Small Integrin-binding Ligand N-linked Glycoprotein Research Articles

Pan-cancer analysis shows that IBSP is a potential prognostic and immunotherapeutic biomarker for multiple cancer types including osteosarcoma.

IBSP is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family that plays a vital role in bone formation, renewal and repair. Emerging evidence revealed that IBSP participated in the tumorigenesis and progression in some cancers. However, its significance in tumour prognosis and immunotherapy is still unknown. In the current study, we studied the role of IBSP in tumorigenesis, tumor diagnosis, genomic heterogeneity, methylation modifications, immune infiltration, and therapy response in pan-cancer. In addition, we constructed a risk score model to assessed the prognostic classification efficiency of IBSP using the co-expression genes of IBSP in osteosarcoma (OS), and analyzed the expression and role of IBSP in OS through a series of assays in vitro. IBSP was upregulated in various cancers compared to the paired normal tissues, and it was strongly correlated with the prognosis, pathological stage, diagnostic accuracy, genomic heterogeneity, methylation modification, immune infiltration, immune and checkpoint. Moreover, the predictive model we established in combination with the clinical characteristics of OS patients showed high survival predictive power in these individuals. The assays in vitro showed that IBSP promoted the proliferation, migration and invasion of OS cells, which further confirmed IBSP's role in cancers. Our research revealed the multifunctionality of IBSP in the tumorigenesis, progression and therapy in various cancers, which demonstrated that IBSP may serve as a potential prognostic biomarker and a novel immunotherapy target in pan-cancer.

Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor indicates a cell-specific response that promotes CD44 activation.

Hearing loss caused by the death of cochlear hair cells (HCs) might be restored through regeneration from supporting cells (SCs) via dedifferentiation and proliferation, as observed in birds. In a previous report, ERBB2 activation in a subset of cochlear SCs promoted widespread down-regulation of SOX2 in neighboring cells, proliferation, and the differentiation of HC-like cells. Here we analyze single cell transcriptomes from neonatal mouse cochlear SCs with activated ERBB2, with the goal of identifying potential secreted effectors. ERBB2 induction in vivo generated a new population of cells with de novo expression of a gene network. Called small integrin-binding ligand n-linked glycoproteins (SIBLINGs), these ligands and their regulators can alter NOTCH signaling and promote cell survival, proliferation, and differentiation in other systems. We validated mRNA expression of network members, and then extended our analysis to older stages. ERBB2 signaling in young adult SCs also promoted protein expression of gene network members. Furthermore, we found proliferating cochlear cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2 signaling in cochlear SCs can alter the microenvironment, promoting proliferation and cell rearrangements. Together these results suggest a novel mechanism for inducing stem cell-like activity in the adult mammalian cochlea.

Does the RGD region of certain proteins affect metabolic activity?

A better understanding of the role of mineralized tissues and their associated factors in governing whole-body metabolism should be of value toward informing clinical strategies to treat mineralized tissue and metabolic disorders, such as diabetes and obesity. This perspective provides evidence suggesting a role for the arginine-glycine-aspartic acid (RGD) region, a sequence identified in several proteins secreted by bone cells, as well as other cells, in modulating systemic metabolic activity. We focus on (a) two of the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family genes/proteins, bone sialoprotein (BSP) and osteopontin (OPN), (b) insulin-like growth factor-binding protein-1 & 2 (IGFBP-1, IGFBP-2) and (c) developmental endothelial locus 1 (DEL1) and milk fat globule–EGF factor-8 (MFG-E8). In addition, for our readers to appreciate the mounting evidence that a multitude of bone secreted factors affect the activity of other tissues, we provide a brief overview of other proteins, to include fibroblast growth factor 23 (FGF23), phosphatase orphan 1 (PHOSPHO1), osteocalcin (OCN/BGLAP), tissue non-specific alkaline phosphatase (TNAP) and acidic serine aspartic-rich MEPE-associated motif (ASARM), along with known/suggested functions of these factors in influencing energy metabolism.

