Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells

The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR‐108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth...

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Veröffentlicht in:Journal of cellular physiology 2001-11, Vol.189 (2), p.216-228
Hauptverfasser: Geng, W.D., Boskovic, G., Fultz, M.E., Li, C., Niles, R.M., Ohno, S., Wright, G.L.
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container_issue 2
container_start_page 216
container_title Journal of cellular physiology
container_volume 189
creator Geng, W.D.
Boskovic, G.
Fultz, M.E.
Li, C.
Niles, R.M.
Ohno, S.
Wright, G.L.
description The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR‐108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC‐α (11.6‐fold), nPKC‐δ (5.3‐fold), and nPKC‐ϵ (22.0‐fold) but not aPKC‐ζ. Northern blot analysis indicated a significant (2.3‐fold) increase in the 10 kb transcript of cPKC‐α, a slight (1.3‐fold) increase in that of nPKC‐ϵ but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC‐α, PKC‐δ, and PKC‐ϵ and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR‐108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme‐selective increases in the protein levels of PKC isozymes of osteoblastic UMR‐108 cells upon growth to confluence which may be regulated at the transcriptional or the post‐transcriptional level. The results from UMR‐108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994. J Bone Miner Res 9(7):999–1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton. © 2001 Wiley‐Liss, Inc.
doi_str_mv 10.1002/jcp.10019
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Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC‐α (11.6‐fold), nPKC‐δ (5.3‐fold), and nPKC‐ϵ (22.0‐fold) but not aPKC‐ζ. Northern blot analysis indicated a significant (2.3‐fold) increase in the 10 kb transcript of cPKC‐α, a slight (1.3‐fold) increase in that of nPKC‐ϵ but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC‐α, PKC‐δ, and PKC‐ϵ and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR‐108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. 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Cell. Physiol</addtitle><description>The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR‐108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC‐α (11.6‐fold), nPKC‐δ (5.3‐fold), and nPKC‐ϵ (22.0‐fold) but not aPKC‐ζ. Northern blot analysis indicated a significant (2.3‐fold) increase in the 10 kb transcript of cPKC‐α, a slight (1.3‐fold) increase in that of nPKC‐ϵ but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC‐α, PKC‐δ, and PKC‐ϵ and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR‐108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme‐selective increases in the protein levels of PKC isozymes of osteoblastic UMR‐108 cells upon growth to confluence which may be regulated at the transcriptional or the post‐transcriptional level. The results from UMR‐108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994. 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Cell. Physiol</addtitle><date>2001-11</date><risdate>2001</risdate><volume>189</volume><issue>2</issue><spage>216</spage><epage>228</epage><pages>216-228</pages><issn>0021-9541</issn><eissn>1097-4652</eissn><abstract>The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR‐108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC‐α (11.6‐fold), nPKC‐δ (5.3‐fold), and nPKC‐ϵ (22.0‐fold) but not aPKC‐ζ. Northern blot analysis indicated a significant (2.3‐fold) increase in the 10 kb transcript of cPKC‐α, a slight (1.3‐fold) increase in that of nPKC‐ϵ but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC‐α, PKC‐δ, and PKC‐ϵ and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR‐108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme‐selective increases in the protein levels of PKC isozymes of osteoblastic UMR‐108 cells upon growth to confluence which may be regulated at the transcriptional or the post‐transcriptional level. The results from UMR‐108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994. J Bone Miner Res 9(7):999–1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton. © 2001 Wiley‐Liss, Inc.</abstract><cop>New York</cop><pub>John Wiley &amp; Sons, Inc</pub><pmid>11598907</pmid><doi>10.1002/jcp.10019</doi><tpages>13</tpages></addata></record>
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subjects Actin Cytoskeleton - ultrastructure
Animals
Cell Culture Techniques - methods
Cell Size
Isoenzymes - biosynthesis
Isoenzymes - genetics
Isoenzymes - metabolism
Microscopy, Confocal
Osteoblasts - cytology
Osteoblasts - enzymology
Osteoblasts - ultrastructure
Protein Kinase C - biosynthesis
Protein Kinase C - genetics
Protein Kinase C - metabolism
Protein Kinase C-alpha
Protein Kinase C-delta
Protein Kinase C-epsilon
Rats
RNA, Messenger - biosynthesis
Stress, Mechanical
Transcription, Genetic
Tumor Cells, Cultured
title Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells
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