Fluid flow-induced tyrosine phosphorylation and participation of growth factor signaling pathway in osteoblast-like cells.
To investigate how mechanical loading stimulates bone cells, we subjected murine osteoblast-like cells, MC3T3E1, to fluid flow generated by shaking culture dishes. Since we had previously found that egr-1 mRNA is up-regulated by the flow, and that the regulation involves tyrosine kinase, we examined which proteins are tyrosine-phosphorylated by flow. Western blotting and immunoprecipitation of cell lysates showed tyrosine phosphorylation enhancement of many proteins, including ERK2 and Shc, and activation of ERK1/2. Although these responses did not occur in serum-free media, addition of EGF or bFGF recovered the responses. AG1478, an inhibitor of EGF receptor kinase activity, abolished tyrosine phosphorylation enhancement, ERK1/2 activation, and egr-1 mRNA accumulation induced by the flow of EGF-containing serum-free media. These results suggest that growth factor signaling pathways are involved in these responses. Repetition of fluid flow induced repeatedly up-regulation of egr-1 mRNA. Such events may also occur in bone under mechanical loading.
Ogata
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Department of Molecular Pharmacology, Division of Functional Disorder Research, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101-0062, Japan. ogatatsk@wa2.so-net.ne.jp
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics; D051766:Early Growth Response Protein 1; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005786:Gene Expression Regulation; D017874:Immediate-Early Proteins; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011799:Quinazolines; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013314:Stress, Mechanical; D014157:Transcription Factors / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D003470:Culture Media; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D011506:Proteins; D011799:Quinazolines; D012333:RNA, Messenger; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D020032:Tyrphostins; C101044:RTKI cpd; D014443:Tyrosine; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1