Major Non-collagenous Protein Research Articles

Porous titanium scaffolds modified with Zeolitic Imidazolate Framework (ZIF-8) with enhanced osteogenic activity for the prevention of implant-associated infections.

In this study, zeolitic imidazolate framework 8 (ZIF-8) was coated on porous Ti6Al4V scaffolds, either bare or previously modified using hydroxyapatite (HA) or HA and gelatin (HAgel), via a growing single-step method in aqueous media using two contact times at 6h and 24h. The coated scaffolds termed ZIF-8@Ti, ZIF-8@HA/Ti, and ZIF-8@HAgel/Ti were characterized via scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), and molecular plasma-atomic emission spectroscopy (MP-AES). In order to assess the cell proliferation rate, the cytocompatibility of the scaffolds was evaluated in primary osteoblasts (hOBs) using alamarBlue assay, while the osteoconductivity was analyzed in hOBs using a real-time approach, evaluating the expression of secreted phosphoprotein 1 (SPP1). Osteopontin, which is the protein encoded by this gene, represents the major non-collagenous bone protein that binds tightly to HA. The scaffolds were shown to be non-cytotoxic based on hOB proliferation at all time points of analysis (24h and 72h). In hOB cultures, the scaffolds induced the upregulation of SPP1 with different fold changes. Some selected scaffolds were assayed in vitro for their antibacterial potential against Staphylococcus epidermidis; the scaffolds coated with ZIF-8 crystals, regardless of the presence of HA and gelatin, strongly inhibited bacterial adhesion to the materials and reduced bacterial proliferation in the culture medium, demonstrating the suitable release of ZIF-8 in a bioactive form. These experiments suggest that the innovative scaffolds, tested herein, provide a good microenvironment for hOB adhesion, viability, and osteoconduction with effective prevention of S. epidermidis adhesion.

Structural role of osteocalcin and its modification in bone fracture.

Osteocalcin (OC), an abundant non-collagenous protein in bone extracellular matrix, plays a vital role in both its biological and mechanical function. OC undergoes post-translational modification, such as glycation; however, it remains unknown whether glycation of OC affects bone's resistance to fracture. Here, for the first time, we demonstrate the formation of pentosidine, an advanced glycation end-product (AGE) cross-link on mouse OC analyzed by ultra-performance liquid chromatography. Next, we establish that the presence of OC in mouse bone matrix is associated with lower interlamellar separation (distance) and thicker bridges spanning the lamellae, both of which are critical for maintaining bone's structural integrity. Furthermore, to determine the impact of modification of OC by glycation on bone toughness, we glycated bone samples in vitro from wild-type (WT) and osteocalcin deficient (Oc-/-) mice, and compared the differences in total fluorescent AGEs and fracture toughness between the Oc -/- glycated and control mouse bones and the WT glycated and control mouse bones. We determined that glycation resulted in significantly higher AGEs in WT compared to Oc-/- mouse bones (delta-WT > delta-OC, p = 0.025). This observed change corresponded to a significant decrease in fracture toughness between WT and Oc-/- mice (delta-WT vs delta-OC, p = 0.018). Thus, we propose a molecular deformation and fracture mechanics model that corroborates our experimental findings and provides evidence to support a 37%-90% loss in energy dissipation of OC due to formation of pentosidine cross-link by glycation. We anticipate that our study will aid in elucidating the effects of a major non-collagenous bone matrix protein, osteocalcin, and its modifications on bone fragility and help identify potential therapeutic targets for maintaining skeletal health.

In Vitro Investigation on Degradable Mg-Based Biomaterial under the Impact of the Serum Glycoprotein Fetuin.

Biomedical applications of magnesium (Mg) and its alloys are generally dependent on their degradation behavior in vivo. Despite its attractive properties, which make Mg suitable for orthopedic applications, the in vivo material-tissue (bone, blood, and lymph tissues) interaction is not yet fully understood. To investigate the influence of major serum proteins on the degradation, this study focused on fetuin, which is one of the major non-collagenous plasma proteins and which is essential for biomineralization. This study used a physiological setup to investigate the influence of fetuin on the degradation behavior of pure Mg in the presence of calcium (Ca). Extruded pure Mg samples were immersed under cell culture conditions in Hank’s balanced salt solution (HBSS) under defined Ca regimes. The results showed a significant decrease in the degradation rate (DR) when both fetuin and Ca were present in an immersion medium as compared to media where they were not simultaneously present. A possible reason for this behavior was the forming of a dense, protein-degradation products protection barrier at the material surface. Furthermore, the limitation of freely available Ca might be a reason for a decreased degradation. The cultivation of primary osteoblasts (pOB) was possible at the fetuin-coated Mg-surface without additional serum supplementation.

