Accumulation, localization, and compartmentation of transforming growth factor beta during endochondral bone development.

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor beta (TGF beta) in the extracts was quantitated by a radioreceptor assay. TGF beta was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF beta was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF beta was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF beta per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF beta was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF beta in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGF beta was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF beta in the mineral phase. In contrast to the delayed expression of TGF beta protein, northern blot analysis showed TGF beta mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGF beta when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF beta may play a role in the regulation of ossification during endochondral bone development

Spag16, an Axonemal Central Apparatus Gene, Encodes a Male Germ Cell Nuclear Speckle Protein that Regulates SPAG16 mRNA Expression

Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9+2” axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions

A Cross-Species Analysis of a Mouse Model of Breast Cancer-Specific Osteolysis and Human Bone Metastases Using Gene Expression Profiling

<p>Abstract</p> <p>Background</p> <p>Breast cancer is the second leading cause of cancer-related death in women in the United States. During the advanced stages of disease, many breast cancer patients suffer from bone metastasis. These metastases are predominantly osteolytic and develop when tumor cells interact with bone. <it>In vivo </it>models that mimic the breast cancer-specific osteolytic bone microenvironment are limited. Previously, we developed a mouse model of tumor-bone interaction in which three mouse breast cancer cell lines were implanted onto the calvaria. Analysis of tumors from this model revealed that they exhibited strong bone resorption, induction of osteoclasts and intracranial penetration at the tumor bone (TB)-interface.</p> <p>Methods</p> <p>In this study, we identified and used a TB microenvironment-specific gene expression signature from this model to extend our understanding of the metastatic bone microenvironment in human disease and to predict potential therapeutic targets.</p> <p>Results</p> <p>We identified a TB signature consisting of 934 genes that were commonly (among our 3 cell lines) and specifically (as compared to tumor-alone area within the bone microenvironment) up- and down-regulated >2-fold at the TB interface in our mouse osteolytic model. By comparing the TB signature with gene expression profiles from human breast metastases and an <it>in vitro </it>osteoclast model, we demonstrate that our model mimics both the human breast cancer bone microenvironment and osteoclastogenesis. Furthermore, we observed enrichment in various signaling pathways specific to the TB interface; that is, TGF-β and myeloid self-renewal pathways were activated and the Wnt pathway was inactivated. Lastly, we used the TB-signature to predict cyclopenthiazide as a potential inhibitor of the TB interface.</p> <p>Conclusion</p> <p>Our mouse breast cancer model morphologically and genetically resembles the osteoclastic bone microenvironment observed in human disease. Characterization of the gene expression signature specific to the TB interface in our model revealed signaling mechanisms operative in human breast cancer metastases and predicted a therapeutic inhibitor of cancer-mediated osteolysis.</p

Calcified Algae for Tissue Engineering.

This book presents the latest advances in marine structures and related biomaterials for applications in both soft- and hard-tissue engineering, as well as controlled drug delivery

Accumulation, localization, and compartmentation of transforming growth factor beta during endochondral bone development.

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor beta (TGF beta) in the extracts was quantitated by a radioreceptor assay. TGF beta was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF beta was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF beta was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF beta per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF beta was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF beta in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGF beta was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF beta in the mineral phase. In contrast to the delayed expression of TGF beta protein, northern blot analysis showed TGF beta mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGF beta when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF beta may play a role in the regulation of ossification during endochondral bone development