Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
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Activation of CREB by PKA Promotes the Chondrogeneic Differentiation of Chick Limb Bud Mesenchymal Cells

  • Kim, Kook-Hee (Department of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Lee, Young-Sup (Department of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University)
  • Published : 2009.09.30
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Abstract

Cyclic AMP-mediated signaling pathways regulate a number of cellular functions. In this study, we examined the regulatory role of cAMP signaling pathways in chondrogenesis of chick limb bud mesenchymal cells in vitro. Forskolin, which increases cellular cAMP levels by the activation of adenylate cyclase, enhanced chondrogenic differentiation. Inhibition of PKA with specific inhibitors (H89 or KT5720) blocked pre-cartilage condensation stage, indicating that chondrogenesis is regulated by the increase in cellular cAMP level and subsequent activation of PKA. Downstream signaling pathway of PKA leading to gene expression was investigated by examination of several nuclear transcription factors. Forskolin treatment increased transcription level for a cartilage-specific marker gene Sox9. However, inhibition of PKA with H89 led to restore expression of Sox9, indicating PKA activity was required to regulate the expression of Sox9 in chondrogenesis. In addition, CREB was highly phosphorylated at early stage of mesenchyme culture, and followed by progressive dephosphorylation. CBP and ATF, another CRE related proteins were transiently expressed at the early stage of chondrogenesis with a pattern similar to CREB phosphorylation. Electrophoretic mobility shift assays confirmed that the binding activity of CREB to the CRE is closely correlated to the phosphorylation pattern of CREB. Therefore, cAMP-mediated signal transduction to nuclear events for the induction of genes appeared to be required at the early stage of chick limb bud chondrogenesis.

