Biomedical subjects

Jake Chen

Publications and source records attributed to Jake Chen.

9 recordsLinked to original sources

PubMed2006

DisProt: the Database of Disordered Proteins.

The Database of Protein Disorder (DisProt) links structure and function information for intrinsically disordered proteins (IDPs). Intrinsically disordered proteins do not form a fixed three-dimensional structure under physiological conditions, either in their entireties or in segments or regions. We define IDP as a protein that contains at least one experimentally determined disordered region. Although lacking fixed structure, IDPs and regions carry out important biological functions, being typically involved in regulation, signaling and control. Such functions can involve high-specificity low-affinity interactions, the multiple binding of one protein to many partners and the multiple binding of many proteins to one partner. These three features are all enabled and enhanced by protein intrinsic disorder. One of the major hindrances in the study of IDPs has been the lack of organized information. DisProt was developed to enable IDP research by collecting and organizing knowledge regarding the experimental characterization and the functional associations of IDPs. In addition to being a unique source of biological information, DisProt opens doors for a plethora of bioinformatics studies. DisProt is openly available at http://www.disprot.org.

Databases, Protein↗

PubMed2006

Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells.

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of alpha1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.

Alkaline Phosphatase↗

PubMed2006

Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo.

Bone morphogenetic protein-2 (BMP-2) plays a key role in osteogenesis. Biomaterials used for the sustained delivery of BMP-2 in vivo have shown therapeutic benefits. In the present study, BMP-2 was loaded in porous silk fibroin scaffolds derived from silkworm cocoons (2.4 +/- 0.14 microg per scaffold). The release profile of BMP-2 under dynamic culture conditions (spinner flasks) showed that after 1 week in culture 25% of the initial BMP-2 was retained adsorbed to the scaffold; up to 4 weeks no additional BMP-2 was released. BMP-2 induced human bone marrow stromal cells (hMSCs) to undergo osteogenic differentiation when the seeded scaffolds were cultured in medium supplemented with osteogenic stimulants for 4 weeks, based on elevated alkaline phosphatase activity, calcium deposition, and transcript levels for bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa-1. Micro-computed tomography revealed densely deposited mineral at the center of the scaffolds. In contrast, hMSCs cultured in control scaffolds (no BMP-2) exhibited limited osteogenesis. When implanted in critical sized cranial defects in mice, scaffolds loaded with BMP-2 and seeded with hMSCs resulted in significant bone ingrowth. These results were qualitatively similar to scaffolds loaded with BMP-2 but no hMSCs or with BMP-2 and hMSCs but not pregrown into bone-like tissue. Bone-related outcomes were improved when compared with the scaffold controls implanted without BMP-2. These studies illustrate the potential use of slow degrading silk fibroin 3-D scaffolds loaded with BMP-2, in combination with hMSCs, in osteogenesis studies in vitro and in vivo, and provide a new range of material properties for these applications.

Adsorption↗

PubMed2006

Disorder and sequence repeats in hub proteins and their implications for network evolution.

Protein interaction networks display approximate scale-free topology, in which hub proteins that interact with a large number of other proteins determine the overall organization of the network. In this study, we aim to determine whether hubs are distinguishable from other networked proteins by specific sequence features. Proteins of different connectednesses were compared in the interaction networks of Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapienswith respect to the distribution of predicted structural disorder, sequence repeats, low complexity regions, and chain length. Highly connected proteins ("hub proteins") contained significantly more of, and greater proportion of, these sequence features and tended to be longer overall as compared to less connected proteins. These sequence features provide two different functional means for realizing multiple interactions: (1) extended interaction surface and (2) flexibility and adaptability, providing a mechanism for the same region to bind distinct partners. Our view contradicts the prevailing view that scaling in protein interactomes arose from gene duplication and preferential attachment of equivalent proteins. We propose an alternative evolutionary network specialization process, in which certain components of the protein interactome improved their fitness for binding by becoming longer or accruing regions of disorder and/or internal repeats and have therefore become specialized in network organization.

Amino Acid Sequence↗

PubMed2005

Silk implants for the healing of critical size bone defects.

