Small Integrin-binding Ligand N-linked Glycoprotein Proteins Research Articles

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.

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.

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.

SB225002 inhibits prostate cancer invasion and attenuates the expression of BSP, OPN and MMP‑2.

The mechanisms of malignant cell metastasis to secondary sites are complex and multifactorial. Studies have demonstrated that small integrin-binding ligand N-linked glycoproteins (SIBLINGs), particularly bone sialoprotein (BSP) and osteopontin (OPN), are involved in neoplastic growth and metastasis. SIBLINGs promote malignant cell invasion and metastasis by enhancing matrix metalloproteinase 2 (MMP-2) and MMP-9 expression. Moreover, BSP and OPN can combine with integrin, which is located on the tumor cell surface, to further promote the malignant behavior of tumor cells. In the present study, we investigated whether SB225002, a specific CXCR2 receptor antagonist, can inhibit prostate cancer cell expression of BSP and OPN and reduce cancer cell invasion ability. A series of experiments showed that after SB225002 treatment, the proliferation, invasion and migration of two androgen-independent prostate cancer cell lines were inhibited, but this inhibitory effect was not observed on androgen-dependent prostate cancer cells. Western blotting showed that the PI3K signaling pathway could regulate the expression of SIBLING and MMP family proteins, and SB22055 could reduce the expression of BSP, OPN and MMP-2 in prostate cancer cells by inhibiting AKT/mTOR phosphorylation. Finally, in vivo experiments confirmed that SB225002 inhibited the proliferation of prostate cancer cells in vivo, and the expression levels of BSP, OPN and MMP-2 were also inhibited.

The role of bone sialoprotein in the tendon–bone insertion

Tendons/ligaments insert into bone via a transitional structure, the enthesis, which is susceptible to injury and difficult to repair. Fibrocartilaginous entheses contain fibrocartilage in their transitional zone, part of which is mineralized. Mineral-associated proteins within this zone have not been adequately characterized. Members of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family are acidic phosphoproteins expressed in mineralized tissues. Here we show that two SIBLING proteins, bone sialoprotein (BSP) and osteopontin (OPN), are present in the mouse enthesis. Histological analyses indicate that the calcified zone of the quadriceps tendon enthesis is longer in Bsp−/− mice, however no difference is apparent in the supraspinatus tendon enthesis. In an analysis of mineral content within the calcified zone, micro-CT and Raman spectroscopy reveal that the mineral content in the calcified fibrocartilage of the quadriceps tendon enthesis are similar between wild type and Bsp−/− mice. Mechanical testing of the patellar tendon shows that while the tendons fail under similar loads, the Bsp−/− patellar tendon is 7.5% larger in cross sectional area than wild type tendons, resulting in a 16.5% reduction in failure stress. However, Picrosirius Red staining shows no difference in collagen organization. Data collected here indicate that BSP is present in the calcified fibrocartilage of murine entheses and suggest that BSP plays a regulatory role in this structure, influencing the growth of the calcified fibrocartilage in addition to the weakening of the tendon mechanical properties. Based on the phenotype of the Bsp−/− mouse enthesis, and the known in vitro functional properties of the protein, BSP may be a useful therapeutic molecule in the reattachment of tendons and ligaments to bone.

Les protéines SIBLING

The small integrin-binding ligand N-linked glycoprotein (SIBLING) family consists of osteopontin (OPN), bonesialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE). These proteins, initially identified in bone and teeth, share many structural characteristics. It is now well established that they are over expressed in many tumors and play a critical role at different steps of cancer development. In this review, we describe the roles of SIBLING proteins at different stages of cancer progression including cancer cell adhesion, proliferation, migration, invasion, metastasis and angiogenesis.

The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling

The small integrin-binding ligand N-linked glycoprotein (SIBLING) family consists of osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein. These proteins share many structural characteristics and are primarily located in bone and dentin. Accumulating evidence has implicated the SIBLING proteins in matrix mineralisation. Therefore, in this review, we discuss the individual role that each of the SIBLING proteins has in this highly orchestrated process. In particular, we emphasise how the nature and extent of their proteolytic processing and post-translational modification affect their functional role. Finally, we describe the likely roles of the SIBLING proteins in clinical disorders of hypophosphataemia and their potential therapeutic use.