Can non-collagenous proteins be employed for the differential diagnosis among fibrous dysplasia, cemento-osseous dysplasia and cemento-ossifying fibroma?

Differential diagnosis among fibrous dysplasias, cemento-ossifying fibromas and cemento-osseous dysplasias is difficult, since there is considerable overlap of histologic features, but also extremely important, since they differ greatly in etiology, clinical behaviour, prognosis and terapeuthic approach. There is no data about the use of immunohistochemistry, a viable and accessible technique, for this purpose. The objective of this study was to investigate, comparatively, the immunohistochemical expression of major non-collagenous proteins (osteonectin [ON], osteopontin [OP], bone sialoprotein [BSP] and osteocalcin [OC]) of mineralized tissue extracellular matrix in 22 cases of fibrous dysplasias, 16 of cemento-ossifying fibromas and 16 of cemento-osseous dysplasias. ON maintained the same expression profile in all cases; the staining for OP was negative in fusiform cells producing cementoid globules and weak, as well as heterogeneous, in high mineralized matrixes; there was negativity for BSP in cementoid globules and in the fusiform cells that produce them, differently from the strong positive expression found in the majority of bone trabeculae and their peripheral cuboidal osteoblasts; and finally, the immuno-reactivity for OC was weak, except in cuboidal osteoblasts and osteocytes. We can conclude that the nature of mineralized structure and the cellular phenotype are much more responsible for variability in immunohistochemical profile than the type of lesion (fibrous dysplasias, cemento-ossifying fibromas and cemento-osseous dysplasias) which makes difficult, at least for a while, the use of these proteins with diagnosis purpose.

Osteocalcin and osteopontin influence bone morphology and mechanical properties.

Osteocalcin (OC) and osteopontin (OPN) are major non-collagenous proteins (NCPs) involved in bone matrix organization and deposition. In spite of this, it is currently unknown whether OC and OPN alter bone morphology and consequently affect bone fracture resistance. The goal of this study is to establish the role of OC and OPN in the determination of cortical bone size, shape, and mechanical properties. Our results show that Oc-/- and Opn-/- mice were no different from each other or wild type (WT) with respect to bone morphology (P > 0.1). Bones from mice lacking both NCPs (Oc-/- Opn-/- ) were shorter, with thicker cortices and larger cortical areas, compared with the WT, Oc-/- , and Opn-/- groups (P < 0.05), suggesting a synergistic role for NCPs in the determination of bone morphology. Maximum bending load was significantly different among the groups (P = 0.024), while tissue mineral density and measures of stiffness and strength were not different (P > 0.1). We conclude that the removal of both OC and OPN from bone matrix induces morphological adaptation at the structural level to maintain bone strength.

Evaluation of Bone Mineral Density in Children with Type I Diabetes Mellitus and Relationship to Serum Levels of Osteopontin.

Type I diabetes mellitus (T1DM) patients are at risk of osteoporosis and fracture due to the osteoblast and osteoclast malfunction. Osteopontin (OPN) as the major non-collagenous bone matrix protein is produced by osteoblasts and osteoclasts and involve in bone resorption, formation and remodeling. To evaluate the serum levels of OPN, bone mineral density (BMD) and correlation in patients with T1DM this study was designed. In this case-control study, 87 children with T1DM and 87 age/sex-matched healthy subjects were enrolled. Blood samples were tested for OPN levels by ELISA. Dual energy X-ray absorptiometry was used to measure BMD. The mean levels of BMD in patients was significantly lower than control group (p<0.0001). There was no significant difference between patients and healthy subjects regarding the levels of OPN. However, in patients with high levels of OPN (mean+1.5 standard deviation) the BMD was significantly lower than other patients (p<0.0001). Totally there was a negative correlation between serum levels of OPN and BMD in patients with T1DM (p<0.016). These results indicated that the BMD in T1DM is less than healthy children and elevated level of OPN in patients could be associated with low BMD. A linear negative correlation between serum OPN and total BMD of T1DM patients compared to control group was noticed in this study indicating that the amount of serum OPN could be effective on BMD and a good predicting factor for osteoporosis in patients.

