Small Integrin-binding Ligand N-linked Glycoproteins Family Research Articles

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.

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.

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.

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.

Knockdown of DSPP inhibits the migration and invasion of glioma cells

Dentin sialophosphoprotein (DSPP) is a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins and has been proved to contribute to the migration of a variety of solid tumor cells. However, whether DSPP participates in the pathogenic process of glioma remains unknown. In this study, we aimed to investigate the expression and biological function of DSPP in human glioma cells. We demonstrated through Western blot that DSPP is overexpressed in glioma tissues comparing to normal brain tissues. To investigate the role of DSPP in glioma carcinogenesis, we reduced the DSPP expression by small interfering RNA (siRNA) and found that DSPP silencing significantly inhibited the migration and invasion of glioma cells, the critical characteristics of glioma. Furthermore, we showed that DSPP down-regulation significantly decreased the activation of the AKT/mTOR/p70S6K pathway in glioma cells. Taken together, these findings indicate that knockdown of DSPP inhibits glioma cells migration and invasion, suggesting that targeting DSPP might be a potentially effective therapeutic strategy for treating glioma.

Elevated serum levels of bone sialoprotein during ICU treatment predict long-term mortality in critically ill patients

Bone sialoprotein (BSP), a member of the SIBLINGs (for Small Integrin-Binding LIgand, N-linked Glycoproteins) family, has recently be associated to inflammatory and infectious diseases. We therefore measured BSP concentrations in 136 patients at admission to the intensive care unit (ICU) and 3 days of ICU. BSP levels were compared to 36 healthy blood donors and correlated to clinical data. In these analysis, BSP serum levels were strongly elevated at the time point of admission to the ICU when compared to healthy controls. Moreover BSP concentrations were significantly elevated after 3 days of treatment on the intensive care unit. A further increase in BSP levels was detected in patients with higher APACHE-II-scores and in patients with septic disease. While in most patients, BSP levels decreased during the first three days of treatment on a medical ICU, patients with persistently elevated BSP levels displayed an unfavorable outcome. In these patients, persistently elevated BSP concentrations were a superior predictor of mortality than established indicators of patient´ prognosis such as the SAPS2 or the APACHE-II score. In summary, our data argue for a novel utility for BSP as a biomarker in patients treated on a medical ICU.

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.

Matrix Metalloproteinase 20 Co-expression With Dentin Sialophosphoprotein in Human and Monkey Kidneys.

We recently reported the expression of matrix metalloproteinase 20 (MMP20), hitherto thought to be tooth specific, in the metabolically active ductal epithelial cells of human salivary glands. Furthermore, our report indicated that MMP20 co-expressed and potentially interacts with dentin sialophosphoprotein (DSPP), a member of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Our earlier reports have shown the co-expression of three MMPs, MMP2, MMP3, and MMP9, with specific members of the SIBLING family: bone sialoprotein, osteopontin, and dentin matrix protein 1, respectively. This study investigated the expression of MMP20 and verified its co-expression with DSPP in human and monkey kidney sections and human mixed renal cells by IHC, in situ proximity ligation assay, and immunofluorescence. Our results show that MMP20 is expressed in all segments of the human and monkey nephron with marked intensity in the proximal and distal tubules, and was absent in the glomeruli. Furthermore, MMP20 co-expressed with DSPP in the proximal, distal, and collecting tubules, and in mixed renal cells. Consistent with other SIBLING-MMP pairs, the DSPP-MMP20 pair may play a role in the normal turnover of cell surface proteins and/or repair of pericellular matrix proteins of the basement membranes in the metabolically active duct epithelial system of the nephrons.

Expression of Matrix Metalloproteinase (MMP)-20 and Potential Interaction with Dentin Sialophosphoprotein (DSPP) in Human Major Salivary Glands.

Matrix metalloproteinase-20 (MMP-20) expression is widely regarded as tooth-specific, with expression limited to dental hard tissues. Necessary for sound enamel formation, MMP-20 and MMP-2 proteolytically process dentin sialophosphoprotein (DSPP) into dentin sialoprotein, dentin phosphoprotein, and dentin glycoprotein during tooth formation. In the mid-2000s, three members of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs) were reported to bind specifically with high affinity (nM) to, and activate, three MMPs in vitro: bone sialoprotein with MMP-2; osteopontin with MMP-3; and dentin matrix protein1 with MMP-9. The SIBLING-MMP interaction was confirmed in biological systems such as the ducts of salivary glands, where all five members of the SIBLINGs are expressed. Recently, we documented MMP-20 expression and interaction with DSPP (another member of the SIBLING family) in human oral squamous cell carcinoma. Here we report the expression of MMP-20, and confirm its co-expression and potential interaction with DSPP in human major salivary gland tissues and cell line using immunohistochemistry, immunofluorescence, western blot, quantitative RT-PCR, and proximity ligation assay. This report reinforces our earlier suggestion that the SIBLING-MMP complexes may be involved in the turnover of extracellular proteins damaged by oxidation byproducts in metabolically active duct epithelial systems.

