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Larry W. Fisher - One of the best experts on this subject based on the ideXlab platform.
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bone Sialoprotein is localized to the root surface during cementogenesis
Journal of Bone and Mineral Research, 2009Co-Authors: R L Macneil, Catherine Strayhorn, N Sheng, Larry W. Fisher, Martha J. SomermanAbstract:: Bone Sialoprotein (BSP), an RGD-containing protein with cell attachment properties, is believed to play a regulatory role in the biomineralization of various connective tissues. To determine its possible role in tooth root formation, murine dentoalveolar tissues at sequential phases of development were analyzed immunohistochemically for the presence of BSP. BSP was localized to alveolar bone and cementum at time points associated with initial mineralization of these tissues. In addition, northern blot analyses of dental follicle tissue at day 27 of tooth development indicated that BSP mRNA is expressed by dental follicle cells at a time point coincident with the initiation of cementogenesis on the peripheral tooth root surface. Collectively, these findings indicate that BSP may play an important role in the formation and mineralization of cementum.
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small integrin binding ligand n linked glycoproteins siblings multifunctional proteins in cancer
Nature Reviews Cancer, 2008Co-Authors: Akeila Bellahcene, Larry W. Fisher, Vincent Castronovo, Kalu U E Ogbureke, Neal S. FedarkoAbstract:Numerous components and pathways are involved in the complex interplay between cancer cells and their environment. The family of glycophosphoproteins comprising osteopontin, bone Sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein - small integrin-binding ligand N-linked glycoproteins (SIBLINGs) - are emerging as important players in many stages of cancer progression. From their detection in various human cancers to the demonstration of their key functional roles during malignant transformation, invasion and metastasis, the SIBLINGs are proteins with potential as diagnostic and prognostic tools, as well as new therapeutic targets.
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elevated serum bone Sialoprotein and osteopontin in colon breast prostate and lung cancer
Clinical Cancer Research, 2001Co-Authors: Neal S. Fedarko, Alka Jain, Abdullah Karadag, Matthew R Van Eman, Larry W. FisherAbstract:Purpose: Histological studies have shown that the two Sialoproteins, bone Sialoprotein (BSP) and osteopontin (OPN), are induced in multiple types of cancer. We have recently found that these proteins are bound in serum to complement factor H and that the complex must be disrupted to generate free protein to measure their total levels. We hypothesized that measuring total BSP and OPN levels would provide informative markers for the detection of cancer. Experimental Design: As a proof of concept study, serum from patients with diagnosed breast, colon, lung, or prostate cancer (n = 20 for each type) as well as normal serum (n = 77) were analyzed using competitive ELISAs developed for BSP and OPN. Sensitivity, specificity, as well as positive and negative predictive values were determined for each Sialoprotein and cancer type. The relationship between sensitivity and specificity was profiled by receiver operating characteristic curves. Results and Conclusions: Determined values for serum BSP in ng/ml were 285 ± 19 for prostate, 373 ± 19 for colon, 318 ± 18 for breast, 155 ± 11 for lung cancer sera, and 154 ± 13 for normal sera. Values of OPN in ng/ml were 653 ± 39 for prostate, 449 ± 22 for colon, 814 ± 53 for breast, 724 ± 33 for lung, and 439 ± 30 for normal sera. The assays provide a high degree of sensitivity and specificity that enables the detection of colon, breast, prostate, and lung cancer.
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Flexible structures of SIBLING proteins, bone Sialoprotein, and osteopontin.
Biochemical and biophysical research communications, 2001Co-Authors: Larry W. Fisher, Dennis A. Torchia, Berthold Fohr, M F Young, Neal S. FedarkoAbstract: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.
