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KMID : 0352219930150010073
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Kyung Hee Dental Journal
1993 Volume.15 No. 1 p.73 ~ p.95
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CHARCTERIZATION OF SECRETED PROTEIN ACIDIC AND RICH IN CYSTEINE, BONE SIALOPROTEIN, OSTEOPONTIN AND ALKALINE PHOSPHATASE ASSOCIATED WITH BONE MATRIX FORMATION AND MINERALIZATION IN OSTEOBLASTIC ROS 17/2.8 CELL CULTURE
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Abstract
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To determine the role of noncollagenous proteins and alkaline phosphatase during matrix formation and mineralization, the biosynthesis of secreted protein acidic and rich in cysteine (SPARC), bone sialoprotein (BSP) and osteopontin (OP), Northern blot analysis of SPARC and alkaline phosphatase (ALP) mRNAs and the specific activity of ALP were studied in cultures of clonal osteoblastic rat osteosarcoma ROS 17/2.8 cells. First, at the culture conditions of confluence, matrix formation and minerlization, [´S-methionine-labelled proteins were specifically immunoprecipitated with antibodies to SPARC, BSP and OP and analysed by SDS/PAGE on 15% cross-linked mini-gels under reducing conditions. The expression of SPARC and ALP mRNAs was estimated by Northern blot analysis and the ALP specific activity (pmol/min/106 cells) was measured by the methods of Lowry et al.´´ and Majeska andRodan.´´´ Second, to determine the inductive effects of dexamethasone (Dex) on the expression of SPARC and ALP mRNAs and the ALP specific activity in mineralizing culture, the confluent cultures of ROS 17/2.8 cells were incubated for 0, 4, 7, and 14 days in contral group [ascorbic acid (AA), J3-glycerophosphate (/3-GP)] and Dex group (AA, /3-GP and Dex). Analysis of mRNA by Northern hybridization using the SPARC and ALP cDNA probes was performed and the ALP specific activity was measured and analysed by spectra-photometry. The results were as follows.
1. ROS 17/2.8 cells incubated in the culture condition for matrix formation exhibited multilayers and in the culture condition for mineralization they indicated many foci-like cell aggregates (bone nodules).
2. SPARC was abundant in culture medium and cell extract, but not detected in E extract of the culture condition for mineralization. BSP was present only in cell extract and OP in medium and cell extract.
3. The strongest band of SPARC was revealed in medium of the culture condition for mineralization.
In BSP, it was present in cell extract of the culture condition for confluence, and in OP, it was in
medium of the culture condition for matrix formation.
4. Northern blot analysis revealed that levels of SPARC mRNA the in culture conditions for matrix forma_ Lion and mineralization comparing with those in culture condition for confluence were shown to be increased by 2.1-and 31-fold, respectively. However, levels of ALP mRNA were increased markedly only in the culture condition for mineralization.
5. In mineralizing culture, Northern hybridization analysis showed a strong induction of SPARC and ALP mRNAs in Dex-treated condition. Especially, maximum effects of Dex were indicated at 7days in both SPARC and ALP mRNAs.
6. ALP specific activites in the culture condition for matrix formation and mineralization comparing with those in the culture condition for confluenece were increased by 1.8-and 3.5-fold, respectively. In Dextreated culture, ALP specific activities at 4, 7 and 14 days were increased by 4.5-, 3.5- and 2.6-fold,
respectively, but cell proliferation was shown to be inhibited.
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KEYWORD
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