The bioactive acidic serine- and aspartate-rich motif peptide.

The organic component of the bone matrix comprises 40% dry weight of bone. The organic component is mostly composed of type I collagen and small amounts of non-collagenous proteins (NCPs) (10-15% of the total bone protein content). The small integrin-binding ligand N-linked glycoprotein (SIBLING) family, a NCP, is considered to play a key role in bone mineralization. SIBLING family of proteins share common structural features and includes the arginine-glycine-aspartic acid (RGD) motif and acidic serine- and aspartic acid-rich motif (ASARM). Clinical manifestations of gene mutations and/or genetically modified mice indicate that SIBLINGs play diverse roles in bone and extraskeletal tissues. ASARM peptides might not be primary responsible for the functional diversity of SIBLINGs, but this motif is suggested to be a key domain of SIBLINGs. However, the exact function of ASARM peptides is poorly understood. In this article, we discuss the considerable progress made in understanding the role of ASARM as a bioactive peptide.

Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate

Native bone tissue is composed of a matrix of collagen, noncollagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite. The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary noncollagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium-binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared, and then (125) I-radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations, all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5, 10, and 24 h at 37°C. The resulting mineral morphology was assessed by atomic force microscopy, and the composition was determined using photochemical assays. Mineralization was seen in the presence of all the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation.

The Roles of Osteocyte Signaling in Bone

Osteocytes are the cells that reside within the bone matrix and make up 90% to 95% of the cellular component of the tissue. Until recently, they were thought to serve as little more than “space holders” in the tissue. However, significant advances have demonstrated that osteocytes are of the utmost importance in regulating the dynamic nature of bone; indeed, it may be that this is their major function. Specifically, osteocyte signaling in skeletal metabolism contributes to (1) regulation of local mineralization, (2) regulation of systemic mineralization, (3) bone formation by osteoblasts, and (4) bone resorption by osteoclasts. Regulation of Local Mineralization Because the osteocyte is, by definition, located within the dense, calcified matrix of bone, it is reasonable to suggest that some part of its repertoire is devoted to developing and maintaining that matrix. Three main proteins have been identified in the mineralization process: dentin matrix acidic phosphoprotein-1 (DMP1), phosphate-regulating neutral endopeptidase (PHEX), and matrix extracellular phosphoglycoprotein (MEPE). As a highly phosphorylated protein, DMP1 seems to be involved in the regulation of hydroxyapatite formation. PHEX is thought to play a similar role and likely interacts extensively with DMP1; indeed, deletion of either gene in mice results in a similar, if not identical, bone phenotype.1 MEPE, along with DMP1, belongs to the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family and is thought to be involved in the regulation of matrix mineralization. Regulation of Systemic Mineralization The transport of inorganic minerals within the skeleton necessarily requires interaction with other organs—particularly the kidneys. One factor that is central to the regulation of the bone-kidney axis is fibroblast growth factor-23 (FGF-23). Abnormal circulating levels of FGF-23, either increased or decreased, can cause a variety of disorders. A fascinating feature of this protein is its predominant production by osteocytes and its regulation by PHEX, DMP1, and MEPE.2 It appears that osteocytes are capable of coordinating bone formation with renal regulation of systemic phosphate homeostasis through the regulated expression of sclerostin, FGF-23, and possibly other factors. Osteocyte Signaling and Bone Formation by Osteoblasts In addition to their role in local and systemic mineral homeostasis, osteocytes have a direct effect on the bone formation activities of osteoblasts. One product in particular that has this effect is sclerostin, which is encoded by the SOST gene. Highly expressed in osteocytes, this factor appears to directly inhibit bone formation.3 By presenting the opportunity to tap directly into bone-building pathway, SOST could yield novel anabolic therapeutic agents to combat such diseases as osteoporosis. Osteocyte Signaling and Bone Formation by Osteoclasts Early discoveries of the relationship between osteocytes and osteoclast activity showed that isolated avian osteocytes could support osteoclast formation in the absence of any other osteotropic factors.4 This fact confirmed that the necessary intracellular machinery exists in osteocytes to produce cytokines related to osteoclastogenesis. Of particular utility in probing the relationship between osteocytes and osteoclastogenesis is the study of their response to localized, noninflammatory microinjury. This response stems from the unique relationship between microdamage in bone and the intrinsic tissue-repair processes. Existing in vivo loading models have been developed, such that microdamage can be induced in localized regions of bone without causing an inflammatory response in the surrounding tissues. In early studies of this kind, fatigueinduced microdamage was shown to cause apoptosis (ie, regulated cell death) in osteocytes nearby, which would subsequently undergo resorption by osteoclasts. Extensive research has been performed since then on the characterization of this system.5 Most recently, it has been shown that apoptotic osteocytes themselves do not upregulate pro-osteoclastogenic markers following fatigue.6 A separate and discrete group of osteocytes surrounding them are the source of this cytokine signaling. This penumbra-like phenomenon of nonapoptotic cells surrounding those undergoing apoptosis has also been reported in other focal injury systems, such as those of the ischemic brain and heart.7 Our knowledge of the contribution of osteocytes to distinct aspects of skeletal metabolism is expanding at an unprecedented rate. The early belief that osteocytes were merely placeholders in the mineralized matrix has been displaced by exciting, and sometimes unexpected, discoveries that reveal this population of cells to be a highly sensitive, active, and responsive network. The recent discovery that apoptosis in osteocytes is essential for osteoclastogenesis, but that it is not those dying osteocytes but rather nearby neighbors that carry out that task, indicates a division of labor among osteocytes in response to damage paralleling that seen in ischemic injury. Such similarities suggest that common pathways and mechanisms probably exist in localized remodeling of many tissue types.

