Characterisation of the constitutive over-expression of AJ18 in a novel rat stromal bone marrow cell line (D8-SBMC).

OBJECTIVE: The elucidation of the molecular pathways involved in osteoblast proliferation and differentiation has been greatly enhanced by the availability of cell culture model systems. However, many of the current bone cell culture systems suffer from disadvantages such as the inability to generate mineralised bone-like nodules, a transformed genetic background, cell heterogeneity, and a relatively long time frame from cell seeding to mineralisation, often in the order of several weeks. Here we describe the establishment and characterisation of a novel bone cell line named D8-SBMC. As a first demonstration of their potential value, D8-SBMC was utilised to further support a role for AJ18 during osteogenesis. DESIGN: D8-SBMC was established from a single cell suspension of the previously characterised long term rat stromal bone marrow cells [Kotev-Emeth S, Pitaru S, Pri-Chen S, Savion N. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect Tissue Res 2002;43(4):606-12; Pitaru S, Kotev-Emeth S, Noff D, Kaffuler S, Savion N. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture. J Bone Miner Res 1993;8(8):919-29]. AJ18 was constitutively and stably over-expressed in D8-SBMC and analysed. RESULTS: D8-SBMC possesses the ability to form robust mineralised bone-like nodules within 8 days proceeding cell confluency. Interestingly, a cement line-like matrix is also generated between the culture dish and a basal monolayer of cells. Constitutive and stable over-expression of AJ18 resulted in an increase in cell proliferation and mineralisation. Expression of bone marker genes, such as bone sialoprotein, osteopontin, osteocalcin, collage type 1, and osteonectin, was up-regulated by AJ18 over-expression. CONCLUSION: A novel bone cell line, D8-SBMC, was established and characterised. D8-SBMC may be a valuable model system for biomineralisation studies. D8-SBMC was utilised to further understand the role of AJ18 in cell proliferation and differentiation during osteogenesis.

Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation.

BACKGROUND: A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the alphavbeta3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface. METHODS: We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model. RESULTS: Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-alphavbeta3 integrin interaction does not induce the expression of MT1-MMP. CONCLUSION: These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2.

Analysis of human bone sialoprotein in normal and pathological tissues using a monoclonal antibody (BSP 1.2 mab).

Bone sialoprotein (BSP), a phosphorylated and sulphated glycoprotein that is expressed by mineralized connective tissues is also produced in tumors that metastasize to bone. To facilitate studies of BSP expression in normal and pathological human tissues a monoclonal antibody (BSP 1.2 mab) was raised against human bone BSP. BSP 1.2 mab was shown by ELISA assays to recognize the epitope "DEYSY" (amino acids 279-283) that is conserved in mammalian BSP sequences. However, whereas the antibody recognized recombinant BSPs expressed in bacteria, it did not recognize native forms of rat or pig BSP in which the first tyrosine of the DEYSY peptide sequence appears to be modified. Immunostaining of embryonic human tibiae and calvariae with BSP 1.2 mab showed strong reaction in osteoblasts and osteocytes with relatively weak staining of the bone matrix, suggesting that the BSP 1.2 mab epitope is partially masked in the bone matrix. BSP 1.2 mab also stained osteosarcoma cells and normal trophoblastic cells in the placenta in areas of microcrystalline deposits. Cancer cells in primary breast tumors, lymph nodes, and secondary bone metastases from individual patients were stained strongly by BSP 1.2 mab. Although BSP 1.2 mab also stained breast cancer carcinoma cell lines and SaOS2 osteosarcoma cells, biosynthesis of radiolabelled BSP could not be demonstrated in breast cancer cells. Notably, the staining of BSP in the breast cancer cells was diffuse contrasting the punctate staining, typical of secreted proteins, in SaOS2 cells. These studies, therefore, have identified a unique epitope in human BSP recognized by a monoclonal antibody, BSP 1.2 mab, which can be used for the unequivocal identification of BSP in normal and pathological human tissues.

Characterization of the 5'-flanking region of the rat AJ18 gene.

Krüppel-associated box (KRAB) domains are present in one-third of all C(2)H(2) zinc finger containing proteins, making the KRAB/C(2)H(2) proteins one of the largest known families of putative transcription repressors. AJ18 has been identified as a novel KRAB/C(2)H(2) gene that is involved in the differentiation of osteogenic cells. To study the regulation of expression of the AJ18 gene, the 5'-flanking region of the AJ18 gene was obtained by screening a rat genomic library. This region was sequenced, and the transcription start site mapped by primer extension. The AJ18 gene consists of at least four exons, the first exon coding for an unusually long 2.3 kb 5'-UTR region. A putative internal ribosome entry site, immediately upstream of the translation initiation site, is indicated from the complementarity of a 12 nucleotide sequence with a region in the rat 18S rRNA. Chimeric constructs encompassing the region surrounding the transcription start site (-77-+171), as well as constructs with additional 1.9 kb upstream from this region revealed strong transcriptional activity when ligated to a luciferase reporter gene and tested in transient transfection assays. This activity was lost on deletion of the 5'-flanking region to -77. In addition, transcriptional activity was progressively lost with the inclusion of downstream sequences extending into the 5'-UTR. Several known response elements for proteins such as Runx2, NFkappaB, Smads, Sp1, and Ets1 are retained within the conserved sequences of rat and mouse AJ18, which was retrieved from mouse genomic libraries. Interestingly, the transcriptional activity was approximately 100-fold higher in the osteocarcinoma cell line ROS 2.8/17 compared to the fibroblast-like C3H10T1/2. Notably, this is the first gene promoter from the large KRAB/C(2)H(2) zinc finger family of proteins to be identified and characterized.

Temporal and spatial expression of a novel zinc finger transcription factor, AJ18, in developing murine skeletal tissues.

Bone morphogenetic proteins (BMPs) are characterized by their ability to induce osteoblastic differentiation. However, the mechanism of osteo-induction by BMPs has yet to be determined. Using differential display we previously identified AJ18, a zinc finger transcription factor, as an immediate-early response gene to BMP-7. AJ18 was shown to bind to the osteoblast-specific element2 (OSE2) and to modulate transactivation by Runx2, a master gene in osteoblastic differentiation. Here we describe the temporal and spatial expression of AJ18 in developing mouse tissues. AJ18 mRNA expression was observed in most tissues, except liver, and was generally highest early in embryonic development, decreasing markedly after parturition. Consistent with immunohistochemical analysis, AJ18 mRNA expression was highest in the brain, kidney, and bone of 17 dpc (days post coitum) embryos. In endochondral bones of embryonic and 4-week-old mice, immunostaining for AJ18 was strong in the nuclei of proliferating and pre-hypertrophic chondrocytes, and osteoblasts, whereas there was low or no staining in hypertrophic chondrocytes. In teeth of embryonic and 4-week-old mice, nuclear staining was observed in precursor and mature ameloblasts, odontoblasts, and cementoblasts, respectively. In addition, in 4-week-old mice staining of AJ18 was observed within alveolar bone cells and periodontal ligament cells. In general, the spatial expression of AJ18 in skeletal and non-skeletal tissues of mouse embryos showed striking similarity to the expression of BMP-7 mRNA. Therefore, the expression of AJ18 is consistent with its perceived role as a transcriptional factor that regulates developmental processes downstream of BMP-7.