Skip to main content
SpringerLink
Log in

Blebbistatin induces chondrogenesis of single mesenchymal cells via PI3K/PDK1/mTOR/p70S6K pathway

  • Published:
Biologia Aims and scope Submit manuscript

Abstract

Rearrangement of the actin cytoskeleton plays an inductive role in chondrogenic differentiation. Our previous study showed that blebbistatin, an inhibitor of myosin II, removes actin stress fibres and induces chondrogenesis of mesenchymal cells in monolayer cultures. In the present study, we investigated signalling pathways implicated in the induction of chondrogenesis by dissolving actin stress fibres after blebbistatin treatment. Blebbistatin increased the activity of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K with LY294002 blocked blebbistatin-induced chondrogenesis without affecting blebbistatin-induced reorganization of actin filaments. Blebbistatin also upregulated the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), and inhibition of PDK1 with GSK2334470 suppressed blebbistatininduced chondrogenesis, indicating that removal of actin stress fibres by blebbistatin induced chondrogenesis by activating PI3K/PDK1. Although inhibition of Akt activity by Akt inhibitor IV blocked blebbistatin-induced chondrogenesis, phosphorylation of Akt was not affected by blebbistatin. Blebbistatin increased the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and p70 ribosomal protein S6 kinase (p70S6K). Inhibition of mTOR with rapamycin almost completely abolished the phosphorylation of p70S6K. Inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) with pp242 diminished phosphorylation of Akt at Ser473, whereas inhibition of mTORC1 with rapamycin did not. However, blebbistatin did not affect the phosphorylation of mTOR at Ser2481. Taken together, the present results suggest that blebbistatin induces chondrogenesis by activating the PI3K/PDK1/mTOR/p70S6K pathway. Our data also indicate that Akt activity is essential for chondrogenesis but is regulated by mTORC2, which is independent of blebbistatin treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BSA:

bovine serum albumin

DAPI:

4’,6-diamidino-2-phenylindole

DMSO:

dimethyl sulfoxide

FBS:

foetal bovine serum

mTOR:

mammalian target of rapamycin

PI3K:

phosphoinositide 3-kinase

PKD1:

phosphoinositidedependent protein kinase 1

p70S6K:

p70 ribosomal protein S6 kinase

References

  • Apsel B., Blair J.A., Gonzalez B., Nazif T.M., Feldman M.E., Aizenstein B., Hoffman R., Williams R.L., Shokat K.M. & Knight Z.A. 2008. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat. Chem. Biol. 4: 691–699.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Avruch J., Hara K., Lin Y., Liu M., Long X., Ortiz-Vega S. & Yonezawa K. 2006. Insulin and amino-acid regulation of mTOR signaling and kinase activity through the Rheb GTPase. Oncogene 25: 6361–6372.

    Article  CAS  PubMed  Google Scholar 

  • Bang O.S., Kim E.J., Chung J.G., Lee S.R., Park T.K. & Kang S.S. 2000. Association of focal adhesion kinase with fibronectin and paxillin is required for precartilage condensation of chick mesenchymal cells. Biochem. Biophys. Res. Commun. 278: 522–529.

    Article  CAS  PubMed  Google Scholar 

  • Bertrand L., Horman S., Beauloye C. & Vanoverschelde J.L. 2008. Insulin signalling in the heart. Cardiovasc. Res. 79: 238–248.

    Article  CAS  PubMed  Google Scholar 

  • Berven L.A. & Crouch M.F. 2000. Cellular function of p70S6K: a role in regulating cell motility. Immunol. Cell Biol. 78: 447–451.

    Article  CAS  PubMed  Google Scholar 

  • Cantley L.C. 2002. The phosphoinositide 3-kinase pathway. Science 296: 1655–1657.

    Article  CAS  PubMed  Google Scholar 

  • Copp J., Manning G. & Hunter T. 2009. TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res. 69: 1821–1827.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Corradetti M.N. & Guan K.L. 2006. Upstream of the mammalian target of rapamycin: do all roads pass through mTOR? Oncogene 25: 6347–6360.

    Article  CAS  PubMed  Google Scholar 

  • Finlay D.K., Rosenzweig E., Sinclair L.V., Feijoo-Carnero C., Hukelmann J.L., Rolf J., Panteleyev A.A., Okkenhaug K. & Cantrell D.A. 2012. PDK1 regulation of mTOR and hypoxiainducible factor 1 integrate metabolism and migration of CD8+ T cells. J. Exp. Med. 209: 2441–2453.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fujita T., Fukuyama R., Enomoto H. & Komori T. 2004. Dexamethasone inhibits insulin-induced chondrogenesis of ATDC5 cells by preventing PI3K-Akt signaling and DNA binding of Runx2. J. Cell. Biochem. 93: 374–383.

    Article  CAS  PubMed  Google Scholar 

  • Gayer C.P., Chaturvedi L.S., Wang S., Craig D.H., Flanigan T. & Basson M.D. 2009. Strain-induced proliferation requires thse phosphatidylinositol 3-kinase/AKT/glycogen synthase kinase pathway. J. Biol. Chem. 284: 2001–2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Greiwe J.S., Kwon G., McDaniel M.L. & Semenkovich C.F. 2001. Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 281: E466–E471.

    Article  CAS  PubMed  Google Scholar 

  • Guan Y., Yang X., Yang W., Charbonneau C. & Chen Q. 2014. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development. FASEB J. 28: 4470–4481.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hay N. & Sonenberg N. 2004. Upstream and downstream of mTOR. Genes Dev. 18: 1926–1945.

