Research progress of bone sialoprotein in osteoclast differentiation and bone resorption

ZENG Jun-ming, HE Xiao-ning

Department of Stomatology, the Second Affiliated Hospital of Hainan Medical University, Haikou 570100, China

Keywords:Bone sialoprotein Bone resorption Osteoclasts Osteoporosis Periodontitis

ABSTRACT

1.Introduction

More than 200 million people now suffer from osteoporosis as population ageing and human life expectancy increases[1], while more than 10% of the world’s adults suffer from severe periodontal disease[2], both reduce the quality of life and increase the financial burden.In essence, both diseases are caused by excessive bone resorption.

As a member of SIBLING (small integrin-binding ligand n-linked glycoprotein) family in mineralized tissues, BSP is a major bone non-collagen extracellular matrix protein that promotes bone formation and bone resorption, it is usually produced by mature osteoblasts, osteocytes, and platelets.BSP has many physiological functions, including matrix mineralization, promoting OC differentiation and attachment, and bone resorption.If BSP levels are abnormal, diseases associated with abnormal osteoclast differentiation and bone resorption activity in humans, such as multiple malignant cancers such as osteoporosis and breast cancer,will occur.

OC is the only cell for bone resorption, which is converted from monocytes/macrophages and plays an important role in bone development and formation as well as bone remodeling.Physiologically, bone remodeling is a five-stage process including quiescence, activation, resorption, formation and mineralization, and OC is a key player in the resorption stage.The abnormal production and activation of OC can lead to the inhibition of osteocyte number and cause related diseases such as Osteoporosis and Periodontal disease.This article reviews the biological function of BSP and the research progress of systemic diseases.

2.The biochemical characteristics and distribution of BSP

BSP is normally found in skeletal tissues such as bone, cartilage,dentin, and cementum, and is synthesized by osteoblasts, OC, and other bone-related cells[3], it is also expressed in salivary glands and aorta[4].

BSP and osteopontin (OPN) , as well as dentin matrix protein-1(DMP1) and dentin phosphate protein (DSPP), together constitute the SIBLING family (small binding ligand n-linked glycoprotein)[5].This member typically contains a cellular site-binding (arginineglycine-L-Aspartic Acid, RGD) amino acid sequence as well as several glycosylation sites and phosphorylation sites[5].All of these genes are found in human chromosome 4(chromosome 5 in mice)[6].BSP accounts for 10% ～ 15% of non-collagen protein.It undergoes a series of intracellular transformation processes, including Tyr Vulcanization, N-and o-glycosylation, Ser phosphorylation, etc.Outside the cell, BSP binds to integrins such as αv β3 on the surface of the cell membrane via the RGD sequence[7] , while other amino acid sequences are also involved in attachment to the cell[7,8].Acidic phosphoproteins play a direct role in the formation of Ha(hydroxylapatite) , and BSP is the most efficient nucleator[9] , but tartrate-resistant Acid phosphatase (TRAP) is also involved in BSP cell attachment through partial phosphorylation[10].The major protein kinase that phosphorylates BSP is classical tyrosine kinase II (FIPK) , with the major phosphorylation site in the n-terminal half and only slight phosphorylation or absence in the C-terminal half[11].These modifications also determine the biological function of BSP to some extent.

3.The biological effects of BSP

3.1 The role of BSP in bone and cementum formation

BSP and OPN are the major phosphorylated proteins in bone and are specific for mineralized tissues.During embryonic development,BSP is expressed at the time of bone formation and reaches its maximum during primary ossification, it can also stimulate osteoblast differentiation by up-regulating CBFA1 and Osterix.Early in vitro studies of purified proteins highlighted the ability of BSP to initiate HAP crystal formation, while immunolabeling and biochemical studies showed that BSP is closely associated with type I collagen scaffolds in the bone matrix[13,14].BSP can induce the appearance of mineralized tissues both in vitro and in vivo[15] , 16 and when BSP is added to a stable agarose gel system containing calcium phosphate (Ca-P) , it is shown to be an effective nucleating agent for the formation of HA[16].In calcified tissues, protein phosphorylation is essential for biomineralization, whereas serine 136 phosphorylation is key for BSP-mediated HA crystallography[11].Therefore, we believe that BSP is one of the important markers of osteoblast[12].