Immunohistochemical Localization of Osteopontin - A Comparative Study on Periodontally Healthy and Diseased Tooth Root Surface

A Great deal of effort has been focused on trying to determine the presence and localization of factors responsible for cementogenesis, as well as in repair and regeneration of periodontal tissues. Bone sialoprotein and osteopontin are the two major non-collagenous proteins in cementum that appeared to have important roles in cementogenesis and regeneration. Aim: To investigate the expression and distribution of osteopontin on diseased root surface in comparison with healthy non-diseased root surface in by immunohistochemical technique using osteopontin mouse monoclonal antibody [NCL-O-PONTIN] (Novocastra) Materials and Methods: Fifty-Five teeth which included 25 permanent healthy human teeth and 30 permanent teeth affected by advanced periodontitis were collected. All teeth were initially fixed in 10% neutral buffered formalin. Following fixation and demineralization, specimens were embedded in paraffin, sectioned and exposed to antibodies against osteopontin. Stained sections were assessed using digital photomicroscope. Results: In healthy root surface staining intensity was found to be mild to moderate in the cemental surface and matrix. Moderate staining was found in relation to periodontal ligament. In diseased root surface there was absence of immunostaining for osteopontin (OPN) in exposed cementum. In, the apical part of the diseased tooth, a strong staining for OPN was seen along the cementum surfaces adjacent to the periodontal ligament. Conclusion: The absence of staining for OPN in all areas of exposed cementum (absence of overlying periodontal ligament) in diseased teeth can affect the ability for regeneration and new connective tissue attachment onto the previously denuded root surface.

Accelerated enamel mineralization in Dspp mutant mice

Dentin sialophosphoprotein (DSPP) is one of the major non-collagenous proteins present in dentin, cementum and alveolar bone; it is also transiently expressed by ameloblasts. In humans many mutations have been found in DSPP and are associated with two autosomal-dominant genetic diseases — dentinogenesis imperfecta II (DGI-II) and dentin dysplasia (DD). Both disorders result in the development of hypomineralized and mechanically compromised teeth. The erupted mature molars of Dspp−/− mice have a severe hypomineralized dentin phenotype. Since dentin and enamel formations are interdependent, we decided to investigate the process of enamel onset mineralization in young Dspp−/− animals. We focused our analysis on the constantly erupting mouse incisor, to capture all of the stages of odontogenesis in one tooth, and the unerupted first molars. Using high-resolution microCT, we revealed that the onset of enamel matrix deposition occurs closer to the cervical loop and both secretion and maturation of enamel are accelerated in Dspp−/− incisors compared to the Dspp+/− control. Importantly, these differences did not translate into major phenotypic differences in mature enamel in terms of the structural organization, mineral density or hardness. The only observable difference was the reduction in thickness of the outer enamel layer, while the total enamel thickness remained unchanged. We also observed a compromised dentin-enamel junction, leading to delamination between the dentin and enamel layers. The odontoblast processes were widened and lacked branching near the DEJ. Finally, for the first time we demonstrate expression of Dspp mRNA in secretory ameloblasts. In summary, our data show that DSPP is important for normal mineralization of both dentin and enamel.

Ultrastructural organization of dentin in mice lacking dentin sialo-phosphoprotein

Dentin Sialophosphoprotein (DSPP) is the major non-collagenous protein of dentin and plays a significant role in dentin mineralization. Recently, animal models lacking DSPP have been developed and the DSPP KO phenotype has been characterized at the histological level. Little is known, however, about the DSPP KO dentin at nano- and meso-scale. Dentin is a hierarchical material spanning from nano- to macroscale, hence information on the effects of DSPP deficiency at the submicron scale is essential for understanding of its role in dentin biomineralization. To bridge this gap, we have conducted ultrastructural studies of dentin from DSPP KO animals. Transmission electron microscopy (TEM) studies of DSPP KO dentin revealed that although the overall ultrastructural organization was similar to the WT, the mineral particles were less organized. Scanning electron microscopy in the back-scattered mode (BS-SEM) of the DSPP KO dentin revealed that circumpulpal dentin comprises large areas of non-mineralized matrix, with numerous spherulitic mineralized inclusions, while the mantle dentin appeared largely unaffected. Analysis of the mineral distribution in the circumpulpal dentin of the DSPP KO mice suggests a reduction in the number of mineral nucleation sites and an increase in the nucleation barrier in DSPP KO dentin. These preliminary results indicate that in addition to the reduction of mineralized and total dentin volume in DSPP KO animals significant changes in the ultrastructural organization exist. These changes are likely related to the role of DSPP in the regulation of mineral formation and organization in dentin.

