ABSTRACT
<p><b>OBJECTIVE</b>To explore the effect of DNAX-associated protein 12 (DAP12) pathway on the transformation from mouse monocytes RAW264.7 to osteoclasts induced by tensile strain.</p><p><b>METHODS</b>DAP12shRNA plasmid was constructed and introduced to RAW264.7 cells. Then we supplied tensile strain to RAW264.7 cells by four-point bending system. The mRNA or protein expression of DAP12, tartrate-resistant acid phosphatase (TRAP), tyrosine kinases Btk and Tec and nuclear facior of activated T cells 1 (NFATc1) was measured by reverse transcription PCR (RT-PCR) and Western blotting respectively.</p><p><b>RESULTS</b>The expression of DAP12 mRNA (0.112 ± 0.025) and protein (0.193 ± 0.015) both declined sharply after plasmid being introduced into monocytes RAW264.7 (P < 0.05). After silencing DAP12 expression in RAW264.7 cells by RNA interference, tensile strain-induced TRAP mRNA expression of RAW264.7 cells increased at 6 h (0.671 ± 0.031) and 12 h (0.800 ± 0.043) (P < 0.05), but it was weaker than non-RNA-interference-groups at each time point (P < 0.05). After silencing DAP12 expression in RAW264.7 cells by RNA interference, the expressions of Btk, Tec, NFATc1 increased as time passed (6, 12 h) (P < 0.05), but the expressions on corresponding time decreased sharply compared with those in control groups (P < 0.05).</p><p><b>CONCLUSIONS</b>DAP12 pathway play an important role in regulating osteoclast differentiation induced by tensile strain.</p>
Subject(s)
Animals , Mice , Acid Phosphatase , Genetics , Metabolism , Adaptor Proteins, Signal Transducing , Genetics , Metabolism , Cell Differentiation , Cell Line , Gene Expression Regulation , Gene Silencing , Isoenzymes , Genetics , Metabolism , Monocytes , Cell Biology , Metabolism , NFATC Transcription Factors , Metabolism , Osteoclasts , Cell Biology , Plasmids , Protein-Tyrosine Kinases , Metabolism , RNA, Messenger , Metabolism , RNA, Small Interfering , Signal Transduction , Tartrate-Resistant Acid Phosphatase , Tensile StrengthABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of parathyroid hormone related protein (PTHrP) on proliferation of human osteoblasts (MG-63) under the circumstance of tension force in vitro.</p><p><b>METHODS</b>An apparatus was designed and fabricated by which force was loaded onto the cultured cells in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) was used for measuring the expression of PTHrP mRNA and c-fos mRNA. The effect of tension force and different PTHrP dose(0, 0.01, 0.1, 1 nmol/L) on the proliferation of human osteoblasts were examined using flow cytometry.</p><p><b>RESULTS</b>Various forces of the mechanical stretching exerted different influences on the intensities of the mRNA' expression. The strain of 12% induced the most remarkable mRNA' expression. The mitogenesis happened in the group with tension force (12%) combined with PTHrP was more active than that in the group with PTHrP or tension' force only. Tension force combined with PTHrP induced significantly more c-fos mRNA than that of tension force only.</p><p><b>CONCLUSION</b>The mechanical stretching can inevitably influence the expression of PTHrP mRNA. The most active mitogenesis happened in the group with tension force combined with PTHrP. The effect may be related with the signaling pathways of c-fos.</p>