INTRA-ARTICULAR TPX-100 SIGNIFICANTLY IMPROVES PAIN MEASURES AND SLOWS PATHOLOGICAL BONE SHAPE CHANGE IN KNEE OA

Purpose: TPX-100 is a 23-amino acid peptide derived from Matrix Extracellular Phosphoglycoprotein (MEPE); a small integrin-binding ligand N-linked glycoprotein (SIBLING) family member highly expressed by osteocytes that may play a role in osteoarthritic bone remodeling. Non-clinical data in experimental knee joint injury in large and small animal species and in OA clinical trials support the safety and efficacy of TPX-100 as a potential disease-modifying OA drug. Study TPX-100-5 was designed to examine MRI-based 3-dimensional bone shape changes after IA TPX-100 or placebo in subjects with moderate to severe knee OA; and to investigate associations between bone shape changes and knee pain measures over 12 months.

IMPROVED WOMAC PHYSICAL FUNCTION IS ASSOCIATED WITH SLOWED PATHOLOGICAL BONE SHAPE CHANGE AFTER TPX-100: TOWARDS A SURROGATE MARKER FOR VIRTUAL KNEE REPLACEMENT?

Purpose: TPX-100 is a 23-amino acid peptide derived from a small integrin-binding ligand N-linked glycoprotein (SIBLING) family member: Matrix Extracellular Phosphoglycoprotein (MEPE). MEPE is highly expressed by osteocytes and may play a role in bone remodeling. Previously reported non-clinical and clinical data support the safety and efficacy of IA TPX-100 in knee OA. In Study TPX-100-5, we were interested in examining clinical and structural measures that have been shown to predict joint failure and total knee replacement (TKR), informed by two recent research streams.

Comprehensive Analysis to Identify SPP1 as a Prognostic Biomarker in Cervical Cancer.

Background: SPP1, secreted phosphoprotein 1, is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. Previous studies have proven SPP1 overexpressed in a variety of cancers and can be identified as a prognostic factor, while no study has explored the function and carcinogenic mechanism of SPP1 in cervical cancer. Methods: We aimed to demonstrate the relationship between SPP1 expression and pan-cancer using The Cancer Genome Atlas (TCGA) database. Next, we validated SPP1 expression of cervical cancer in the Gene Expression Omnibus (GEO) database, including GSE7803, GSE63514, and GSE9750. The receiver operating characteristic (ROC) curve was used to evaluate the feasibility of SPP1 as a differentiating factor by the area under curve (AUC) score. Cox regression and logistic regression were performed to evaluate factors associated with prognosis. The SPP1-binding protein network was built by the STRING tool. Enrichment analysis by the R package clusterProfiler was used to explore potential function of SPP1. The single-sample GSEA (ssGSEA) method from the R package GSVA and TIMER database were used to investigate the association between the immune infiltration level and SPP1 expression in cervical cancer. Results: Pan-cancer data analysis showed that SPP1 expression was higher in most cancer types, including cervical cancer, and we got the same result in the GEO database. The ROC curve suggested that SPP1 could be a potential diagnostic biomarker (AUC = 0.877). High SPP1 expression was associated with poorer overall survival (OS) (P = 0.032). Further enrichment and immune infiltration analysis revealed that high SPP1 expression was correlated with regulating the infiltration level of neutrophil cells and some immune cell types, including macrophage and DC. Conclusion: SPP1 expression was higher in cervical cancer tissues than in normal cervical epithelial tissues. It was significantly associated with poor prognosis and immune cell infiltration. Thus, SPP1 may become a promising prognostic biomarker for cervical cancer patients.

Dentine sialophosphoprotein signal in dentineogenesis and dentine regeneration.

Dentineogenesis starts on odontoblasts, which synthesise and secrete non-collagenous proteins (NCPs) and collagen. When dentine is injured, dental pulp progenitors/mesenchymal stem cells (MSCs) can migrate to the injured area, differentiate into odontoblasts and facilitate formation of reactionary dentine. Dental pulp progenitor cell/MSC differentiation is controlled at given niches. Among dental NCPs, dentine sialophosphoprotein (DSPP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members share common biochemical characteristics such as an Arg-Gly-Asp (RGD) motif. DSPP expression is cell- and tissue-specific and highly seen in odontoblasts and dentine. DSPP mutations cause hereditary dentine diseases. DSPP is catalysed into dentine glycoprotein (DGP)/sialoprotein (DSP) and phosphoprotein (DPP) by proteolysis. DSP is further processed towards active molecules.DPP contains an RGD motif and abundant Ser-Asp/Asp-Ser repeat regions. DPP-RGD motif binds to integrin αVβ3 and activates intracellular signalling via mitogen-activated protein kinase (MAPK) and focal adhesion kinase (FAK)-ERK pathways. Unlike other SIBLING proteins, DPP lacks the RGD motif in some species. However, DPP Ser-Asp/Asp-Ser repeat regions bind to calcium-phosphate deposits and promote hydroxyapatite crystal growth and mineralisation via calmodulin-dependent protein kinase II (CaMKII) cascades.DSP lacks the RGD site but contains signal peptides. The tripeptides of the signal domains interact with cargo receptors within the endoplasmic reticulum that facilitate transport of DSPP from the endoplasmic reticulum to the extracellular matrix. Furthermore, the middle- and COOH-terminal regions of DSP bind to cellular membrane receptors, integrin β6 and occludin, inducing cell differentiation. The present review may shed light on DSPP roles during odontogenesis.