Bone (re)-generation and bone fixation strategies utilize biomaterial implants, which are gradually replaced by autologous tissues. Ideally, these biomaterials should be biodegradable, osteoconductive, and provide mechanical strength and integrity until newly formed host tissues can maintain function. Some protein-based biomaterials such as collagens are promising because of their biological similarities to natural proteins on bone surfaces. However, their use as bone implant materials is largely hampered by poor mechanical properties. In contrast, silks offer distinguishing mechanical properties that are tailorable, along with slow degradability to permit adequate time for remodeling. To assess the suitability of silk-based biomaterials as implants for bone healing, we explored the use of novel porous silk fibroin scaffolds as templates for the engineering of bone tissues starting from human bone marrow derived stem cells cultured under osteogenic conditions for up to 5 weeks. The slowly degrading protein matrix permitted adequate temporal control of hydroxyapatite deposition and resulted in the formation of a trabecular-like bone matrix in bioreactor studies. The organic and inorganic components of the engineered bone tissues resembled those of bone, as shown by gene expression analysis, biochemical assays, and X-ray diffractometry. Implantation of the tissue-engineered bone implants (grown in bioreactors for 5 weeks prior to implantation) into calvarial critical size defects in mice demonstrated the capacity of these systems to induce advanced bone formation within 5 weeks, whereas the implantation of stem cell loaded silk scaffolds, and scaffolds alone resulted in less bone formation. These results demonstrate the feasibility of silk-based implants with engineered bone for the (re-)generation of bone tissues and expand the class of protein-based bone-implant materials with a mechanically stable and durable option.

Animals↗

PubMed2005

Cloning of hamster osteopontin and expression distribution in normal tissues and experimentally induced oral squamous-cell carcinoma.

Osteopontin (OPN) is a non-collagenous extracellular matrix (ECM) protein expressed and secreted by several human cancers. This study investigated the expression pattern of OPN during development of oral squamous-cell carcinoma by using 7,12-dimethylbenz[a]anthracene (DMBA)-induced squamous-cell carcinomas in buccal pouch of syrian golden hamsters. We first identified the hamster OPN cDNA sequence by screening of a hamster calvariae cDNA library with a rat OPN cDNA probe. The resulting 1,449 bp of hamster OPN cDNA led to a deduced protein sequence of 305 amino acids containing several putative binding sites to integrins, CD44 receptors, calcium ions and hydroxyapatite, as well as multiple sites for phosphorylation, glycosylation and sulphation. Hamster OPN cDNA was then used as a probe to analyze the expression of OPN mRNA by Northern blot and in situ hybridization analyses of normal and malignant tissues. OPN mRNA was detected in several non-mineralized tissues as well as in mineralized tissues, but was not present in normal hamster buccal epithelium. DMBA-treated hamster buccal pouches expressed OPN mRNA as early as 4 weeks and displayed the highest level of expression at 15 weeks. The specimens treated with DMBA for 15 weeks exhibited histological features of squamous-cell carcinoma, presented microcrystalline deposits and showed OPN expression associated with malignant epithelium and tumor-associated macrophages. To summarize, our results suggest that buccal-pouch carcinogenesis of Syrian golden hamster may constitute an excellent experimental model to study the mechanisms by which OPN is associated with oral cancer pathogenesis, and to validate OPN-based therapeutic approaches to ameliorate oral cancer progression and metastasis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2005

BSP and RANKL induce osteoclastogenesis and bone resorption synergistically.

