BSP Protein Research Articles

Discoidin domain receptor 2 activation of p38 mitogen-activated protein kinase as an important pathway for osteonectin-regulating osteoblast mineralization

ObjectiveThe present study aimed to determine the role of the discoidin domain receptor 2 (DDR2) in the osteonectin (ON) regulation of osteoblast mineralization through the activation of p38 mitogen-activated protein kinase (MAPK).MethodsFour groups were established: the ON group, the inhibitor group, the Ddr2-small interfering ribonucleic acid (siRNA) group, and the control group. Osteoblasts from the parietal bones of neonatal Sprague–Dawley rats were isolated and cultured. In the ON group, 1 µg/mL ON was added to the osteoblasts. The gene expressions of collagen 1 (Col 1) and Ddr2 were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the inhibitor group, the osteoblasts were added to WRG-28 (a specific DDR2 inhibitor), and in the Ddr2-siRNA group, the osteoblasts were transfected with Ddr2-siRNA. The gene and protein expressions of DDR2, bone sialoprotein, osteocalcin, osteopontin, and p38 MAPK were determined using RT-qPCR and western blot analysis. Alizarin red staining and transmission electron microscopy were used to detect mineralization.ResultsThe results showed that ON enhanced the osteoblast Col 1 and Ddr2 gene expressions, while the use of a Ddr2-siRNA/DDR2-blocker decreased the OPN, BSP, OCN, and P38 gene and protein expressions and reduced osteoblast cellular activity and mineralized nodules.ConclusionThe present study demonstrated that DDR2 activation of p38 MAPK is an important approach to ON-regulating osteoblast mineralization.

Semaphorin 3A promotes the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in inflammatory environments by suppressing the Wnt/β-catenin signaling pathway.

After periodontal treatment, the local inflammatory environment surrounding periodontal tissues cannot be entirely eliminated. The means by which alveolar bone repair and regeneration are promoted in inflammatory environments have important clinical significance. As a powerful protein that promotes the differentiation of osteocytes, semaphorin 3A (Sema3A) shows potential for bone regeneration therapy. However, the effect of Sema3A on osteogenic differentiation in an inflammatory environment, as well as the underlying mechanism, have not yet been explored. We used lentivirus to transduce rat bone marrow-derived mesenchymal stem cells (rBMSCs) to stably overexpress Sema3A. Lipopolysaccharide from Escherichia coli (E. coli LPS) was used to stimulate rBMSCs to establish an inflammatory environment. ALP staining, Alizarin red staining, ALP activity tests, quantitative RT-PCR (qRT-PCR), and Western blotting were used to elucidate the effect of Sema3A on the osteogenesis of rBMSCs in inflammatory environments. XAV939 and LiCl were used to determine whether the Wnt/β-catenin signaling pathway was involved in attenuating the inhibition of Sema3A-induced osteogenic differentiation by LPS. The qRT-PCR and Western blot results demonstrated that the lentiviral vector (LV-NC) and lentiviral-Sema3A (LV-Sema3A) were successfully transduced into rBMSCs. An inflammatory environment could be established by stimulating rBMSCs with 1 μg/ml E. coli LPS. After Sema3A overexpression, mineral deposition was exacerbated, and the BSP and Runx2 gene and protein expression levels were increased. Furthermore, E. coli LPS activated the Wnt/β-catenin signaling pathway and decreased rBMSC osteogenesis, but these effects were attenuated by Sema3A. In conclusion, Sema3A could protect BMSCs from LPS-mediated inhibition of osteogenic differentiation in inflammatory environments by suppressing the Wnt/β-catenin pathway.