Overexpression of DSPP promotes the proliferation and migration of astrocytes

Astrocytes are activated after central nervous system (CNS) injury, such as spinal cord injury (SCI). Activated astrocytes can form glial scar to block nerve regeneration. Dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family, has been reported to contribute to the proliferation and migration of different types of tumor cells, including glioma. However, the functions of DSPP in reactive astrocytes after CNS injury remain unknown. In this study, starvation-serum stimulation model in astrocytes was conducted to explore this issue. Our results showed that DSPP expression was increased in reactive astrocytes comparing to normal ones. Meanwhile, up-regulation of DSPP was accompanied with PCNA and GFAP. To explore the role of DSPP in astrocytes, we overexpressed DSPP with recombinant GFP-DSPP plasmid and the results showed that overexpression of DSPP could promote the proliferation and migration of the cells, the important characteristics of reactive astrocytes. In addition, overexpression of DSPP obviously increased the activation of Akt/mTOR pathway in astrocytes. Taken together, we demonstrated that DSPP may play a key role in the proliferation and migration of astrocytes, suggesting that targeting DSPP might be a promising therapeutic strategy for treating CNS injury which characterized by glia scar formation.

Spatial survey of non-collagenous proteins in mineralizing and non-mineralizing vertebrate tissues ex vivo

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca10(PO4)6(OH)2) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.

Differential intolerance to loss of function and missense mutations in genes that encode human matricellular proteins.

Targeted gene disruption in mice has provided valuable insights into the functions of matricellular proteins. Apart from missense and loss of function mutations that have been associated with inherited diseases, however, their functions in humans remain unclear. The availability of deep exome sequencing data from over 140,000 individuals in the Genome Aggregation Database provided an opportunity to examine intolerance to loss of function and missense mutations in human matricellular genes. The probability of loss-of-function intolerance (pLI) differed widely within members of the thrombospondin, CYR61/CTGF/NOV (CCN), tenascin, small integrin-binding ligand N-linked glycoproteins (SIBLING), and secreted protein, acidic and rich in cysteine (SPARC) gene families. Notably, pLI values in humans had limited correlation with viability of the corresponding homozygous null mice. Among the thrombospondins, only THBS1 was highly loss-intolerant (pLI = 1). In contrast, Thbs1 is not essential for viability in mice. Several known thrombospondin-1 receptors were similarly loss-intolerant, although thrombospondin-1 is not the exclusive ligand for some of these receptors. The frequencies of missense mutations in THBS1 and the gene encoding its signaling receptor CD47 indicated conservation of some residues implicated in specific receptor binding. Deficits in missense mutations were also observed for other thrombospondin genes and for SPARC, SPOCK1, SPOCK2, TNR, and DSPP. The intolerance of THBS1 to loss of function in humans and elevated pLI values for THBS2, SPARC, SPOCK1, TNR, and CCN1 support important functions for these matricellular protein genes in humans, some of which may relate to functions in reproduction or responding to environmental stresses.

Serum level of osteopontin in patients with alopecia areata and its correlation with ocular changes