Keywords

References

  1. Akiyama H, Chaboissier MC, Martin JF, Schedl A, and Crombrugghe B (2002) The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 16: 2813-2828 https://doi.org/10.1101/gad.1017802
  2. Biddulph DM and Dozier MM (1989) Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMPl. Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMPl. Exp Cell Res 185:541-545 https://doi.org/10.1016/0014-4827(89)90323-6
  3. Biddulph DM, Sawyer LM, Dozier MM (1988) Chondrogenesis in chick limb bud mesemchyme in vitro derived from distal limb bud tips: Changes in cyclic AMP and in prostaglandin responsiveness. J Cell Physiol 136: 81-87 https://doi.org/10.1002/jcp.1041360110
  4. Bobick BE and Kulyk WM (2004) The MEK-ERK signaling pathway is a negative regulator of cartilage-specific gene expression in embryonic limb mesenchyme. J Biol Chem 279:4588-4595 https://doi.org/10.1074/jbc.M309805200
  5. Borrelli E, Montmayeur JP, Foulkes NS, and Sassone-Corsi P (1992) Signal transduction and gene control: the cAMP pathway. Crit Rev Oncog 3: 321-338
  6. Chang SH, Oh CD, Yang MS, Kang SS, Lee YS, Sonn JK, and Chun JS (1998) Protein kinase C regulates chondrogenesis of mesenchymes via mitogen-activated protein kinase signaling. J Biol Chem 273: 19213-19219 https://doi.org/10.1074/jbc.273.30.19213
  7. Chen P, Yu YM, and Reddi AH (1993) Chondrogenesis in chick limb bud mesodermal cells: reciprocal modulation by activin and inhibin. Exp Cell Res 206: 119-127 https://doi.org/10.1006/excr.1993.1127
  8. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, and Hidaka H (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMPdependent protein kinase, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J BioI Chem 265: 5267-5272
  9. Chrivia JC, Kwok RPS, Lamb N, Hagiwara M, Montminy MR, and Goodman RH (1993) Phosphorylated CREB binds specifically to the nuclear protein CBP.Nature 365: 855-859 https://doi.org/10.1038/365855a0
  10. Dean DC, Blakely MS, Newby RF, Ghazal P, Henninghauser L, and Bourgeois S (1989) Forskolin inducibility and tissuespecific expression of the fibronectin promoter. Mol Cell Biol 9: 1498-1506
  11. Gadbois DM, Crissman HA, Tobey RA, and Bradbury EM (1992) Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells Proc Natl Acad Sci USA 89: 8626-8630 https://doi.org/10.1073/pnas.89.18.8626
  12. Gonzalez GA and Montminy MR (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 77: 713-725 https://doi.org/10.1016/0092-8674(94)90055-8
  13. Habener JF (1990) CyclicAMP response element binding proteins:a cornucopia oftranscription factors. Mol Endocrinol 4: 1087-1094 https://doi.org/10.1210/mend-4-8-1087
  14. Huang W, Zhou X, Lefebvre v., and Crombrugghe B (2000) P~lOsphorylation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 chondrocyte-specific enhancer. Mol Cell BioI 20: 4149-4158 https://doi.org/10.1128/MCB.20.11.4149-4158.2000
  15. Hunter T, and Karin M (1992) The regulation of transcription by phosphorylation. Cell 70: 375-388 https://doi.org/10.1016/0092-8674(92)90162-6
  16. Kulyk WM, Franklin JL, and Hoffman LM (2000) Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme. Exp Cell Res 255: 327-332 https://doi.org/10.1006/excr.1999.4784
  17. Kulyk WM, Upholt WB, and Kosher RA (1989) Fibronectin gene expression during limb cartilage differentiation. Develop 106:449-455
  18. Kwok RP, Lundbald JR, Chrivia JC, Richards JP, Bachinger HP, Brennan Ro, Roberts So, Green MR, and Goodman RH (1994) Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370: 223-226 https://doi.org/10.1038/370223a0
  19. Lalli E and Sassone-Corsi P (1994) Signal transduction and gene regulation: the nuclear response to cAMP. J Biol Chem 269:17359-17362
  20. Lee YS and Chuong CM (1997) Activation ofprotein kinase A is a pivotal step involved in both BMP-2 and cyclic AMPinduced chondrogenesis. J Cell Physiol 170: 153-165 https://doi.org/10.1002/(SICI)1097-4652(199702)170:2<153::AID-JCP7>3.0.CO;2-N
  21. Lim YB, Kang SS, Park TK, Lee YS, Chun JS, and Sonn JK (2000) Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-a signaling. Biochem Biophy Res Commun 273: 609-613 https://doi.org/10.1006/bbrc.2000.2987
  22. Montminy MR and Bilezikjian LM (1987) Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328: 175-178 https://doi.org/10.1038/328175a0
  23. Oh CD, Chang SH. Yoon YM, Lee SJ, Lee YS, Kang SS, and Chun JS (2000) Opposing role of mitogen-activated protein kinase subtypes, erk-l/2 and p38, in the regulation of chondrogenesis of mesenchymes. J Biol Chem 275: 5613-5619 https://doi.org/10.1074/jbc.275.8.5613
  24. Park SY, Jung JC, Kim SD, Lee YS, Park TK, and Kang SS (1995) cAMP induces phosphorylation of a 40-kDa nuclear protein which is distinct from CREB during chondrogenesis of chick limb bud mesenchymal cells in vitro. Biochem Biophy Res Commun 212: 16-20 https://doi.org/10.1006/bbrc.1995.1929
  25. Rodgers BJ, Kulyk WM, and Kosher RA (1989) Stimulation of limb cartilage differentiation by cyclic AMP is dependent on cell density. Cell Differen Dev. 28: 179-188 https://doi.org/10.1016/0922-3371(89)90003-8
  26. Solursh M (1989) Differentiation of cartilage and bone. Curr Opin Cell BioI 1: 989-994 https://doi.org/10.1016/0955-0674(89)90070-7
  27. Wadzinski BE, Wheat WH, Jaspers S, Peruski LF, Lickteig RL, Johnson GL, and Klemm DJ (1993) Nuclear protein phosphatase 2A dephosphorylates protein kinase Aphosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell BioI 13: 2822-2834
  28. Wheat WH, Roesler WJ, and Klemm DJ (1994) Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 14: 5881-5890 https://doi.org/10.1128/MCB.14.9.5881
  29. Widnell KL, Russell DS, and Nestler EJ (1994) Regulation of expression of cAMP response element-binding protein in the locus coeruleus in vivo and in a locus coeruleus-like cell line in vitro. Proc Natl Acad Sci USA 91: 10947-10951 https://doi.org/10.1073/pnas.91.23.10947
  30. Yoon YM, Oh CD, Kang SS, and Chun JS (2000a) Protein kinase A regulates chondrogenesis ofmesenchymal cells at the postprecartilage condensation stage via protein kinase C-alpha signaling. J Bone Miner Res 15: 2197-2205 https://doi.org/10.1359/jbmr.2000.15.11.2197
  31. YoonYM, Oh CD, Kim DY,Lee YS, Park JW, Huh TL, Kang SS, and Chun JS (2000b) Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-a, Erk-l, and p38 MAPK signaling pathways. J BioI Chem 275: 12353-12359 https://doi.org/10.1074/jbc.275.16.12353
  32. Zheng S, Brown MC, and Taffet SM (1993) Lipopolysaccharide stimulates both nuclear localization of the nuclear factor kappa B 50-kDa subunit and loss of the 105-kDa precursor in RAW264 macrophage-like cells. J Biol Chem 268: 17233-17239