Early B-cell factor 1 is an essential transcription factor for postnatal glomerular maturation

The coordination of multiple cytokines and transcription factors with their downstream signaling pathways have been shown to be integral to nephron maturation. Here we present a completely novel role for the helix-loop-helix transcription factor Early B cell Factor 1 (Ebf1), originally identified for B cell maturation, for the proper maturation of glomerular cells from mesenchymal progenitors. The expression of Ebf1 was both spatially and temporally regulated within the developing cortex and glomeruli. Using Ebf1-null mice we then identified biochemical, metabolic, and histological abnormalities in renal development that arose in the absence of this transcription factor. In the Ebf1 knockout mice the developed kidneys show thinned cortices and reduced glomerular maturation. The glomeruli showed abnormal vascularization and severely effaced podocytes. The mice exhibited early albuminuria and elevated blood urea nitrogen levels. Moreover, the GFR was reduced over 66 percent and the expression of podocyte-derived VEGF-A was decreased compared to wild type control mice. Thus, Ebf1 has a significant and novel role in glomerular development, podocyte maturation, and the maintenance of kidney integrity and function.

Dentin Phosphophoryn Activates Smad Protein Signaling through Ca2+-Calmodulin-dependent Protein Kinase II in Undifferentiated Mesenchymal Cells

Dentin phosphophoryn (DPP) is a major noncollagenous protein in the dentin matrix. In this study, we demonstrate that pluripotent stem cells such as C3H10T1/2 and human bone marrow cells can be committed to the osteogenic lineage by DPP. Treatment with DPP can stimulate the release of intracellular Ca(2+). This calcium flux triggered the activation of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII induced the phosphorylation of Smad1 and promoted nuclear translocation of p-Smad1. Inhibition of store Ca(2+) depletion by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or down-regulation of CaMKII by KN-62, a selective cell-permeable pharmacological inhibitor or a dominant negative plasmid of CaMKII, blocked DPP-mediated Smad1 phosphorylation. Activation of Smad1 resulted in the expression of osteogenic markers such as Runx2, Osterix, DMP1, Bone sialoprotein, Osteocalcin, NFATc1, and Schnurri-2, which have been implicated in osteoblast differentiation. These findings suggest that DPP is capable of triggering commitment of pluripotent stem cells to the osteogenic lineage.

Bioluminescence imaging of bone formation using hairless osteocalcin-luciferase transgenic mice

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells during the late stage of maturation. We introduced a 10kb human osteocalcin enhancer/promoter (OC)-luciferase (Luc) construct into a hairless mouse line. Examination of tissue RNAs from these transgenic mice showed a predominant restriction of Luc mRNA expression to bone-associated tissues. Immunohistochemical staining of calvaria tissue sections revealed the localization of Luc protein to osteoblasts. Utilizing in vivo bioluminescence imaging, supplementation of 1α,25-dihydroxyvitamin D3 increased Luc activity throughout the skeleton, consistent with in vitro transient transfection studies in osteoblast-like cells. Moreover, we observed an abrupt decrease in bioluminescence activity as the mice reached puberty, and a further decrease gradually thereafter. Using a radius skeletal repair model, we observed enhanced bioluminescence at the fracture site in both young (14–22weeks old) and aged (50–66weeks old) mice. However, peak bioluminescence was delayed in aged mice compared with young mice, suggesting retarded osteocalcin expression with aging. Our in vivo imaging system may contribute to the therapy and prevention of various bone metabolic disorders through its effective monitoring of the bone formation process.

Immunohistochemical localization of the NH2-terminal and COOH-terminal fragments of dentin sialoprotein in mouse teeth

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH(2)-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH(2)-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH(2) antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH(2)-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN.

