Osteoblast Extracellular Matrix Research Articles

The hyalectan degrading ADAMTS-1 enzyme is expressed by osteoblasts and up-regulated at regions of new bone formation

During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.

Retracted: Effects of Vitamin K2 on Osteoporosis

1. Iwamoto J, Takeda T, Ichimura S. Combined treatment with vitamin K2 and bisphosphonate in postmenopausal women with osteoporosis. Yonsei Med J 2003; 44: 751-6. Available at: https://eymj.org/DOIx.php?id=10.3349/ymj.2019.60.1.115. It is a condition of publication that manuscripts submitted to this journal have not been published and will not be simultaneously submitted or published elsewhere. Furthermore, any data, illustration, structure or table that has been published elsewhere must be reported, and copyright permission for reproduction must be obtained. Plagiarism is strictly forbidden, and by submitting the article for publication the authors agree that the publishers have the legal right to take appropriate action against the authors, if plagiarism or fabricated information is discovered. By submitting a manuscript, the authors agree that the copyright of their article is transferred to the publishers if and when the article is accepted for publication.

4P-1132 Human evidence that the cystatin C gene is implicated in focal progression of coronary artery disease

During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.

Vitamin K2 enhances osteocalcin accumulation in the extracellular matrix of human osteoblasts in vitro.

The role of vitamin K in osteocalcin accumulation in the extracellular matrix of normal human osteoblasts in culture was investigated by using a human intact osteocalcin-specific assay system. Human osteoblasts produced osteocalcin by treatment with 10(-9) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 20 days in culture. With the addition of vitamin K2 (1.5-5.0 microM), osteocalcin accumulation in the extracellular matrix of the osteoblasts was increased, but the osteocalcin content in the conditioned medium decreased, in comparison with that treated with 10-9 M 1,25(OH)2D3 alone. The enhancement of osteocalcin accumulation induced by vitamin K2 was dependent on the duration of the treatment. The vitamin K2 plus 1,25(OH)2D3-induced osteocalcin accumulation was blocked by the addition of warfarin 2 days before the vitamin treatment. At that time, warfarin significantly reduced the mineralization by osteoblasts in vitro. Osteocalcin accumulated in the extracellular matrix was almost completely precipitated by a low concentration of hydroxyapatite, 10 mg/ml. Moreover, the gamma-carboxyglutamic acid (Gla)-containing osteocalcin level was increased by the vitamin K2 plus 1,25(OH)2D3 treatment. These results proved that vitamin K2 increased Gla-containing osteocalcin, which accumulated osteocalcin in the extracellular matrix, and facilitated mineralization in vitro. Vitamin K2 also enhanced the 1,25(OH)2D3-induced osteocalcin mRNA level, but vitamin K2 alone did not show osteocalcin mRNA expression. We thus demonstrated that vitamin K2 enhanced not only the accumulation of Gla osteocalcin, but also the osteocalcin production induced by 1,25(OH)2D3 in human osteoblasts in culture.

Osteoclast resorption — stimulating activity is associated with the osteoblast cell surface and/or the extracellular matrix

Osteoblasts mediate much of the hormonal responsiveness of osteoclasts. We and others have found that one mechanism through which this regulation is effected is by release of osteoclast resorption-stimulating activity (ORSA) into culture supernatants. However, although hormonal responsiveness is regularly observed in co-cultures, ORSA is not always detectable in conditioned media. We show here that one explanation for this finding is that ORSA may be retained by heparin-like glycosaminoglycans (GAGs) of the cell surface or extracellular matrix of osteoblasts. We found that protease-sensitive ORSA could be extracted from monolayers of the osteoblastic cell line UMR 106 with 2M NaCl or collagenase. Production of this activity was increased in response to 1,25(OH)2D3. The presence of the GAG heparin was required to reveal ORSA. Immobilisation of ORSA by GAGs may assist osteoblastic cells in the regulation of the complex patterns of osteoclastic activity observed during skeletal morphogenesis and restructuring.

The Onset and Progression of Osteoblast Differentiation is Functionally Related to Cellular Proliferation

The relationship of proliferation to the developmental sequence associated with bone cell differentiation was examined in primary osteoblast cultures derived from fetal rat and embryonic chick calvaria. A reciprocal and functional relationship exists between the decline in proliferative activity which occurs during the initial stages of the developmental sequence and the induction of genes encoding osteoblast phenotype proteins associated with matrix maturation and mineralization. This relationship is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase (AP) and osteopontin (OP) genes immediately following the proliferative period and expression of osteocalcin with the onset of mineralization, and 2) increases in AP and OP when DNA synthesis is inhibited. By determining cellular mRNA levels and rates of mRNA synthesis in isolated nuclei, we found that the down-regulation of cell growth-related genes is modified at both the levels of transcription and mRNA stability. For a histone gene where down-regulation is transcriptionally mediated, we have observed that the shutdown of osteoblast proliferation is associated with the selective loss of the interaction of a promoter binding factor (HiNF-D) with a proximal regulatory element (Site II). A relationship between Site II occupancy by HiNF-D and the onset of osteoblast differentiation is supported by the persistence of Site II-HiNF-D interactions when proliferating rat osteoblasts are growth arrested under conditions that do not induce differentiation; and additionally, by the loss of Site II-HiNF-D interactions during the shut-down of proliferation when HL60 promyelocytic leukemia cells are induced to differentiate into monocytes. Our results are consistent with a requirement of proliferation for expression of genes involved with production, deposition and possibly organization of the osteoblast extracellular matrix. It is also reasonable to postulate that properties of the mineralizing matrix are related to the shut-down of proliferation.