Because BSP contains multiple amino acid sequences, they bind to integrins on the surface of the cell membrane to complete cell attachment and cell movement, a series of biological behaviors such as cell mechanical conduction and cell transmembrane information conduction[17].BSP purified from bone can promote osteoblast differentiation and new bone deposition in vitro and in vivo[23,26] , and these effects may be associated with glycosylation of its n-terminal[19].Overexpression of BSP can promote the expression of osteoblast-associated genes and nodule formation,whereas inhibition of BSP expression can also inhibit osteoblastlike cell marker production and nodule appearance[20,21].During root development, BSP is secreted by odontoblasts and deposited in acellular and cellular cementum, and plays an important role in cementum, alveolar bone formation and mineralization, and periodontal function, lack of BSP reduces acellular cementum formation in molar and incisor roots and reduces cementum mineralization[17-22].And in cementum, alveolar bone formation and mineralization the BSP provides insertion of Sharpey fibers by promoting mineralization of the root surface, thereby making the tooth firmly attached to the alveolar bone and acellular fiberderived cementum (AEFC) , therefore, it plays an important role in alveolar bone formation.Osteoblasts adjacent to the new cementum also showed high expression of BSP[23].Mice lacking BSP protein exhibit severe periodontal breakdown, including osteopenia, PDL disturbance, and alveolar bone destruction[24], all of which suggest that BSP plays an indispensable role in bone formation.

3.2 The role of BSP in osteoclast differentiation and bone resorption

3.2.1 BSP and osteoclast

Bone resorption is controlled by a complex network of molecules,including hormones, chemokines, and cytokines.Rankl (receptor activator of the nuclear factor kappa B (RANK)) , a key molecule linking the bone and the immune system described above, regulates osteoclast maturation to control bone resorption by stimulating the activation of signaling pathways downstream of RANK[25] , it is an important differentiation factor of osteoclasts.On the other hand,OPG (osteoprotegerin), a soluble protein that binds to RANKL and prevents its interaction with RANK, thereby inhibiting OC cell maturation, is a negative regulator of osteoclast maturation[26].Rank is expressed in osteoclasts and their precursors, and RANKL promotes gene expression in osteoclast precursor cells through regulation of RANK, a process that typically originates from osteoblasts[27].BSP can prompt osteoclasts to produce NFAT-2(activated T cell nuclear factor-2)[28] , whereas NFAT-2 plays an indispensable role in RANKL-induced transcriptional programs during terminal differentiation of osteoclasts.A series of stimuli lead to OC cell generation and regulate cell survival and bone resorption activity.BSP can increase the bone resorption capacity of RANKL-induced osteoclast precursor and increase the intracellular calcium level, and the activation of osteoclast can also increase the intracellular calcium level, the Calcineurin-NFAT pathway maintains a balance between osteoclast and osteoblast activity[29].

Valverde et al found that BSP promotes osteoclast differentiation and bone resorption and synergizes with RANKL to perform these functions[30].These in vitro findings suggest that: (1) BSP enhances the ability of RANKL to regulate OC cell differentiation by binding to the OC surface receptor integrin αv Β3 and RANKL;(2) BSP also interacts with other molecules.

3.2.2 The role of BSP in bone resorption

Knockdown of the BSP gene results in reduced bone formation with reduced bone resorption[31] , but leads to an increase in OPN(osteopontin)[32].Bsp-splenocyte culture indicates that lack of BSP leads to impaired osteoclast precursor (POC) and OC formation,along with decreased expression of osteoclast markers, and results in developmental defects in ossification[34].In the trabecular bone of mice overexpressing BSP, the surface and number of OCs doubled,and in transgenic bone marrow cultures, the number of OCs and their reabsorption activity in vitro increased[33].In the culture of BSP-splenocytes, the addition of exogenous BSP or other RGDcontaining proteins can obviously regenerate multinucleated osteoclasts through the αvβ3 integrin signaling pathway, however,it failed to restore preosteoclast numbers or their Gene expression profiling, suggesting that endogenous BSP is more effective than exogenous BSP in regulating osteoclasts[33].help to establish new preoperative stratification strategies, to better screen patients who require tumor resection[45].The expression of BSP in the above mentioned malignant tumors indicates that BSP plays a very important role in bone metastasis.