P53 and MDM2 are involved in the regulation of osteocalcin gene expression

Osteocalcin (OC) is a major noncollagenous bone matrix protein and an osteoblast marker whose expression is limited to mature osteoblasts during the late differentiation stage. In previous studies we have shown osteosarcomas to lose p53 function with a corresponding loss of osteocalcin gene expression. Introduction of wild type p53 resulted in re expression of the osteocalcin gene. Using gel shift and chromatin immunoprecipitation assays, we have identified a putative p53 binding site within the rat OC promoter region and observed an increase in OC promoter activity when p53 accumulates using a CAT assay. The p53 inducible gene Mdm2 is a well-known downstream regulator of p53 levels. Our results showed a synergistic increase in the OC promoter activity when both p53 and MDM2 were transiently overexpressed. We further demonstrate that p53 is not degraded during overexpression of MDM2 protein. Increased OC expression was observed with concomitantly increased p53, VDR, and MDM2 levels in ROS17/2.8 cells during treatment with differentiation promoting (DP) media, but was significantly decreased when co-treated with DP media and the small molecule inhibitor of MDM2–p53 interaction, Nutlin-3. We have also observed a dramatic increase of the OC promoter activity in the presence of p53 and Mdm2 with inclusion of Cbfa-1 and p300 factors. Our results suggest that under some physiological conditions the oncoprotein MDM2 may cooperate with p53 to regulate the osteocalcin gene during osteoblastic differentiation.

Dentin Phosphoprotein (DPP) Activates Integrin-mediated Anchorage-dependent Signals in Undifferentiated Mesenchymal Cells

Dentin phosphoprotein (DPP), a major noncollagenous protein of the dentin matrix, is a highly acidic protein that binds Ca(2+) avidly and is thus linked to matrix mineralization. Here, we demonstrate that the RGD domain in DPP can bind to integrins on the cell surface of undifferentiated mesenchymal stem cells and pulp cells. This coupling generates intracellular signals that are channeled along cytoskeletal filaments and activate the non-receptor tyrosine kinase focal adhesion kinase, which plays a key role in signaling at sites of cellular adhesion. The putative focal adhesion kinase autophosphorylation site Tyr(397) is phosphorylated during focal adhesion assembly induced by DPP on the substrate. We further demonstrate that these intracellular signals propagate through the cytoplasm and activate anchorage-dependent ERK signaling. Activated ERK translocates to the nucleus and phosphorylates the transcription factor ELK-1, which in turn coordinates the expression of downstream target genes such as DMP1 and dentin sialoprotein (DSP). These studies suggest a novel paradigm demonstrating that extracellular DPP can induce intracellular signaling that can be propagated to the nucleus and thus alter gene activities.

Acidic bone matrix proteins and their roles in calcification

Mammalian bones are composed of calcium phosphate crystals in a protein matrix. The major form of the calcium phosphate is hydroxyapatite. The most abundant matrix protein in bone is type I collagen. Collagen contributes to the mechanical properties of bone and is necessary for calcification of the tissue. In addition to collagen, several acidic proteins are present as minor components. Osteocalcin is a gamma-carboxyglutamic acid-containing protein of bone, which has an affinity to hydroxyapatite and can prevent crystal growth. Bone sialoprotein (BSP) and osteopontin are acidic glycophosphoproteins of bone. These proteins have RGD cell-attachment sequences and consecutive sequences of acidic amino acids. The poly glutamic acid sequences of BSP act as possible nucleation sites for hydroxyapatite crystals. Dentin phosphoprotein is the major non-collagenous protein of dentin. This protein has (Asp-Ser-Ser) repeat sequences, in which most of the Ser residues are phosphorylated. Some of these acidic matrix proteins are immobilized on the collagen fibrils and induce nucleation of hydroxyapatite crystals. They can also modulate crystal shape by adsorption on a specific face of the crystals.

