, signaling proteins, some critical membrane receptors, chemokines and extracellular matrix proteins to prevent the proteins from enzymatic degradation and mediate outside-in transition of proteins, thereby balancing the concentrations of intracellular and extracellular proteins [[12], [13], [14]]. On the other hand, HSPGs can be rapidly transferred on cell surfaces and modify their structures in responding to the extracellular signal molecules [15,16]. The components of HSPGs can also participate in the formation and regulation of bone matrix, thus, stabilizing the internal structures and functions of bone tissue [17,18]. This structural feature of HSPGs is crucial for HS to induce various regulatory osteogenesis effects through multi-pathway signaling. However, the biological impacts of HS on RANKL/RANK/OPG signal pathway is still poorly understood, leaving the detailed regulatory mechanism to be further uncovered.
ER-β is a nuclear protein which belongs to the nuclear hormone receptor family of Nuclear Receptor Subfamily 3, and expressed in many tissues at a lower level [23]. The primary structure of ER includes six functional areas (A/B, C, D, E/F), of which the function of the D region is to bind DNA, and the E/F region of ligand binding domain (LBD) plays a regulatory role for transcriptional activation. Windahl et al. found ER-β expressed in chondrocytes and osteoblasts of growth plate, suggesting it can affect the growth of long bone and bone metabolism of adults [24]. Vidal et al. found that estrogen effect was poor in ER-β silenced cells, so they believed that ER-β participated in bone formation and bone absorption [25]. In the process of differentiation and maturation of rat osteoblasts, the expression level of ER-α was very low at the beginning, then increased at the matrix maturation stage and decreased at the mineral formation stage, while the expression of ER-β was stable during the whole differentiation process, continuously being kept at a high level [26].
In the current work, by studying the interactions between of HS and ER-β, we aim to reveal the roles of HS on regulating the expression of RANKL and OPG in osteoblasts, thus providing better understanding of the impacts of HS on mediating RANKL/RANK/OPG signaling pathway. In addition, we used Discovery Studio, a Pipeline Pilot-based comprehensive molecular modeling and simulative platform for life sciences, to simulate the docking of HS fragment and ER-β at the molecular level. By placing the ligand molecule in the active site of the receptor, the interaction between sulfated HS fragments and ER-β can be evaluated in real time. Furthermore, we experimentally confirmed the binding between ER-β and HS using SPR analysis, which is preferred for biological interactions for its free-labelling, small sample size required and operational convenience. From the combined results of computer simulation and SPR experiment, it was revealed that the inhibitory effect of HS on osteoclast formation is mostly likely achieved through its direct binding to ER-β.
Section snippets
Materials
All animal experiments in this investigation were performed according to the Guide for the Care and Use of Laboratory Animals published by the Chinese National Institutes of Health. A total of 8 Sprague-Dawley rats, born within 24 h, regardless of weight and gender, were obtained from China Changzhou cavens Lab animal Co., Ltd.
HS (cat#HY-101916) was purchased from MedChem Express (USA) which was extracted from bovine lung mucosa, with a potency of 58 IU/mg. The molecular formula of HS monomer
Simulative docking of HS and ER-β
Discovery Studio 3.0 was used to detect the interaction between ER-β and HS fragments with different sulfation degrees and positions. After running the program, the two-dimensional plan views of the spatial conformation interactions and the corresponding docking scores were obtained. As shown in Fig. 2A–D, all the tested sulfated HS fragments showed strong binding to ER-β, with multiple van der Waals forces, hydrogen bonding, electrostatic attractions and π‑sulfur interactions illustrated with
Discussion
As a linear polysaccharide, HS has various biological functions, such as promoting osteoblast differentiation, converting proliferative organelles into differentiative organelles [27,28], regulating the secretion of bone growth factors in the epiphysis to stimulate bone formation and regulating BMP-4 (bone morphogenetic protein-4) signaling transduction. It can also stimulate the proliferation of bone marrow stromal stem cells and the differentiation of stem cells into osteoblasts [29]. HSPGs
Conclusions
In conclusion, we have demonstrated that HS, as a functional component suppressing osteoclastogenesis, played a vital role in regulating bone mass, and this effect was most likely owing to the interaction between HS and ER-β. Moreover, our study showed that ER-β, which widely considered as a cell proliferation-related receptor [40,41], was also a receptor that regulated RANKL/RANK/OPG axis and inhibited osteoclast formation to a certain extent. These findings may pave a promising new path in
CRediT author statement
Yi Liu –Conceptualization, Methodology, Investigation, Writing - Original Draft.
Zhujie Xu – Conceptualization, Methodology Investigation, Writing - Original Draft.
Qiqi Wang – Methodology, Investigation.
Yuyu Jiang – Methodology, Investigation.
Rui Wang – Methodology, Investigation.
Shayang Chen – Methodology, Investigation.
Jingyu Zhu – Investigation for molecular simulation.
Yan Zhang – Writing - Review & Editing, Supervision.
Jinghua Chen – Writing - Review & Editing, Supervision, Resources and
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgments
This study was supported by National Natural Science Foundation of China [grant number 21574059]; National First-class Discipline Program of Light Industry Technology and Engineering [grant number LITE2018-20]; Fundamental Research Funds for the Central Universities [grant number JUSRP51709A]; Wuxi Funds for Science and Technology Development [grant number WX0302B010507180087PB].
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Yi Liu and Zhujie Xu contributed equally to this work.