UNLABELLED: RANKL and BSP are upregulated in several bone resorptive disorders. However, the mechanisms by which these two factors might induce osteoclastogenesis and bone resorption synergistically under pathological conditions remain largely unknown. INTRODUCTION: RANKL and bone sialoprotein II (BSP) have been shown to be upregulated in the serum of individuals with abnormally high osteoclastogenic and bone resorptive activities. Here we provide experimental evidence that RANKL and BSP induce osteoclastogenesis and bone resorption synergistically but mediate opposite effects in osteoclast survival and apoptosis. MATERIALS AND METHODS: RAW264.7 cells and mouse bone marrow-derived monocytes/macrophages were treated with human recombinant BSP in the presence and absence of RANKL. TRACP stainings, bone resorption assays, Western blotting, immunoprecipitation analyses, and semiquantitative RT-PCR were used to evaluate the effects of BSP in osteoclast differentiation and bone resorption. Survival, DNA condensation, and caspase activity assays were used to determine the putative effects of BSP in osteoclast survival and apoptosis. RESULTS AND CONCLUSIONS: RANKL induced osteoclast differentiation and bone resorption at a higher extent in the presence than in the absence of BSP in RAW264.7 cells and bone marrow-derived monocytes/macrophages. c-Src-dependent c-Cbl phosphorylation was 8-fold higher in RAW264.7 cells treated with BSP and RANKL than in those treated with RANKL alone. Furthermore, BSP and RANKL activated the master regulator of osteoclastogenesis nuclear factor of activated T cells (NFAT)-2 and increased the mRNA expression of other differentiation markers such as cathepsin K or TRACP. Inhibition of c-Src activity or chelating intracellular calcium inhibited the synergistic effects in bone resorption and the phosphorylation of the c-Src substrate c-Cbl. Inhibition of calcineurin or intracellular calcium elevation inhibited the synergistic effects in osteoclastogenesis and decreased NFAT-2 nuclear levels. On the other hand, BSP and RANKL mediated opposite effects in osteoclast survival and apoptosis. Thus, BSP increased survival and decreased apoptosis markers in differentiated RANKL-treated RAW267.5 cells and RANKL/macrophage-colony stimulating factor (M-CSF)-treated bone marrow-derived monocytes/macrophages. In addition, RAW267.5 cells treated with BSP and RANKL exhibited decreased activation of the proapoptotic Jun N-terminal kinase pathway and increased activation of anti-apoptotic AKT pathway than cells treated with RANKL or BSP alone. Taken together, our findings suggest that BSP contributes to RANKL-mediated bone resorption by inducing osteoclastogenesis and osteoclast survival and decreasing osteoclast apoptosis.

Animals↗

PubMed2005

An in vivo model to study osteogenic gene regulation: targeting an avian retroviral receptor (TVA) to bone with the bone sialoprotein (BSP) promoter.

UNLABELLED: To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. INTRODUCTION: Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation. MATERIALS AND METHODS: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. RESULTS AND CONCLUSION: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased approximately 15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo.

Animals↗

PubMed2003

Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse model.

Bone sialoprotein (BSP) is a major non-collagenous protein found almost exclusively in bone and other mineralized tissues including enamel, dentin and cementum. Although a role for BSP in mineralization has been indicated, BSP also appears to function in patho-physiological processes, including the metastasis of breast and prostate cancer cells to bone. The purpose of this study was to determine the role of BSP in the homing of cancer cells and to provide insights into the role of BSP in physiological as well as pathological processes. We established cultures of MDA-231 breast cancer cells stably transfected with DNA constructs of pIRES2-EGFP (green fluorescent protein) expressing human BSP (hBSP) cDNA (231BSP) under a CMV promoter, or with an antisense sequence of hBSP cDNA (231BSPAS), or with an empty vector as a control (231EV). These 3 cell groups were selected for neomycin resistance using G418 and analyzed by flow cytometry for GFP expression. The resultant cultured cells expressed different levels of hBSP as detected by RT-PCR and Western blot. Among the three, 231BSP expressed the highest levels of hBSP while 231BSPAS expressed the lowest. The capacity of the tumor cells to metastasize to bone was determined in nude mice (5 in each group) by intra-cardiac injection of the cells from the 3 different groups. Four weeks after inoculation, radiological examination revealed that all the 5 mice in the 231BSP cell group had developed osteolytic bone metastases. In the 231BSPAS group only 1 mouse demonstrated metastatic bone lesions while 3 out of 5 mice in the control group (231EV) developed metastatic lesions in the bone. These results strongly suggest that BSP over-expression in human tumor cells can enhance bone metastasis of MDA-231 cells whereas repressed expression of BSP, using antisense BSP cDNA, inhibits this effect in a mouse model.

Animals↗