Background Alopecia areata (AA) is a type of nonscarring hair loss affecting anagen-stage hair follicles with a multifactorial autoimmune pathogenesis and an unknown etiology. Osteopontin (OPN), also known as bone sialoprotein I and early T-lymphocyte activation-1, is a member of the small integrin-binding ligand N-linked glycoprotein family. OPN is widely distributed in human tissues and expressed in several cell types. The significant increase in the immunopositivity of the perifollicular inflammatory cells for OPN in AA cases as well as its strong expression in the hair follicles, especially the miniaturized ones, offers further proof for its role in AA. OPN was found to be strongly expressed in retina, trabecular meshwork, optic nerve, iris, and vitreous humour. Ocular findings associated with AA, the most prevalent of these, are cataracts and retinal pigment epithelial changes. Aim The aim of the present study is to evaluate serum level of OPN in patients with AA and correlate it with ocular changes to assess its possible role in the pathogenesis of the disease. Patients and methods This case–control study included 40 patients who presented with AA diagnosed on the basis of typical clinical features, representing the patient group. Moreover, 40 age-matched and sex-matched apparently healthy individuals were also included, representing the control group. All patients were recruited (according to inclusion and exclusion criteria) from the Outpatient Clinic of Dermatology and Venereology of Al-Zahra University Hospital during the period from February 2017 to February 2018. Results In our study, there was a highly statistically increase in serum level of OPN in the patient group. There was no significant difference between male and female regarding serum levels of OPN. There was a statistically significant positive correlation between serum OPN level in patient group and duration of disease. There was no correlation between serum OPN and age and severity of alopecia tool score. There was more significant increase in eye manifestations in patients than controls. Mean break-up time was statistically higher among patients than controls. There was a statistically highly significant positive correlation between serum OPN level and break-up time, lens abnormalities, and fundus abnormalities. Conclusion Patients with alopecia may have more lenticular and retinal abnormal findings than individuals without alopecia, but those findings do not interfere with visual acuity.

Osteopontin in Bone Metabolism and Bone Diseases.

Osteopontin (OPN), a secreted phosphoprotein, is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of cell matrix proteins and participates in many biological activities. Studies have shown that OPN plays a role in bone metabolism and homeostasis. OPN not only is an important factor in neuron-mediated and endocrine-regulated bone mass, but also is involved in biological activities such as proliferation, migration, and adhesion of several bone-related cells, including bone marrow mesenchymal stem cells, hematopoietic stem cells, osteoclasts, and osteoblasts. OPN has been demonstrated to be closely related to the occurrence and development of many bone-related diseases, such as osteoporosis, rheumatoid arthritis, and osteosarcoma. As expected, the functions of OPN in the bone have become a research hotspot. In this article, we try to decipher the mechanism of OPN-regulated bone metabolism and bone diseases.

Hypophosphatemic osteosclerosis, hyperostosis, and enthesopathy associated with novel homozygous mutations of DMP1 encoding dentin matrix protein 1 and SPP1 encoding osteopontin: The first digenic SIBLING protein osteopathy?

The SIBLINGs are a subfamily of the secreted calcium-binding phosphoproteins and comprise five small integrin-binding ligand N-linked glycoproteins [dentin matrix protein-1 (DMP1), secreted phosphoprotein-1 (SPP1) also called osteopontin (OPN), integrin-binding sialoprotein (IBSP) also called bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (DSPP)]. Each SIBLING has at least one "acidic, serine- and aspartic acid-rich motif" (ASARM) and multiple Ser-x-Glu/pSer sequences that when phosphorylated promote binding of the protein to hydroxyapatite for regulation of biomineralization. Mendelian disorders from loss-of-function mutation(s) of the genes that encode the SIBLINGs thus far involve DSPP causing various autosomal dominant dysplasias of dentin but without skeletal disease, and DMP1 causing autosomal recessive hypophosphatemic rickets, type 1 (ARHR1). No diseases have been reported from gain-of-function mutation(s) of DSPP or DMP1 or from alterations of SPP1, IBSP, or MEPE. Herein, we describe severe hypophosphatemic osteosclerosis and hyperostosis associated with skeletal deformity, short stature, enthesopathy, tooth loss, and high circulating FGF23 levels in a middle-aged man and young woman from an endogamous family living in southern India. Both shared novel homozygous mutations within two genes that encode a SIBLING protein: stop-gain ("nonsense") DMP1 (c.556G>T,p.Glu186Ter) and missense SPP1 (c.769C>T,p.Leu266Phe). The man alone also carried novel heterozygous missense variants within two additional genes that condition mineral homeostasis and are the basis for autosomal recessive disorders: CYP27B1 underlying vitamin D dependent rickets, type 1, and ABCC6 underlying both generalized arterial calcification of infancy, type 2 and pseudoxanthoma elasticum (PXE). By immunochemistry, his bone contained high amounts of OPN, particularly striking surrounding osteocytes. We review how our patients' disorder may represent the first digenic SIBLING protein osteopathy.

Osteonectin regulates the extracellular matrix mineralization of osteoblasts through P38 signaling pathway.