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expression of bone Sialoprotein in human lung cancer
Calcified Tissue International, 1997Co-Authors: Akeila Bellahcene, Larry W. Fisher, Sylvie Menard, Naima Maloujahmoum, H Pastorino, Elda Tagliabue, Vincent CastronovoAbstract:Lung cancer belongs to the group of malignant lesions that specifically select bone as secondary implantation site. The molecular bases for this property, defined as osteotropism, is still largely unknown. The recent demonstration that human breast cancer cells express and attach to bone Sialoprotein (BSP), a sulfated phosphoprotein rich in bone and other mineralized tissues, could provide a clue to elucidating bone metastases formation. BSP contains the integrin binding peptide Arg-Gly-Asp (RGD), as well as non-RGD cell attachment domain. Using an immunoperoxidase technique and a specific polyclonal antibody directed against a BSP synthetic peptide, we examined the expression of BSP in 48 lung lesions including 25 squamous carcinoma, 21 adenocarcinoma, and 2 bronchioloalveolar cancers, as well as 38 human ovarian carcinoma that constitute a group of generally nonosteotropic cancers. BSP was not specifically detected in normal lung tissue with the exception of cartilage associated with bronchi. Most of the adenocarcinoma (74%) and all squamous carcinoma of the lung examined exhibited detectable levels of BSP. Staining was mainly cytoplasmic and membrane associated. The two bronchioloalveolar lung cancers examined did not show detectable amounts of BSP. When microcalcifications were observed in pulmonary malignant lesions, they were usually associated with cancer cells expressing BSP. Only 21% of the ovarian cancers examined contained malignant cells with 2+ or 3+ positivity for BSP. We further demonstrated that in 8 of 10 additional lung cancers, BSP was detected at the mRNA level. Our observation is the first demonstration that BSP is expressed in non-small cell lung carcinoma. Lung cancer cells are now the second type of osteotropic malignant cells described to express BSP. Added to the observation that BSP expression is not frequent in ovarian carcinoma, a low osteotropic cancer, our study supports our hypothesis that BSP could play a role in determining the affinity of cancer cells to bone.
Jaro Sodek - One of the best experts on this subject based on the ideXlab platform.
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expression of rat bone Sialoprotein promoter in transgenic mice
Journal of Bone and Mineral Research, 2009Co-Authors: Jinkun Chen, Huw F Thomas, Hangqing Jin, Heping Jiang, Jaro SodekAbstract:Bone Sialoprotein (BSP) is a major protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite crystals. Our previous studies have demonstrated that BSP mRNA is expressed by differentiated osteoblasts, odontoblasts, and cementoblasts involved in de novo mineralized tissue formation in a tissue-specific and developmentally regulated manner. To determine the basis of the selective expression of the BSP gene, we have generated four transgenic mouse lines in which ∼2.7 kb of the rat BSP promoter ligated to a luciferase reporter gene has been stably integrated into the mouse genome. Assays of luciferase activities in 5-day-old animals has revealed consistently high levels in bone tissues with negligible activities in various other organs including kidney, liver, stomach, intestine, and spleen. In some animals, variable expression was observed in brain and skin. Temporal analyses revealed the highest luciferase expression in neonatal bones, with expression decreasing markedly with subsequent growth and development, as observed previously for the endogenous gene in rats. Immunohistochemical analysis of luciferase activity and in situ hybridization of luciferase mRNA in bone tissues show that differentiated osteoblasts express the highest levels of luciferase, consistent with the induction of endogenous gene expression. These studies demonstrate that the regulation of the BSP gene during osteoblastic differentiation, together with its tissue-specific, developmentally regulated expression, is primarily mediated within the ∼2.7 kb region of the promoter.
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human osteocalcin and bone Sialoprotein mediating osteomimicry of prostate cancer cells role of camp dependent protein kinase a signaling pathway
Cancer Research, 2005Co-Authors: Wenchin Huang, Jaro Sodek, Zhihui Xie, Hiroyuki Konaka, Haiyen E Zhau, Leland W K ChungAbstract:Osteocalcin and bone Sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone Sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone Sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone Sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone Sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone Sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone Sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone Sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone Sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone Sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.