Comparative Study of Human Dental Follicle Cell Sheets and Periodontal Ligament Cell Sheets for Periodontal Tissue Regeneration

Periodontal ligament cell (PDLC) sheets have been shown to contribute to periodontal tissue regeneration. Dental follicle cells (DFCs), acknowledged as the precursor cells of PDLCs, have demonstrated stemness, embryonic features, heterogeneity, and pluripotency. Therefore, we hypothesized that DFC sheets might be more effective and suitable for periodontal tissue regeneration than PDLC sheets. In this study, we compared the biological characteristics of DFC sheets and PDLC sheets in vitro. To investigate the potential for periodontal tissue regeneration in vivo, complexes composed of two types of cell sheets combined with dentin matrix were implanted subcutaneously into nude mice for 6 weeks. Our results showed that, when forming cell sheets, DFCs secreted richer extracellular matrix than PDLCs. And compared to DFCs, DFC sheets expressed high levels of calcification-related genes, including alkaline phosphatase (alp), bone sialoprotein (bsp), osteopontin (opn), runt-related transcription factor (runx2), as well as the periodontal ligament-specific genes collagen III (col III) and periostin, while the gene expression of bsp, osteocalcin (ocn), and opn were greatly increased in PDLC sheets, when compared to PDLCs. col I expression did not change significantly. However, cementum protein 23 (cp-23) expression increased several fold in PDLC sheets compared to PDLCs but decreased in DFC sheets compared to DFCs. DFC and PDLC sheets were both positive for Collagen I (Col I), cementum attachment protein (CAP), ALP, BSP, OCN, and OPN protein expression, and Col I, ALP, BSP, and OPN expression were increased after cell sheets were formed. Furthermore, the levels of laminin and fibronectin were higher in DFCs and DFC sheets than that of PDLCs and PDLC sheets, respectively. In vivo, DFC and PDLC sheets could both regenerate periodontal tissue-like structures, but DFC sheets demonstrated stronger periodontal regeneration potential than PDLC sheets. Therefore, DFC sheets derived from discarded dental follicle tissue after tooth extraction may be more advantageous for clinical periodontal tissue regeneration in the future.

In Vivo Biological Responses to Silk Proteins Functionalized with Bone Sialoprotein

Recombinant 6mer + BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein-based biomaterial further for in vivo behavior related to biocompatibility. 6mer + BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer + BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications.

Preparation and characterization of mesoporous bioactive glass/polycaprolactone nanofibrous matrix for bone tissues engineering

A polycaprolactone (PCL) nanofibrous composite matrix having mesoporous bioactive glass nanoparticles (MBG) was fabricated using the electrospinning method, and the microstructural, physical and biological properties of the composite matrix were characterized. The fiber diameters of PCL, 5 % MBG/PCL (5 M-PCL) and 10 % MBG/PCL (10 M-PCL) were 575 ± 162 nm, 312 ± 134 nm and 321 ± 144 nm, respectively. The bioactivity of the composite matrix was evaluated by soaking the matrix in 1.5× simulated body fluid; the MBG/PCL matrix showed a better biomineralization capability than did the PCL matrix. The biological performance of the PCL and the MBG/PCL were evaluated using an in vitro culture of MG63 osteoblast-like cells. We found that the cell attachment and proliferation rates were significantly higher on the 10 M-PCL than on the PCL. Moreover, the expression of several genes, including ANX-V, type I collagen and OCN, ALP activity, the deposition of calcium, and the BSP protein, were also significantly higher on 10 M-PCL than PCL. These results indicated that MBG/PCL has the ability to support cell attachment, growth, and differentiation and can also yield high bioactivity. Therefore, MBG/PCL could be potentially applied in bone implants.

New insights towards understanding the mechanisms of sperm protection by egg yolk and milk

Mammalian sperm preservation in extenders containing egg yolk (EY) and/or milk has been used for over half a century. However, the mechanism by which EY or milk protects sperm during storage remains elusive. Studies conducted over the past two decades in our laboratory have revealed that a family of lipid-binding proteins (BSP proteins) present in bull seminal plasma is detrimental to sperm preservation since these proteins induce cholesterol and phospholipid removal from the sperm membrane. Interestingly, these detrimental factors of seminal plasma interact with the low-density lipoproteins (LDL) present in EY. This interaction minimizes lipid removal from the sperm membrane, which positively influences sperm storage in liquid or frozen states. Based on several lines of evidence, we suggest that the sequestration of BSP proteins by LDL (BSP proteins: lipoprotein interaction) is the major mechanism of sperm protection by EY. Skimmed milk, which is devoid of lipoproteins, also protects sperm during storage. Several studies indicate that the active components involved in sperm protection by milk are casein micelles. Thus, it appears that the mechanism by which milk protects sperm involves a BSP protein: casein micelle interaction. In view of these new insights, novel strategies have been suggested to improve the efficiency of semen preservation.