Osteonectin binds strongly to type I collagen and hydroxyapatite and plays a crucial role in extracellular matrix mineralization. Previous studies have also shown that p38 signaling pathway is an important regulator for osteoblast mineralization. This study focused on the role of osteonectin in regulating extracellular matrix mineralization via the p38 signaling pathway. Osteoblasts were isolated and cultured from parietal bones of neonatal Sprague-Dawley rats. The gene and protein expressions of noncollagen proteins (BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin), p38 mitogen-activated protein kinase, and SIBLINGs (Small Integrin-Binding LIgand N-linked Glycoproteins) members (DMP1, dentine matrix protein 1, DSPP, dentin sialophosphoprotein, and MEPE, matrix extracellular phosphoglycoprotein) were detected by reverse-transcription quantitative polymerase chain reaction and western blot analysis. Alizarin red staining, intracellular calcium assay, and transmission electron microscopy were used to detect mineralization. Initially, by adding osteonectin at different concentrations in osteoblasts and detecting the above mineralization indexes, 1 µg/ml was determined to be the optima osteonectin concentration, which significantly increased gene expressions of BSP, OPN, OCN, DMP1, MEPE, DSPP, and p38 in osteoblasts, p38 and p-p38 protein expressions were also significantly increased, mineralized nodules were significantly enhanced; when added with SB203580 (a specific inhibitor for p38) these effects were inhibited. Furthermore, osteoblasts transfected with Ad-p38 also significantly upregulated the protein and gene expressions of noncollagens and SIBLINGs members, whereas transfection of p38-rhRNA showed the opposite effect. Our data suggest that osteonectin regulates the extracellular matrix mineralization of osteoblasts through the P38 signaling pathway.

Abstract 4658: Cigarette smoke extract and benzo(a)pyrene induce osteopontin expression in lung cancer and promote mesenchymal stem cell recruitment

Abstract Cigarette smoking produces many organic compounds, including the carcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene, (BAP), which is linked to lung cancer. Tumor tissue recruits mesenchymal stem cells (MSCs; undifferentiated multipotent adult stem cells) to increase cancer cell proliferation, invasion and metastasis. We sought to determine the effects of cigarette smoke extract (CSE) and BAP stimulation on lung cancer cells and MSCs. We found that CSE and BAP increased mRNA and protein expression of osteopontin (OPN), a member of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs) family, but had no such effects on any other SIBLINGs proteins. A National Center for Biotechnology Information (NCBI) analysis of data from the Gene Expression Omnibus (GEO) database (GSE31210) identified much higher OPN expression in current and ex-cigarette smokers with lung cancer than in lung cancer patients who were nonsmokers. Our analysis found that stimulating lung cancer cells with CSE and BAP led to the recruitment of MSCs, which adhered to lung cancer cells via OPN production. Next, we used a gene set from the BioCarta database to investigate signaling pathways associated with lung cancer. We found that the MAPK, JAK/STAT and RAS signaling pathways had the highest repeatability in lung cancer patients who were cigarette smokers. We discovered that CSE and BAP only activated the JAK2/STAT3 pathway and that transfection of lung cancer cells with JAK2 and STAT3 small interfering RNAs interrupted CSE- and BAP-induced OPN expression, inhibiting MSC recruitment and adhesion. We are now investigating whether MSCs link back to lung cancer cells and further induce migration and invasion. In summary, our data indicate that CSE and BAP increase OPN expression in lung cancer cells through the JAK2/STAT3 signaling pathway and thereby recruit MSCs and their adhesion to cancer cells. Citation Format: Ya-Jing Jiang, An-Chen Chang, Po-Chun Chen, Chih-Hsin Tang. Cigarette smoke extract and benzo(a)pyrene induce osteopontin expression in lung cancer and promote mesenchymal stem cell recruitment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4658.