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delineation of the hydroxyapatite nucleating domains of bone Sialoprotein
Journal of Biological Chemistry, 2003Co-Authors: Coralee E Tye, Jaro Sodek, Graeme K Hunter, Kevin J Warner, Kevin R Rattray, Jonathan A R Gordon, Harvey A GoldbergAbstract:Bone Sialoprotein (BSP) is a highly modified, anionic phosphoprotein that is expressed almost exclusively in mineralizing connective tissues and has been shown to be a potent nucleator of hydroxyapatite (HA). Two polyglutamic acid (poly[E]) regions, predicted to be in an α-helical conformation and located in the amino-terminal half of the molecule, are believed to be responsible for this activity. Using a prokaryotic expression system, full-length rat BSP was expressed and tested for HA nucleating activity in a steady-state agarose gel system. The unmodified protein is less potent than native bone BSP, indicating a role for the post-translational modifications in HA nucleation. Site-directed mutagenesis of the poly[E] regions in full-length BSP was performed, replacing the poly[E] with either polyaspartic acid (poly[D]) or polyalanine (poly[A]) to examine role of charge and conformation, respectively, in HA nucleation. Replacement of single domains with either poly[A] or poly[D] did not alter nucleating activity nor did replacement of both domains with poly[D]. Replacement of both domains with poly[A], however, significantly decreased nucleating activity. In addition, two recombinant peptides, each encompassing one of the two poly[E] domains, were expressed and tested for nucleating activity. Whereas the peptide encompassing the second poly[E] domain was capable of nucleating HA, the first domain peptide showed no activity. The conformation of the wild-type and mutated proteins and peptides were studied by circular dichroism and small angle x-ray scattering, and no secondary structure was evident. These results demonstrate that a sequence of at least eight contiguous glutamic acid residues is required for the nucleation of HA by BSP and that this nucleating "site" is not α-helical in conformation.
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glucocorticoid regulation of bone Sialoprotein bsp gene expression identification of a glucocorticoid response element in the bone Sialoprotein gene promoter
FEBS Journal, 1995Co-Authors: Yorimasa Ogata, Masato Yamauchi, Richard H Kim, Leonard P Freedman, Jaro SodekAbstract:Glucocorticoids modulate the development and growth of many organs through interactions with a specific intracellular receptor (glucocorticoid receptor) that regulates gene transcription through a cognate element, the glucocorticoid response element (GRE), in the promoter of target genes. In bone formation glucocorticoids stimulate osteoblast differentiation and the formation of bone matrix. Recent studies have demonstrated that the induction of the bone Sialoprotein (BSP) gene is associated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP expression, we have analyzed the effects of the synthetic glucocorticoid, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bone tissue formation by confluent fetal rat calvarial cells and adult rat marrow cells and also stimulated BSP expression up to sixfold in osteoblastic cells (UMR 106–6 and ROS 17/2.8 cells). Most of the stimulation was blocked by cycloheximide, indicating direct and indirect mechanisms of BSP gene regulation. Nuclear ‘run-on’ transcription analysis revealed an up to twofold increase in transcription corresponding to the increase in mRNA that was unaffected by cycloheximide. Analysis of BSP mRNA in the presence of a transcription inhibitor (5,6–dichloro-1-β-d-ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by dexamethasone, indicating that the major, indirect, stimulation of BSP expression involves a nuclear post-transcriptional mechanism. To study the direct effects of dexamethasone, nucleotide sequence analysis of the rat BSP promoter was extended upstream to position−2992 and downstream to +2282 in the first intron. Transient transfection analyses, using various rat BSP promoter constructs linked to a luciferase reporter gene, and gel mobility shift assays were used to identify a putative glucocorticoid response unit comprising three GRE half-sites and a putative AP-1 site, located within positions –906 to –931 upstream from the translation start site of the BSP gene promoter. BSP transcription was stimulated ≈1.5-fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoid receptor binding to this site. These studies, therefore, have identified both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in the rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated.