Cirrhosis is a common end-stage of chronic liver disease, and most patients in compensatory stage have no obvious symptoms, studies have shown that BSP serum levels are negatively correlated with portal pressure and its substitutes (eg, platelet count, portal vein cross-sectional area) and positively correlated with portal vein flow velocities[46].BSP may represent a previously unrecognized portal hypertension marker in cirrhotic patients.In critically ill patients,however, circulating levels of BSP are also markedly elevated[47],suggesting that BSP has previously unrecognized functions in critically ill Pathophysiology.

3.3 BSP and systemic diseases

As a pivotal gene, BSP has also been shown to play an important role in common mechanisms with osteoporosis patients as well as arteriosclerosis[35].Aggressive periodontitis (AgP) is often associated with severe periodontal attachment and bone loss and can lead to early tooth loss, often affecting young adults, and can be found in alveolar bone biopsies of patients with aggressive periodontitis;m-RNA levels of BSP and RANKL are upregulated [36].Osteoclast function can be inhibited by initial periodontal treatment to prevent bone loss, and BSP levels decrease significantly after initial periodontal treatment[36],which also suggests that BSP affects osteoclast differentiation and regulation.

BSP mRNA expression may be involved in the development of diabetes and osteoporosis[48].Abnormal expression of BSP is associated with poor prognosis of malignant tumors in patients with breast, prostate, bladder, and non-small-cell lung carcinoma cancer[37,39,40].For example, the mean serum BSP concentration in patients with Paget’s disease was 32.3 ± 17.3 μg/L[41].As bone mass decreases, so does serum BSP content[39],and most theories suggest that BSP promotes bone metastasis in breast cancer by binding to αvβ3 integrin.At the same time, the ability of invasion and migration decreased significantly after αvβ3 integrin was knocked out.Therefore, we propose that BSP can regulate bone metastasis by regulating the expression of this receptor and by binding to it[42].In particular, in ER breast cancer with bone metastases, breast cancer cells secrete BSP and thereby recruit osteoclast precursors, which can induce bone metastasis in patients rather than directly affecting cancer cell growth or motility; This process can be inhibited by chlorogenic acid (CGA)[43].At present, there is no effective and safe program to prevent breast cancer bone metastasis and recurrence.CGA is abundant in plant extracts and has the least side effect on long-term prophylactic treatment.In prostate cancer with bone metastasis, BSP is one of the most important expressed genes[44].The number of patients with pancreatic cancer who relapse after tumor resection remains high, and the use of BSP as a biomarker in patients with pancreatic cancer undergoing tumor resection could

4.Summary and Prospect

BSP is a multifunctional major extracellular matrix non-collagen protein that interacts with αvβ3, αvβ5 and RANKL to promote OC adhesion and differentiation and bone resorption, thus, it affects a series of bone resorption diseases such as periodontitis, osteoporosis,and bone metastasis of various malignant tumors.It can also be used as one of the serum markers for the diagnosis of liver cirrhosis and emergency patients.However, the specific mechanism is not clear at present, so to clarify the specific molecular mechanism of BSP promoting OC differentiation and bone resorption,appropriately hindering BSP function, can achieve the regulation of osteoclast maturation and differentiation, and then prevent and treat osteoporosis and periodontal disease and other bone resorptionrelated diseases, inhibit breast cancer, prostate cancer and other malignant bone metastasis possibility.In the application research,the present research mainly adopts the BSP which is synthesized in vitro, but the utility is still weaker than the endogenous BSP.However, there are still many problems in large-scale production,purification, preparation and preservation of clinical-grade natural BSP, improving the production system of BSP and carrying out strict verification is also the focus of future development.