IMMUNOHISTOCHEMICAL DISTRIBUTION OF THE SIBLINGS IN AMELOBLASTOMA AND ODONTOGENIC KERATOCYST

Objective Ameloblastomas are benign but aggressive neoplasms of the jaw. The odontogenic keratocysts (OKC) are aggressive jaw cysts of odontogenic origin. A recent WHO classification designated OKC as a neoplasm but a current edition re-designated it as a cyst. The small integrin-binding ligand n-linked glycoproteins (SIBLINGs) are a family of molecules that some epithelial neoplasms use in furthering their progression. Members of the SIBLING family are bone sialoprotein (BSP), dentin matrix protein1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN). The objective of this study was to compare the expression of the SIBLINGs in ameloblastomas and odontogenic keratocysts. Findings Using immunohistochemistry, with appropriate controls, 49 cases of ameloblastomas and 35 cases of OKCs were screened for the expression of all five SIBLINGs in a retrospective study on archived paraffin sections. Immunoreactivity was scored as positive if > 10% of tumor/cyst cells stained for a SIBLING and negative if Conclusion While all members of the SIBLINGs are expressed in ameloblastomas and OKCs, DSPP expression in OKCs is significantly higher than in ameloblsatomas, whereas, MEPE expression in OKCs is considerably lower than in ameloblastomas. The differences in the expression of DSPP and MEPE between ameloblastomas and OKCs may indicate differences in the degree of aggressive behaviors.

Proteomics and N-glycoproteomics analysis of an extracellular matrix-based scaffold-human treated dentin matrix.

Extracellular matrix (ECM)-based biomaterials developed from mammalian tissues have been successfully used in preclinical and clinical tissue engineering applications. We have previously reported about the applicability of dentin-based scaffold, treated dentin matrix (TDM), for tooth root regeneration. However, TDM protein composition has not been characterized. Here, we used a shotgun proteomic strategy to profile human TDM proteome. N-glycoproteins were enriched by lectin affinity chromatography and identified by mass spectrometry. The total human TDM proteome was compared with the previously published human dentin proteome, and bioinformatics analysis were performed accordingly. In total, 708 proteins were identified by mass spectrometry in human TDM, of which 208 were N-glycoproteins with 318 identified glycosylation sites. Collagens, proteoglycans, small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and growth factors, such as COL1A1, biglycan, dentin sialoprotein, and transforming growth factor beta 1, were identified. Glycoproteins were enriched in "biological processes" Gene Ontology terms such as cellular process, biological regulation, response to stimulus, metabolic process, immune system process, and biological adhesion. Thus, our comprehensive study of the human TDM proteome revealed that dentin proteins are more heterogeneous than previously documented. Our findings provide clues for designing new biomaterials for tooth root regeneration and understanding dentin formation.

Survey of dentin sialophosphoprotein and its cognate matrix metalloproteinase-20 in human cancers.

BackgroundMatrix metalloproteinases‐20 (MMP20) expression is widely regarded as tooth specific, with expression limited to dental hard tissues. Recently, we reported MMP20 expression and interaction with dentin sialophosphoprotein (DSPP), a member of the Small Integrin Binding Ligand N‐linked Glycoproteins (SIBLINGs), in human oral squamous cell carcinoma (OSCC) and dysplastic oral premalignant lesions (OPLs), suggesting a role for MMP20‐DSPP interaction in oral carcinogenesis.MethodsThis study aimed to survey the expression of MMP20 and its cognate DSPP partner in the breast, colon, prostate, thyroid, and cervical neoplasms. Using commercially available tissue microarrays (TMAs) and cell lines, we performed immunohistochemistry, immunofluorescence, proximity ligation assay, and western blot experiments to determine the expressions of MMP20 and DSPP in the breast, colon, prostate, thyroid, cervical neoplasms, and their normal counterparts.ResultsSignificantly high expression levels of MMP20 and DSPP were observed in the malignant breast, colon, prostate, thyroid, and cervical neoplasms compared with their benign and normal counterparts. Furthermore, MMP20 levels increased with advanced stages of colon and thyroid cancers. DSPP expression increased significantly with tumor stage in all cancers examined.ConclusionsThe co‐localization and potential MMP20‐DSPP interaction previously reported in oral cancers are present in other cancers. These results suggest MMP20‐DSPP pairing as a potential marker of disease activity in some epithelial cancers with diagnostic and prognostic implications.

Isolation of SIBLING Proteins from Bone and Dentin Matrices.

The extracellular matrix of the bone and dentin contains several non-collagenous proteins (NCPs). One category of NCPs is termed the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family, which includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), etc. These proteins have abundant phosphoserines, aspartic acids, and glutamic acids. In this protocol, we describe the extraction of NCPs from the bone and dentin matrices using guanidine-HCl/EDTA and the isolation of polyanionic SIBLINGs from NCPs using ion-exchange fast protein liquid chromatography (FPLC) to separate the differentially charged proteins into different fractions through a gradient elution by NaCl.

Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.