Bone sialoprotein is predictive of bone metastases in resectable non-small cell lung cancer: A case-control study and prevalence data

7049 Background: Bone metastases (BM) in non small cell lung cancer (NSCLC) may be present at diagnosis or develop in the follow up, are associated with a worse prognosis, and currently there are no chemical or biological markers predicting their clinical onset. Methods: Thirty cases of resected NSCLC which subsequently develop BM (group A - mean follow up time 27.2 months) were matched for several clinico-pathological parameters (including age, sex, stage of the disease, histology, differentiation grade, adjuvant therapy) to 30 cases of resected NSCLC without any metastases (group B - mean follow up time 75.1 months) and 26 resected NSCLC with non-bone metastatic (group C - mean follow up 21.1 months). Primary tumor samples were investigated by immunohistochemistry for 10 markers previously recognized to be involved in bone resorption or metastatization process (cathepsin K, bone sialoprotein [BSP], VEGF, MMP-2, p53, RECK, TIMP-1, CD-117, Ki-67 and TRAcP). For statistical analysis the intensity of the staining was assessed by a semi-quantitative score (0, <10%, 10–50%, >50% +ve tumor cells). Differences among groups were estimated by X-square test, whereas the prognostic impact of clinico-pathological parameters and markers expression was evaluated by univariate and multivariate analyses. Results: Among the different markers investigated, BSP was strongly associated to bone dissemination (p < 0.001) and, independently, to poor outcome (p = 0.02 by Mantel-Cox test). None of the other markers was differentially expressed within the groups or demonstrated a prognostic impact, both in terms of overall survival and of time interval to metastases. Based on these findings, the prevalence of BSP in NSCLC was further estimated in a large series of 120 resected lung carcinomas (M:F ratio 3:1; mean age 67 years; adenocarcinomas 55%, squamous cell carcinoma 39%, others 6%; stages: I 54%, II 17%, III 29%). BSP prevalence reached 40%, without any statistically significant difference according to histotype or other clinico-pathological parameters. Conclusions: BSP protein expression in resected NSCLC strongly predicts bone dissemination, and may therefore be useful in selecting patients for treatments targeted to inhibit bone metastatic spread. [Table: see text]

Bone Sialoprotein Mediates the Tumor Cell–Targeted Prometastatic Activity of Transforming Growth Factor β in a Mouse Model of Breast Cancer

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

Bovine seminal plasma proteins and their relatives: A new expanding superfamily in mammals

BSP proteins represent three major proteins of bovine seminal plasma: BSP-A1/-A2, -A3 and -30 kDa. The BSP protein signature is characterized by two tandemly repeated fibronectin type 2 (Fn2) domains. Although classical affinity chromatography and protein sequencing have proven that the BSP protein homologs may be ubiquitous in mammals and functionally related to sperm capacitation, only the three bovine genes have been reported thus far. In this study, we report three new BSP protein-related genes from bovine, as well as other BSP protein-related DNA sequences from human, chimpanzee, mouse, rat, dog, horse and rabbit. Analysis of the relationships between all Fn2 domain-containing proteins revealed that the Fn2 domains found in BSP-related proteins have special features that distinguish them from non-BSP-related proteins. These features can be used to identify new BSP protein-related sequences. Further molecular evolutionary analysis of the BSP protein lineage revealed that all BSP proteins and their related sequences can be grouped into three subfamilies: BSPH4, BSPH5 and BSPH6, which indicates that the BSP protein family is much bigger than previously envisioned. More interestingly, the three BSP proteins in bovine within the BSPH4-subfamily were shown to evolve rapidly. The ratio of nonsynonymous to synonymous substitutions was higher than 1. The analysis also indicated that the rate of evolution was heterogeneous between the first and second Fn2 domains of the genes. These data may reflect that some amino acids in BSP proteins are under a strong positive selection after gene duplication and that each BSP protein evolves rapidly, possibly to acquire new functions.