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expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro inductive effects of dexamethasone on the osteoblastic phenotype
Journal of Cellular Physiology, 1991Co-Authors: Shohei Kasugai, Reynaldo Todescan, William T Butler, Toshihiko Nagata, Jaro SodekAbstract:The nature and tissue distribution of non-collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and β-glycerophosphate (β-GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone Sialoprotein (BSP) and osteocalcin in DEX-treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP-1), type I collagen, SPARC, and alkaline phos-phatase was stirnulated 6-, 5-, 3-, and 2.5- fold, respectively. Metabolic labeling of proteins showed that the Sialoproteins (SPP-1 and BSP) were mostly secreted into the culture medium in the non-mineralizing (-β-GP) cultures, but were the predominant non-collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+β-GP). Extraction of the tissue matrix with 4 M GuHCI and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non-covalently and covalently with the collagenous matrix. SPP-1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de-mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP-1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the Sialoproteins; the 44 kDa SPP-1 incorporating significantly more [32PO4] than the 55 kDa SPP-1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non-collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.
Helena H Ritchie - One of the best experts on this subject based on the ideXlab platform.
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the functional significance of dentin Sialoprotein phosphophoryn and dentin Sialoprotein
International Journal of Oral Science, 2018Co-Authors: Helena H RitchieAbstract:Phosphophoryn (PP) and dentin Sialoprotein (DSP) are the most dominant non-collagenous proteins in dentin. PP is an extremely acidic protein that can function as a mineral nucleator for dentin mineralization. DSP was first identified in 1981, yet its functional significance is still controversial. Historically, these two proteins were considered to be independently synthesized and secreted by dental pulp cells into the developing dentin matrix. However, with the identification of the DSP coding sequence in 1994, followed 2 years later by the finding that the PP coding sequence was located immediately downstream from the DSP sequence, it became immediately clear that DSP and PP proteins were derived from a single DSP-PP (i.e., dentin sialophosphoprotein, DSPP) transcript. Since DSPP cDNA became available, tremendous progress has been made in studying DSP-PP mRNA distribution and DSP generation from the DSP-PP precursor protein at specific cleavage sites by protease tolloid-related-1 (TLR1) or bone morphogenetic protein 1 (BMP1). The functions of DSP-PP and DSP were investigated via DSP-PP knockout (KO) and DSP knockin in DSP-PP KO mice. In addition, a number of in vitro studies aimed to elucidate DSPP and DSP function in dental pulp cells.
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Phosphophoryn and Dentin Sialoprotein Effects on Dental Pulp Cell Migration, Proliferation, and Differentiation
MDPI AG, 2018Co-Authors: Shu-feng Chuang, Yu-hsuan Chen, Helena H RitchieAbstract:Phosphophoryn (PP) and dentin Sialoprotein (DSP) are two of the most abundant dentin matrix non-collagenous proteins, and are derived from dentin Sialoprotein-phosphophoryn (DSP-PP) mRNA. Mutations in the DSP-PP gene are linked to dentinogenesis imperfecta II and III. Previously, we reported transient DSP-PP expression in preameloblast cells first, followed by co-expression in preameloblasts and young odontoblasts, and finally sustained expression in odontoblasts. This phenomenon raised the possibility that DSP/PP proteins secreted by preameloblasts might promote dental pulp cell migration toward the dental pulp border and promote dental pulp cell differentiation. To examine the effects of DSP/PP proteins on dental pulp cell development, we investigated:(1) native PP effects on dental pulpcell migration and matrix protein expression; and (2) recombinant DSP/PP protein effects on cell proliferation and differentiation. We found that PP promoted cell migration and the expression of high levels of Col type I and PP in dental pulp cells. The addition of recombinant DSP/PP proteins affected cell proliferation and differentiation in a dental pulp cell line. These findings strongly suggest that DSP/PP may modulate cell migration, cell proliferation and differentiation, thus leading to dentin formation. DSP/PP protein may be useful clinically for pulp tissue regeneration
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dynamic processing of recombinant dentin Sialoprotein phosphophoryn protein
Journal of Biological Chemistry, 2007Co-Authors: Valentina Godovikova, Helena H RitchieAbstract:Dentin Sialoprotein (DSP) and phosphophoryn (PP) are the two noncollagenous proteins classically linked to dentin but more recently found in bone, kidney, and salivary glands. These two proteins are derived from a single copy DSP-PP gene. Although this suggests that the DSP-PP gene is first transcribed into DSP-PP mRNAs, which later undergo processing to yield the DSP and PP proteins, this mechanism has not yet been demonstrated because of the inability to identify a DSP-PP precursor protein from any cell or tissue sample. To study this problem, we utilized a baculovirus expression system to produce recombinant DSP-PP precursor proteins from a DSP-PP240 cDNA, which represents one of several endogenous DSP-PP transcripts that influence various tooth mineralization phases. Our in vitro results demonstrate that DSP-PP240 precursor proteins are produced by this system and are capable of self-processing to yield both DSP and PP proteins. We further demonstrated that purified recombinant DSP-PP240, purified recombinant PP240, and the native highly phosphorylated protein (equivalent to the PP523 isoform) have proteolytic activity. These newly identified tissue proteases may play key roles in tissue modeling during organogenesis.