Expression and distribution of SIBLING proteins in the predentin/dentin and mandible of hyp mice

Human X-linked hypophosphatemia (XLH) and its murine homologue, Hyp are caused by inactivating mutations in PHEX gene. The protein encoded by PHEX gene is an endopeptidase whose physiological substrate(s) has not been identified. Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP), two members of the Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family are proteolytically processed. It has been speculated that PHEX endopeptidase may be responsible for the proteolytic cleavage of DMP1 and DSPP. To test this hypothesis and to analyse the distribution of SIBLING proteins in the predentin/dentin complex and mandible of Hyp mice, we compared the expression of four SIBLING proteins, DMP1, DSPP, bone sialoprotein (BSP) and osteopontin (OPN) between Hyp and wild-type mice. These SIBLING proteins were analysed by protein chemistry and immunohistochemistry. (1) Dentin matrix protein 1 and DSPP fragments are present in the extracts of Hyp predentin/dentin and bone; (2) the level of DMP1 proteoglycan form, BSP and OPN is elevated in the Hyp bone. The PHEX protein is not the enzyme responsible for the proteolytic processing of DMP1 and DSPP. The altered distribution of SIBLING proteins may be involved in the pathogenesis of bone and dentin defects in Hyp and XLH.

Distribution of Small Integrin-Binding LIgand, N-linked Glycoproteins (SIBLING) in the condylar cartilage of rat mandible

The Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family is one category of non-collagenous proteins closely related to osteogenesis. In this study, the authors systematically evaluated the presence and distribution of four SIBLING family members, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (OPN), in rat mandibular condylar cartilage using protein chemistry and immunohistochemistry. For protein chemistry, SIBLING proteins in the dissected condylar cartilage were extracted with 4M guanidium-HCl, separated by ion-exchange chromatography, and analyzed by Western immunoblotting. Immunohistochemistry was employed to assess the distribution of these four SIBLING proteins in the condylar cartilage of 2-, 5- and 8-week-old rats. Results from both approaches showed that all four members are expressed in the condylar cartilage. DSPP, unlike that observed in dentin and bone, exists as a full-length form (uncleaved) in the condylar cartilage. The NH(2)-terminal fragment of DMP1 is mainly detected in the matrix of the cartilage while the COOH-terminal fragment is primarily localized in the nuclei of cells in the chondroblastic and hypertrophic layers. The data obtained in this investigation provide clues about the potential roles of these SIBLING proteins in chondrogenesis.

Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.

Expression and processing of small integrin-binding ligand N-linked glycoproteins in mouse odontoblastic cells

Objective Small integrin-binding ligand N-linked glycoproteins (SIBLINGs) are expressed in dentin and believed to control dentinogenesis. Five members of SIBLING family include bone sialoprotein (BSP), osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). These genes are clustered on chromosome 4q in humans and share similar biological features. DSPP and DMP1 are processed into given structural/functional fragments in rat and porcine. It still remains unclear whether these evidences occur in mouse and other SIBLING members are also processed into given fragments from their parent precursors. The aim of this study was to identify expression and processing of the five proteins in two mouse odontoblastic cell lines. Design Two mouse odontoblastic cells were used to study expression and processing of the five SIBLING proteins by immunohistochemistry and Western blot analyses. Results Immunohistochemistry study showed that all of the five SIBLING members were expressed within the cytoplasm and cellular processes in the mouse odontoblastic cell lines. Expression levels of DMP1 and DSPP were higher in differentiated mouse odontoblasts than undifferentiated mouse odontoblasts. Immunolabelling signal of DSP and MEPE was also detected within the nucleus in the two cell lines. Western blot assay indicated that all five members were processed into at least two fragments in these cells. Conclusions These results suggest that different processed products and expression levels of the SIBLING proteins may play distinct biological functions in tooth development and mineralisation.