Isolation and characterization of the major proteins of ram seminal plasma

Mammalian seminal plasma contains among others, two major families of proteins, namely spermadhesins and those proteins that contain fibronectin type II domains. Spermadhesins are the major proteins of boar and stallion seminal plasma and homologous proteins have been identified in the bull. These proteins appear to be involved in capacitation and sperm-egg interaction. In bovine seminal plasma, proteins containing fibronectin type II domains are the major proteins and are designated BSP proteins. These proteins play a role in sperm capacitation. In this study, we present the isolation and characterization of the major proteins of ram seminal plasma. Precipitated proteins from Suffolk ram seminal plasma were loaded onto a gelatin-Agarose column. The unadsorbed (fraction A) and retarded proteins (fraction B) were removed by washing the column with phosphate buffered-saline and the adsorbed proteins (fraction C) were eluted with 5 M urea. SDS-PAGE of fraction B indicated the presence of a 15.5 kDa protein, which is the major protein of ram seminal plasma (approximately 45% of total protein by weight) and was identified as a spermadhesin by N-terminal sequencing. SDS-PAGE analysis of fraction C revealed the presence of four proteins, which represented approximately 20% of total ram seminal plasma proteins by weight, and were identified as proteins of the BSP family and named RSP proteins. These RSP proteins were designated RSP-15 kDa, RSP-16 kDa, RSP-22 kDa, and RSP-24 kDa. Only RSP-15 kDa and -16 kDa proteins cross-reacted with antibodies against BSP proteins. Ram spermadhesin and RSP proteins interact with heparin but only RSP proteins bind to hen's egg yolk low-density lipoprotein. In conclusion, spermadhesin is the major protein of ram seminal plasma and other major proteins belong to the BSP protein family.

Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk.

Over the past 60 years, egg yolk (EY) has been routinely used in both liquid semen extenders and those used to cryopreserve sperm. However, the mechanism by which EY protects sperm during liquid storage or from freezing damage is unknown. Bovine seminal plasma contains a family of proteins designated BSP-A1/-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). These proteins are secretory products of seminal vesicles that are acquired by sperm at ejaculation, modifying the sperm membrane by inducing cholesterol efflux. Because cholesterol efflux is time and concentration dependent, continuous exposure to seminal plasma (SP) that contains BSP proteins may be detrimental to the sperm membrane, which may adversely affect the ability of sperm to be preserved. In this article, we show that the BSP proteins bind to the low-density fraction (LDF), a lipoprotein component of the EY extender. The binding is rapid, specific, saturable, and stable even after freeze-thawing of semen. Furthermore, LDF has a very high capacity for BSP protein binding. The binding of BSP proteins to LDF may prevent their detrimental effect on sperm membrane, and this may be crucial for sperm storage. Thus, we propose that the sequestration of BSP proteins of SP by LDF may represent the major mechanism of sperm protection by EY.

Influence of proteins Bsp and FemH on cell shape and peptidoglycan composition in group B streptococcus.

Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp. The bsp gene was located close to another gene, designated femH, and reverse transcription-PCR revealed a bicistronic transcriptional organization for both genes. The Bsp protein was detected in the culture supernatant from 31 tested clinical isolates of GBS, suggesting a wide distribution of Bsp in these bacteria. Overexpression of bsp resulted in lens-shaped GBS cells, indicating a role for bsp in controlling cell morphology. Insertional disruption of femH resulted in a reduction of the L-alanine content of the peptidoglycan, suggesting that femH is involved in the incorporation of L-alanine residues in the interpeptide chain of the peptidoglycan of GBS.

Complementary deoxyribonucleic acid cloning and tissue expression of BSP-A3 and BSP-30-kDa: phosphatidylcholine and heparin-binding proteins of bovine seminal plasma.

BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa are four major proteins of bovine seminal plasma (BSP protein family). These heparin- and phosphatidylcholine-binding proteins potentiate the capacitation of spermatozoa. Here we determined the complete sequences of the two cDNAs coding for the BSP-A3 and BSP-30-kDa proteins. Degenerate oligonucleotides designed on the basis of the primary sequences of the proteins were used as primers in reverse transcription-polymerase chain reaction, with cDNA preparations of bovine seminal vesicles as templates, to amplify an internal fragment of each BSP cDNA. Specific oligonucleotides designed on the basis of these partial cDNA sequences were used to clone the two complete cDNAs by using the 3' rapid amplification of cDNA ends (RACE) and 5' RACE methods. We also verified the expression of all members of the bovine BSP protein family in several adult bovine tissues by RNase protection assays. The results indicated that each BSP protein mRNA is expressed only in seminal vesicles and in the ampullae. Homologous genes were detected in human, rat, hamster, and rabbit genomic DNA, using high-stringency Southern hybridization with a specific BSP-30-kDa cDNA probe.

Major proteins of bovine seminal plasma inhibit phospholipase A2

We have recently shown that the major proteins of bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called BSP proteins) bind to spermatozoa and that the binding sites on the plasma membrane of spermatozoa are choline phospholipids. In view of the fact that these phospholipids are substrates for phospholipase A2 (PLA2), a key enzyme in sperm capacitation and the acrosome reaction, the effect of BSP proteins on this enzyme activity was investigated. Since these BSP proteins are ubiquitous, the effect on pig pancreatic PLA2 was also studied. In contrast with control proteins, when preincubated with phosphatidylcholine as substrate, all BSP proteins inhibited both pancreatic and sperm PLA2 activity in a dose-dependent manner and in the presence of 1-6 microM BSP protein the enzyme activity was completely abolished. When phosphatidylethanolamine was used as substrate, only pancreatic PLA2 was inhibited. On the other hand, when the BSP proteins were preincubated with the enzyme followed by addition of substrate, a biphasic effect was observed; there was stimulation of enzyme activity below 1.3 microM BSP followed by an inhibition above this concentration. The inhibitory activity was trypsin-sensitive but heat-resistant. The effect of co-incubation of heparin, which is implicated in sperm capacitation and which also interacts with BSP proteins, was studied. Heparin (10 microM) had no effect on the PLA2 inhibitory activity exhibited by all BSP proteins. The PLA2 inhibitory effect exhibited by BSP proteins was abolished with excess substrate. The BSP proteins were adsorbed on PLA2-agarose and could be affinity cross-linked to the enzyme, indicating a direct interaction of enzyme with the inhibitor. These results suggest that these BSP proteins modulate PLA2 activity and therefore, phospholipid metabolism.

Characterization of the major proteins of bovine seminal fluid by two‐dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.

Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid.

A group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), are the major proteins found in bovine seminal fluid. These proteins are secretory products of seminal vesicles, and they bind to spermatozoa upon ejaculation, suggesting that there are binding sites for these proteins on the spermatozoa. It was of interest to characterize these binding sites on spermatozoa which may help in the elucidation of the biological function of BSP proteins. The binding sites on spermatozoa are resistant to protease or acid treatment and are heat-stable but extractable with organic solvents. The solvent-extractable material, when coated on plastic microtitration wells, binds radiolabeled BSP proteins thus indicating the lipid nature of the BSP binding sites on spermatozoa. We investigated the specificity of interaction of BSP proteins with lipids using liposomes of phospholipids, solid-phase, and thin-layer chromatography-overlay techniques. Results showed that BSP-A1, -A2, and -A3 proteins bound specifically to those phospholipids which contain the phosphorylcholine group. In contrast, BSP-30-kDa protein preferentially bound to phospholipids containing the phosphorylcholine moiety but also interacted with phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Furthermore, of those lipids that were extracted from spermatozoa, only phospholipids which contain the phosphorylcholine moiety bound radiolabeled BSP proteins. These data suggest that the BSP protein binding sites on spermatozoa are phospholipids. We propose that this specific interaction plays an important role in the membrane modification of spermatozoa that occurs during capacitation and/or acrosome reaction.