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cloning and sequence determination of rat dentin Sialoprotein a novel dentin protein
Journal of Biological Chemistry, 1994Co-Authors: Helena H Ritchie, Hui Hou, Arthur Veis, William T ButlerAbstract:Dentin Sialoprotein (DSP) is a 53-kDa protein isolated from rat dentin. It contains 29.6% carbohydrate (including 9% sialic acid) and has an overall composition similar to that of the bone Sialoproteins osteopontin and bone Sialoprotein (i.e. rich in Asp, Ser, Glu, and Gly). Using a monospecific anti-DSP polyclonal antibody to screen a rat incisor odontoblast cDNA library, a cDNA clone was isolated and sequenced. This approximately 750-base pair clone contained a DNA sequence corresponding to the NH2-terminal 9 amino acids of DSP. A second cDNA clone was isolated by using the first cDNA as a probe to rescreen the library. This second clone had the full-length DSP coding region. From the sequence, we deduced that the DSP cDNA coded for 366 amino acids, predominantly Asp, Ser, Glu, and Gly. The amino acid composition calculated for this sequence was very similar to that for purified DSP reported earlier; likewise the deduced molecular weight (53,045) was essentially identical to that determined by sedimentation equilibrium. Six potential N-linked glycosylation sites were present in the predicted DSP sequence. No Arg-Gly-Asp sequence was found, and the sequence for DSP was dissimilar to those of osteopontin and bone Sialoprotein. Multiple transcripts near 4.6 and 1.5 kilobases were detected by Northern blot analysis in the incisor of 21-day-old rat and the tooth germ of the newborn rat. Consistent with previous immunohistochemical findings, no transcripts were detected in brain, salivary gland, heart, muscle, spleen, kidney, intestine, lung, liver, pancreas, tibia, calvaria, or osteoblast-like osteosarcoma (ROS 17/2.8) cells, indicating that DSP is specifically expressed by odontoblasts and related cells.
William T Butler - One of the best experts on this subject based on the ideXlab platform.
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colocalization of dentin matrix protein 1 and dentin Sialoprotein at late stages of rat molar development
Matrix Biology, 2004Co-Authors: Otto Baba, Chunlin Qin, Jan C. Brunn, James N. Wygant, Bradley W. Mcintyre, William T ButlerAbstract:Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin Sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone Sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.
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Detection of dentin Sialoprotein in rat periodontium
European Journal of Oral Sciences, 2004Co-Authors: Otto Baba, Chunlin Qin, Jan C. Brunn, Jarrod E. Jones, James N. Wygant, Bradley W. Mcintyre, William T ButlerAbstract:Cloning and sequencing of the cDNA indicates that dentin sialophosphoprotein (DSPP) is a precursor of both dentin Sialoprotein (DSP) and dentin phosphoprotein (DPP). Dentin sialophosphoprotein must be proteolytically processed to form these two extracellular matrix (ECM) proteins. Numerous studies led us to conclude that DSP (and DSPP) are exclusively expressed by odontoblasts and preameloblasts. However, recent observations suggest a wider distribution. To test this hypothesis, we conducted systematic studies on rat first molar during root formation with immunohistochemical techniques using specific anti-DSP polyclonal and monoclonal antibodies. We also performed in situ hybridization, using high-stringency RNA probes to detect DSP transcripts. Immunohistochemical studies demonstrated that DSP is not only localized in odontoblasts, dentin ECM and preameloblasts, but also in alveolar bone, cellular cementum, osteocytes, cementocytes, and their matrices. The results of in situ hybridization were consistent with those from immunohistochemistry, showing the expression of DSP transcripts in osteoblasts of alveolar bone, fibroblasts in periodontal ligament and cementoblasts in cellular cementum. Together, these observations suggest that DSP is involved in formation of the periodontium as well as tooth structures.