Expression of mineralized tissue associated proteins: Dentin sialoprotein and phosphophoryn in rodent hair follicles

Mammalian hair development and tooth development are controlled by a series of reciprocal epithelial-mesenchymal interactions. Similar growth factors and transcription factors, such as fibroblast growth factor (FGF), sonic hedgehog homolog (SHH), bone morphogenetic proteins (BMPs) and Wnt10a, were reported to be involved in both of these interactions. Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two major non-collagenous proteins secreted by odontoblasts that participate in dentin mineralization during tooth development. Because of striking similarities between tooth development and hair follicle development, we investigated whether DSP and/or PP proteins may also play a role in hair follicle development. In this study, we examined the presence and location of DSP/PP proteins during hair follicle development. Rat PP proteins were detected using immunohistochemical/immunofluorescent staining. DSP-PP mRNAs were detected by in situ hybridization with riboprobes. LacZ expression was detected in mouse tissues using a DSP-PP promoter-driven LUC in transgenic mice. We found that PP proteins and DSP-PP mRNAs are present in rat hair follicles. We also demonstrate that an 8 kb DSP-PP promoter is able to drive lacZ expression in hair follicles. We have firmly established the presence of DSP/PP in mouse and rat hair follicles by immunohistochemical/immunofluorescent staining, in situ hybridization with riboprobes and transgenic mice studies. The expression of DSP/PP in hair follicles is the first demonstration that major mineralization proteins likely may also contribute to soft tissue development. This finding opens a new avenue for future investigations into the molecular-genetic management of soft tissue development.

Biochemical characterization of major bone-matrix proteins using nanoscale-size bone samples and proteomics methodology.

There is growing evidence supporting the need for a broad scale investigation of the proteins and protein modifications in the organic matrix of bone and the use of these measures to predict fragility fractures. However, limitations in sample availability and high heterogeneity of bone tissue cause unique experimental and/or diagnostic problems. We addressed these by an innovative combination of laser capture microscopy with our newly developed liquid chromatography separation methods, followed by gel electrophoresis and mass spectrometry analysis. Our strategy allows in-depth analysis of very limited amounts of bone material, and thus, can be important to medical sciences, biology, forensic, anthropology, and archaeology. The developed strategy permitted unprecedented biochemical analyses of bone-matrix proteins, including collagen modifications, using nearly nanoscale amounts of exceptionally homogenous bone tissue. Dissection of fully mineralized bone-tissue at such degree of homogeneity has not been achieved before. Application of our strategy established that: (1) collagen in older interstitial bone contains higher levels of an advanced glycation end product pentosidine then younger osteonal tissue, an observation contrary to the published data; (2) the levels of two enzymatic crosslinks (pyridinoline and deoxypiridinoline) were higher in osteonal than interstitial tissue and agreed with data reported by others; (3) younger osteonal bone has higher amount of osteopontin and osteocalcin then older interstitial bone and this has not been shown before. Taken together, these data show that the level of fluorescent crosslinks in collagen and the amount of two major noncollagenous bone matrix proteins differ at the level of osteonal and interstitial tissue. We propose that this may have important implications for bone remodeling processes and bone microdamage formation.

Two models of Smad4 and Hoxa9 Complex are Proposed: Structural and Interactional Perspective

Transforming growth factor-beta superfamily growth factors (TGF-β) regulate a diverse range of cellular functions, including proliferation, differentiation, extracellular matrix secretion and cell adhesion. TGF-β is also one of the most abundant of the known growth factors. Osteopontin (OPN), the major non-collagenous bone matrix protein is a secreted, arginine-glycine-aspertate containing phosphorylated glycoprotein. Analysis of the OPN promoter sequence reveals both Hoxc8 and Hoxa9 (mouse homeotic gene) recognize and utilize the same consensus TAAT motif in the binding sequence to mediate the repression. Hoxa9 functions as a strong transcriptional repressor, similar to Hoxc-8 (X. Shi, X. Yang, D. Chen, Z. Chang, and X. Cao, J Biol Chem 274, 13711-13717, 1999). The DNA-binding protein Hoxa9 interacts with Smad4 (X. Shi, S. Bai, L. Li and X, and Cao X, J Biol Chem 276, 850-855, 2001), but not with Smad3 (which binds to OPN promoter), and the interaction between Smad4 and Hoxa9 results in the transcriptional activation of OPN in response to TGF- stimulation. In this paper we have proposed two possible model structures of Hoxa9 and Smad4 complex. These have been modeled based on homology modeling and a new method has been used to model the flexible loop part. Manual docking has been used to achieve the final model involving the Hoxa9 -Smad4 complex which tallies with the experimental results. We have mutated some selective important residues and looked at their effect in terms of interaction energy in complex formation in both the models.

The Canonical BMP Signaling Pathway Plays a Crucial Part in Stimulation of Dentin Sialophosphoprotein Expression by BMP-2

Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (-791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.