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.

Dentin Matrix Protein 1 (DMP1) Signals via Cell Surface Integrin

Dentin matrix phosphoprotein 1 (DMP1) is a non-collagenous, acidic extracellular matrix protein expressed chiefly in bone and dentin. We examined the DMP1 ability to engage cell-surface receptors and subsequently activate intracellular signaling pathways. Our data indeed show that the presence of extracellular DMP1 triggers focal adhesion point formation in human mesenchymal stem cells and osteoblast-like cells. We determine that DMP1 acts via interaction with αvβ3 integrin and stimulates phosphorylation of focal adhesion kinase. Further biochemical characterization confirms the activation of downstream effectors of the MAPK pathways, namely ERK and JNK, after DMP1 treatment. This activation is specifically inhibitable and can also be blocked by the addition of anti-αvβ3 integrin antibody. Furthermore, we show that extracellular treatment with DMP1 stimulates the translocation of phosphorylated JNK to the nucleus and a concomitant up-regulation of transcriptional activation by phosphorylated c-Jun. The evidence presented here indicates that DMP1 is specifically involved in signaling via extracellular matrix-cell surface interaction. Combined with the published DMP1-null data (Feng, J. Q., Ward, L. M., Liu, S., Lu, Y., Xie, Y., Yuan, B., Yu, X., Rauch, F., Davis, S. I., Zhang, S., Rios, H., Drezner, M. K., Quarles, L. D., Bonewald, L. F., and White, K. E. (2006) Nat. Genet. 38, 1310-1315) it can be hypothesized that DMP1 could be a key effector of ECM-osteocyte signaling.

Primary Structure and Phosphorylation of Dentin Matrix Protein 1 (DMP1) and Dentin Phosphophoryn (DPP) Uniquely Determine Their Role in Biomineralization.

The SIBLING (small integrin-binding ligand N-linked glycoproteins) family is the major group of noncollagenous proteins in bone and dentin. These extremely acidic and highly phosphorylated extracellular proteins play critical roles in the formation of collagenous mineralized tissues. Whereas the lack of individual SIBLINGs causes significant mineralization defects in vivo, none of them led to a complete cessation of mineralization suggesting that these proteins have overlapping functions. To assess whether different SIBLINGs regulate biomineralization in a similar manner and how phosphorylation impacts their activity, we studied the effects of two SIBLINGs, dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP), on mineral morphology and organization in vitro. Our results demonstrate distinct differences in the effects of these proteins on mineralization. We show that phosphorylation has a profound effect on the regulation of mineralization by both proteins. Specifically, both phosphorylated proteins facilitated organized mineralization of collagen fibrils and phosphorylated DMP1-induced formation of organized mineral bundles in the absence of collagen. In summary, these results indicate that the primary structure and phosphorylation uniquely determine functions of individual SIBLINGs in regulation of mineral morphology and organization.

Dentin Sialophosphoprotein (DSPP) Gene-Silencing Inhibits Key Tumorigenic Activities in Human Oral Cancer Cell Line, OSC2

BackgroundWe determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells.Methodology/Principal FindingsMultiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y = 0.850x, p<0.001) (y = 1.156x, p<0.001)}, MMP-3 {(y = 0.994x, p<0.001) (y = 1.324x, p = 0.004)}, and MMP-9 {(y = 1.248x, p = 0.005, y = 0.809, p = 0.013)}.Conclusions/Significance DSPP-silencing in OSC2 cell decreased salient hallmarks of oral tumorigenesis and provides the first functional evidence of a potential key role for DSPP in oral cancer biology. The down-regulation of MMP-2, MMP-3, MMP-9, p53 and VEGF in DSPP-silenced OSC2 cells provides a significant functional/molecular framework for deciphering the mechanisms of DSPP activities in oral cancer biology.