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dentin Sialoprotein dsp has limited effects on in vitro apatite formation and growth
Calcified Tissue International, 2000Co-Authors: Adele L Boskey, Lyudmila Spevak, M Tan, S B Doty, William T ButlerAbstract:Sialoproteins such as bone Sialoprotein (BSP) and dentin Sialoprotein (DSP) accumulate at the mineralization fronts in bone and dentin, respectively, suggesting they have some function in the mineralization process. BSP, a highly phosphorylated protein rich in polyglutamate repeats, is an effective nucleator of hydroxyapatite (HA) formation in vitro. The present study examines the effect of DSP, a low phosphorylated but related Sialoprotein, on the formation and growth of HA. In vitro, in a gelatin gel diffusion system, DSP at low concentrations (<25 μg/ml) slightly increased the yield of HA formed at 3.5 and 5 days, while at higher concentrations (50–100 μg/ml) it slightly inhibited accumulation. Fewer mineral crystals were formed in the presence of high concentrations of DSP but they tended to aggregate (making them appear larger by electron microscopic analysis) than those formed in DSP-free gels. X-ray diffraction line broadening analysis failed to show significant changes in c-axis crystal dimensions with increasing DSP concentration. When HA-seed crystals were coated with DSP before inclusion in the gelatin gel there was a reduction in mineral accumulation relative to HA-seeds which had not been coated with DSP, but the extent of inhibition was significantly less than that seen in this system with other mineralized tissue matrix Sialoproteins, such as osteopontin or BSP. The low affinity of DSP for well-characterized seed crystals and the limited effect of this protein on HA formation and growth suggest that the role of DSP in dentin is not primarily that of a mineralization regulator.
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cloning and sequence determination of rat dentin Sialoprotein a novel dentin protein
Journal of Biological Chemistry, 1994Co-Authors: Helena H Ritchie, Hui Hou, Arthur Veis, William T ButlerAbstract:Dentin Sialoprotein (DSP) is a 53-kDa protein isolated from rat dentin. It contains 29.6% carbohydrate (including 9% sialic acid) and has an overall composition similar to that of the bone Sialoproteins osteopontin and bone Sialoprotein (i.e. rich in Asp, Ser, Glu, and Gly). Using a monospecific anti-DSP polyclonal antibody to screen a rat incisor odontoblast cDNA library, a cDNA clone was isolated and sequenced. This approximately 750-base pair clone contained a DNA sequence corresponding to the NH2-terminal 9 amino acids of DSP. A second cDNA clone was isolated by using the first cDNA as a probe to rescreen the library. This second clone had the full-length DSP coding region. From the sequence, we deduced that the DSP cDNA coded for 366 amino acids, predominantly Asp, Ser, Glu, and Gly. The amino acid composition calculated for this sequence was very similar to that for purified DSP reported earlier; likewise the deduced molecular weight (53,045) was essentially identical to that determined by sedimentation equilibrium. Six potential N-linked glycosylation sites were present in the predicted DSP sequence. No Arg-Gly-Asp sequence was found, and the sequence for DSP was dissimilar to those of osteopontin and bone Sialoprotein. Multiple transcripts near 4.6 and 1.5 kilobases were detected by Northern blot analysis in the incisor of 21-day-old rat and the tooth germ of the newborn rat. Consistent with previous immunohistochemical findings, no transcripts were detected in brain, salivary gland, heart, muscle, spleen, kidney, intestine, lung, liver, pancreas, tibia, calvaria, or osteoblast-like osteosarcoma (ROS 17/2.8) cells, indicating that DSP is specifically expressed by odontoblasts and related cells.