MC3T3-E1 cell adhesion to hydroxyapatite with adsorbed bone sialoprotein, bone osteopontin, and bovine serum albumin

Native bone tissue is composed of a complex matrix of collagen, non-collagenous proteins, and hydroxyapatite (HAP). Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the non-collagenous protein family termed the SIBLING (small integrin-binding ligand, N-linked glycoproteins) proteins, which are primarily found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen, while the MC3T3-E1 cell adhesion was shown to be dependant on the conformational flexibility of BSP specifically bound to collagen. Additionally, OPN was shown to play a greater role than BSP for cell binding to collagen. In this work, the orientations and conformations of BSP and OPN specifically bound to HAP are probed under similar conditions. Radiolabeled adsorption isotherms were obtained for BSP and OPN on HAP formed from a simulated body fluid, and the results show that HAP has the capacity to bind significantly more BSP than OPN. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of adsorbed BSP and OPN specifically bound to HAP. It was found that there is a preference for cell binding to HAP with adsorbed BSP as compared to OPN, but not to a statistically significant level. However, the maximum cell binding was observed on HAP substrates with adsorbed heat denatured bovine serum albumin (BSA). The influence of BSA on cell binding was shown to be concentration dependant and it is believed that the adsorbed BSA modulates the proliferation state of the bound cells.

Immunohistochemical study of small integrin‐binding ligand, N‐linked glycoproteins in reactionary dentin of rat molars at different ages

Small integrin-binding ligand, N-linked glycoproteins (SIBLING) are believed to play key roles in the process of biomineralization. Reactionary dentin (RD), formed by odontoblasts in response to external stimuli, differs morphologically from primary dentin (PD). To test our hypothesis that the microscopic changes reflect variations in molecular mechanisms involved in formation of the two forms of dentin, and to characterize RD further, we compared the distributions of four SIBLING proteins [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP)] in naturally occurring RD with those in PD. Molars of rats aged 12, 18, 24 and 36 wk were analyzed using immunohistochemistry with antibodies against BSP, OPN, DMP-1, and dentin sialoprotein (a fragment of DSPP). Differences in the distribution of the four SIBLING proteins were evident. Bone sialoprotein, not seen in PD, was consistently observed in RD. Osteopontin, almost absent from PD, was clearly observed in RD. The expression levels of DMP-1 and DSP in RD were lower than in PD. Elevated expression of BSP and OPN, along with a marked decrease of dentin sialoprotein and DMP-1 in RD, suggests a difference in the mechanism of formation of the two forms of dentin.

Transglutaminase Crosslinking of SIBLING Proteins in Teeth

Transglutaminase 2 (TG2), a protein-crosslinking enzyme, participates in extracellular matrix maturation and cell adhesion in cartilage and bone. We hypothesized that TG2 has similar roles in teeth. A TG activity assay and immunoblotting of rat tooth extracts showed TG activity and the presence of high-molecular-weight forms of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) proteins: dentin matrix protein 1 (DMP1), dentin phosphoprotein (DPP), and bone sialoprotein (BSP). DMP1 and BSP, each containing both glutamine and lysine residues critical for crosslink formation, readily formed polymers in vitro when incubated with TG2. The ability of glutamine-lacking DPP to form polymers in vitro and in vivo demonstrates that it could act as a lysine donor for crosslinking, potentially having protein crosslinking partner(s) in teeth. Consistent with a role in cell adhesion, the TG2 isoform was co-localized by immunohistochemistry with its substrates at cell-matrix adhesion sites, including along odontoblast tubules (DMP1 and DPP), in the pericellular matrix of cementocytes (DMP1), and in predentin (BSP).

Renal expression of SIBLING proteins and their partner matrix metalloproteinases (MMPs)