Distribution of Small Integrin-binding Ligand, N-linked Glycoproteins (SIBLING) in the Articular Cartilage of the Rat Femoral Head

The small integrin-binding ligand, N-linked glycoprotein (SIBLING) family is closely related to osteogenesis. Until recently, little was known about their existence in articular cartilage. In this study, we systematically evaluated the presence and distribution of four SIBLING family members in rat femoral head cartilage: dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and dentin sialophosphoprotein (DSPP). First, non-collagenous proteins were extracted and then separated by ion-exchange chromatography. Next, the protein extracts eluted by chromatography were analyzed by Stains-all staining and Western immunoblotting. IHC was used to assess the distribution of these four SIBLING family members in the femoral head cartilage. Both approaches showed that all the four SIBLING family members are expressed in the femoral head cartilage. IHC showed that SIBLING members are distributed in various locations throughout the articular cartilage. The NH₂-terminal fragments of DMP1, BSP, and OPN are present in the cells and in the extracellular matrix, whereas the COOH-terminal fragment of DMP1 and the NH₂-terminal fragment of DSPP are primarily intracellularly localized in the chondrocytes. The presence of the SIBLING family members in the rat femoral head cartilage suggests that they may play important roles in chondrogenesis.

Selective osteopontin knockdown exerts anti‐tumoral activity in a human glioblastoma model

Osteopontin (OPN), a member of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) family, is overexpressed in human glioblastoma. Higher levels of OPN expression correlate with increased tumor grade and enhanced migratory capacity of tumor cells. Based on these observations, we explored the possibility that knocking down OPN expression in glioblastoma cells could exert an anti-tumoral activity using an avian in vivo glioblastoma model that mimics closely human gliobastoma. Human U87-MG glioma cells transfected with specific anti-OPN small interfering RNAs (siRNAs) were grafted onto the chicken chorio-allantoic membrane (CAM). OPN-deficient U87-MG cells gave rise to tumors that were significantly smaller than tumors formed from untransfected cells (paired t-test, p < 0.05). Accordingly, the amount of proliferating cells in OPN-deficient tumors showed a six-fold reduction when compared to control tumors. However, OPN inhibition did not affect significantly tumor-associated angiogenesis. In vitro, OPN-silenced U87-MG and U373-MG cells showed decreased motility and migration. This is the first demonstration that OPN inhibition blocks glioma tumor growth, making this invasion-related protein an attractive target for glioma therapy.

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.

Osteopontin selectively regulates p70S6K/mTOR phosphorylation leading to NF-κB dependent AP-1-mediated ICAM-1 expression in breast cancer cells

BackgroundBreast cancer is one of the most frequently diagnosed cancer and accounts for over 400,000 deaths each year worldwide. It causes premature death in women, despite progress in early detection, treatment, and advances in understanding the molecular basis of the disease. Therefore, it is important to understand the in depth mechanism of tumor progression and develop new strategies for the treatment of breast cancer. Thus, this study is aimed at gaining an insight into the molecular mechanism by which osteopontin (OPN), a member of SIBLING (Small Integrin Binding LIgand N-linked Glycoprotein) family of protein regulates tumor progression through activation of various transcription factors and expression of their downstream effector gene(s) in breast cancer.ResultsIn this study, we report that purified native OPN induces ICAM-1 expression in breast cancer cells. The data revealed that OPN induces NF-κB activation and NF-κB dependent ICAM-1 expression. We also observed that OPN-induced NF-κB further controls AP-1 transactivation, suggesting that there is cross talk between NF-κB and AP-1 which is unidirectional towards AP-1 that in turn regulates ICAM-1 expression in these cells. We also delineated the role of mTOR and p70S6 kinase in OPN-induced ICAM-1 expression. The study suggests that inhibition of mTOR by rapamycin augments whereas overexpression of mTOR/p70S6 kinase inhibits OPN-induced ICAM-1 expression. Moreover, overexpression of mTOR inhibits OPN-induced NF-κB and AP-1-DNA binding and transcriptional activity. However, rapamycin further enhanced these OPN-induced effects. We also report that OPN induces p70S6 kinase phosphorylation at Thr-421/Ser-424, but not at Thr-389 or Ser-371 and mTOR phosphorylation at Ser-2448. Overexpression of mTOR has no effect in regulation of OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424. Inhibition of mTOR by rapamycin attenuates Ser-371 phosphorylation but does not have any effect on Thr-389 and Thr-421/Ser-424 phosphorylation of p70S6 kinase. However, OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424 is being controlled by MEK/ERK pathway.ConclusionThese results suggest that blocking of OPN-induced ICAM-1 expression through mTOR/p70S6 kinase signaling pathway may be an important therapeutic strategy for the treatment of breast cancer.