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expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro inductive effects of dexamethasone on the osteoblastic phenotype
Journal of Cellular Physiology, 1991Co-Authors: Shohei Kasugai, Reynaldo Todescan, William T Butler, Toshihiko Nagata, Jaro SodekAbstract:The nature and tissue distribution of non-collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and β-glycerophosphate (β-GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone Sialoprotein (BSP) and osteocalcin in DEX-treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP-1), type I collagen, SPARC, and alkaline phos-phatase was stirnulated 6-, 5-, 3-, and 2.5- fold, respectively. Metabolic labeling of proteins showed that the Sialoproteins (SPP-1 and BSP) were mostly secreted into the culture medium in the non-mineralizing (-β-GP) cultures, but were the predominant non-collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+β-GP). Extraction of the tissue matrix with 4 M GuHCI and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non-covalently and covalently with the collagenous matrix. SPP-1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de-mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP-1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the Sialoproteins; the 44 kDa SPP-1 incorporating significantly more [32PO4] than the 55 kDa SPP-1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non-collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.
Neal S. Fedarko - One of the best experts on this subject based on the ideXlab platform.
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small integrin binding ligand n linked glycoproteins siblings multifunctional proteins in cancer
Nature Reviews Cancer, 2008Co-Authors: Akeila Bellahcene, Larry W. Fisher, Vincent Castronovo, Kalu U E Ogbureke, Neal S. FedarkoAbstract:Numerous components and pathways are involved in the complex interplay between cancer cells and their environment. The family of glycophosphoproteins comprising osteopontin, bone Sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein - small integrin-binding ligand N-linked glycoproteins (SIBLINGs) - are emerging as important players in many stages of cancer progression. From their detection in various human cancers to the demonstration of their key functional roles during malignant transformation, invasion and metastasis, the SIBLINGs are proteins with potential as diagnostic and prognostic tools, as well as new therapeutic targets.
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elevated serum bone Sialoprotein and osteopontin in colon breast prostate and lung cancer
Clinical Cancer Research, 2001Co-Authors: Neal S. Fedarko, Alka Jain, Abdullah Karadag, Matthew R Van Eman, Larry W. FisherAbstract:Purpose: Histological studies have shown that the two Sialoproteins, bone Sialoprotein (BSP) and osteopontin (OPN), are induced in multiple types of cancer. We have recently found that these proteins are bound in serum to complement factor H and that the complex must be disrupted to generate free protein to measure their total levels. We hypothesized that measuring total BSP and OPN levels would provide informative markers for the detection of cancer. Experimental Design: As a proof of concept study, serum from patients with diagnosed breast, colon, lung, or prostate cancer (n = 20 for each type) as well as normal serum (n = 77) were analyzed using competitive ELISAs developed for BSP and OPN. Sensitivity, specificity, as well as positive and negative predictive values were determined for each Sialoprotein and cancer type. The relationship between sensitivity and specificity was profiled by receiver operating characteristic curves. Results and Conclusions: Determined values for serum BSP in ng/ml were 285 ± 19 for prostate, 373 ± 19 for colon, 318 ± 18 for breast, 155 ± 11 for lung cancer sera, and 154 ± 13 for normal sera. Values of OPN in ng/ml were 653 ± 39 for prostate, 449 ± 22 for colon, 814 ± 53 for breast, 724 ± 33 for lung, and 439 ± 30 for normal sera. The assays provide a high degree of sensitivity and specificity that enables the detection of colon, breast, prostate, and lung cancer.
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Flexible structures of SIBLING proteins, bone Sialoprotein, and osteopontin.
Biochemical and biophysical research communications, 2001Co-Authors: Larry W. Fisher, Dennis A. Torchia, Berthold Fohr, M F Young, Neal S. FedarkoAbstract: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.