Three members of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of proteins have recently been shown to bind and activate specific promatrix metalloproteinases (MMPs) and to overcome the inhibition of tissue inhibitors of MMPs (TIMPs). Although usually associated with mineralized tissues, we have shown that the SIBLINGs and their MMP partners, when known, are coexpressed in salivary gland ductal cells. The present study examined the expression patterns of both the SIBLINGs and their MMP partners in adult kidney. The expression patterns of all five SIBLINGs known to date, and their MMP partners were determined in monkey kidney using immunohistochemistry and in situ hybridization techniques. Bone sialoprotein (BSP) and its partner, MMP-2, were coexpressed in both the proximal and distal tubules. Osteopontin, as previously shown, was expressed in the distal tubules while its partner MMP-3 was expressed in both the proximal tubule and distal tubles. Dentin matrix protein-1 (DMP1) and MMP-9 were coexpressed throughout the nephron, including both parietal cells of Bowman's capsule and the thin limb of the loop of Henle. Dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE) were expressed in the proximal tubule and distal tubule, and proximal tubule, respectively. In contrast to salivary gland in which all SIBLINGs and their MMP partners were coexpressed throughout the length of the ducts, these proteins were differentially expressed within the normal adult nephron. We hypothesize that the cells use the SIBLING/MMP pairs in the normal turnover of cell surface proteins and/or pericellular matrix proteins such as those in basement membranes.

Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis.

The extracellular matrix (ECM) of bone and dentin contains several non-collagenous proteins. One category of non-collagenous protein is termed the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family, that includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). These polyanionic SIBLING proteins are believed to play key biological roles in the mineralization of bone and dentin. Although the specific mechanisms involved in controlling bone and dentin formation are still unknown, it is clear that some functions of the SIBLING family members are dependent on the nature and extent of post-translational modifications (PTMs), such as phosphorylation, glycosylation, and proteolytic processing, since these PTMs would have significant effects on their structure. OPN and BSP are present in the ECM of bone and dentin as full-length forms, whereas amino acid sequencing indicates that DMP1 and DSPP exist as proteolytically processed fragments that result from scission of X-Asp bonds. We hypothesized that the processing of DMP1 and DSPP is catalyzed by the PHEX enzyme, since this protein, an endopeptidase that is predominantly expressed in bone and tooth, has a strong preference for cleavage at the NH2-terminus of aspartyl residue. We envision that the proteolytic processing of DMP1 and DSPP may be an activation process that plays a significant, crucial role in osteogenesis and dentinogenesis, and that a failure in this processing would cause defective mineralization in bone and dentin, as observed in X-linked hypophosphatemic rickets.

Dentin matrix protein 1 is expressed in human lung cancer.

We have previously shown that breast and prostate cancers express bone matrix proteins. DMP1 expression was evaluated in 59 human lung cancer samples at the protein and mRNA levels. It was detectable in 80% of the cases, suggesting a potential role for DMP1 in tumor progression and bone metastasis. Previously, we and others have shown that bone extracellular matrix proteins such as bone sialoprotein (BSP) and osteopontin (OPN) are expressed in various types of cancer that are characterized by a high affinity for bone including breast, prostate, and lung adenocarcinoma. Based on biochemical and genetic features, BSP, OPN, dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP) have been recently classified in a unique family named SIBLING (small integrin-binding ligand, N-linked glycoprotein). Therefore, we investigated whether DMP1 could also be detected in osteotropic cancers. We first used a cancer array for evaluating the relative abundance of DMP1 transcript in a broad spectrum of human cancer tissues. This screening showed that DMP1 was strongly detectable in lung tumors compared with normal corresponding tissue. In a second step, we used an immunophosphatase technique and a specific polyclonal antibody directed against DMP1 to examine the expression of DMP1 in 59 human non-small cell lung cancer samples, including 29 squamous carcinoma, 20 adenocarcinoma, and 10 bronchioloalveolar carcinoma. Student's t-test was used to determine the statistical significance of immunostaining scores between the lung cancer histological groups studied and between cancer and normal lung tissues. Our results show that DMP1 is detectable in 90% of the adenocarcinoma and squamous carcinoma analyzed while 8 of 10 bronchioloalveolar specimens were negative. DMP1 immunostaining intensity and extent scores were significantly higher in adenocarcinoma (p = 0.0004) and squamous carcinoma (p < 0.0001) samples compared with adjacent normal lung tissue. In situ hybridization experiments confirmed that DMP1 mRNA is localized in lung cancer cells. In this study, we show that a third SIBLING protein is ectopically expressed in lung cancer. The role of DMP1 in lung cancer is largely unknown. Further studies are required to determine the implication of this protein, next to its sisters SIBLING proteins, in tumor progression and bone metastasis development.

Flexible Structures of SIBLING Proteins, Bone Sialoprotein, and Osteopontin

Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.