    Article  CAS  PubMed  Google Scholar 

  • Hidaka K., Kanematsu T., Takeuchi H., Nakata M., Kikkawa U. & Hirata M. 2001. Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGFI- induced chondrogenesis of the mouse embryonal carcinomaderived cell line ATDC5. Int. J. Biochem. Cell Biol. 33: 1094–1103.

    Article  CAS  PubMed  Google Scholar 

  • Hirsch E., Costa C. & Ciraolo E. 2007. Phosphoinositide 3-kinases as a common platform for multi-hormone signaling. J. Endocrinol. 194: 243–256.

    Article  CAS  PubMed  Google Scholar 

  • Hoshino Y., Nishimura K. & Sumpio B.E. 2007. Phosphatase PTEN is inactivated in bovine aortic endothelial cells exposed to cyclic strain. J. Cell. Biochem. 100: 515–526.

    Article  CAS  PubMed  Google Scholar 

  • Katso R., Okkenhaug K., Ahmadi K., White S., Timms J. & Waterfield M.D. 2001. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 17: 615–675.

    Article  CAS  PubMed  Google Scholar 

  • Kim M.J., Kim S., Kim Y., Jin E.J. & Sonn J.K. 2012. Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells. Biochem. Biophys. Res. Commun. 418: 500–505.

    Article  CAS  PubMed  Google Scholar 

  • Krasilnikov M.A. 2000. Phosphatidylinositol-3 kinase dependent pathways: the role in control of cell growth, survival, and malignant transformation. Biochemistry (Moscow) 65: 59–67.

    CAS  Google Scholar 

  • Krause U., Bertrand L., Maisin L., Rosa M. & Hue L. 2002. Signalling pathways and combinatory effects of insulin and amino acids in isolated rat hepatocytes. Eur. J. Biochem. 269: 3742–3750.

    Article  CAS  PubMed  Google Scholar 

  • Langelier E., Suetterlin R., Hoemann C.D., Aebi U. & Buschmann M.D. 2000. The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments. J. Histochem. Cytochem. 48: 1307–1320.

    Article  CAS  PubMed  Google Scholar 

  • Lee H.H., Chang C.C., Shieh M.J., Wang J.P., Chen Y.T., Young T.H. & Hung S.C. 2013. Hypoxia enhances chondrogenesis and prevents terminal differentiation through PI3K/Akt/FoxO dependent anti-apoptotic effect. Sci. Rep. 3. 2683.

    Article  PubMed  PubMed Central  Google Scholar 

  • Lim Y.B., Kang S.S., Park T.K., Lee Y.S., Chun J.S. & Sonn J.K. 2000. Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-alpha signaling. Biochem. Biophys. Res. Commun. 273: 609–613.

    Article  CAS  PubMed  Google Scholar 

  • Mora A., Komander D., van Aalten D.M. & Alessi D.R. 2004. PDK1, the master regulator of AGC kinase signal transduction. Semin. Cell Dev. Biol. 15: 161–170.

    Article  CAS  PubMed  Google Scholar 

  • Najafov A., Sommer E.M., Axten J.M., Deyoung M.P. & Alessi D.R. 2011. Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem. J. 433: 357–369.

    Article  CAS  PubMed  Google Scholar 

  • Oh C.D. & Chun J.S. 2003. Signaling mechanisms leading to the regulation of differentiation and apoptosis of articular chondrocytes by insulin-like growth factor-1. J. Biol. Chem. 278: 36563–36571.

    Article  CAS  PubMed  Google Scholar 

  • Oh C.D., Kim S.J., Ju J.W., Song W.K., Kim J.H., Yoo Y.J. & Chun J.S. 2001. Immunosuppressant rapamycin inhibits protein kinase C alpha and p38 mitogen-activated protein kinase leading to the inhibition of chondrogenesis. Eur. J. Pharmacol. 427: 175–185.

    Article  CAS  PubMed  Google Scholar 

  • Park E.H., Kang S.S., Lee Y.S., Kim S.J., Jin E.J., Tak E.N. & Sonn J.K. 2008. Integrity of the cortical actin ring is required for activation of the PI3K/Akt and p38 MAPK signaling pathways in redifferentiation of chondrocytes on chitosan. Cell Biol. Int. 32: 1272–1278.

    Article  CAS  PubMed  Google Scholar 

  • Peyrollier K., Hajduch E., Blair A.S., Hyde R. & Hundal H.S. 2000. L-leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the Lleucine-induced up-regulation of system A amino acid transport. Biochem. J. 350: 361–368.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Phornphutkul C., Lee M., Voigt C., Wu K.Y., Ehrlich M.G., Gruppuso P.A. & Chen Q. 2009. The effect of rapamycin on bone growth in rabbits. J. Orthop. Res. 27: 1157–1161.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Phornphutkul C., Wu K.Y., Auyeung V., Chen Q. & Gruppuso P.A. 2008. mTOR signaling contributes to chondrocyte differentiation. Dev. Dyn. 237: 702–712.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qian Y., CorumL., Meng Q., Blenis J., Zheng J.Z., Shi X., Flynn D.C. & Jiang B.H. 2004. PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration. Am. J. Physiol. 286: C153–C163.

    Article  CAS  Google Scholar 

  • Rodriguez-Viciana P., Warne P.H., Dhand R., Vanhaesebroeck B., Gout I., Fry M.J., Waterfield M.D. & Downward J. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370: 527–532.

    Article  CAS  PubMed  Google Scholar 

  • Sanez Canedo C., Demeulder B., Ginion A., Bayascas J.R., Balligand J.L., Alessi D.R., Vanoverschelde J.L., Beauloye C., Hue L. & Bertrand L. 2010. Activation of the cardiac mTOR/p70(S6K) pathway by leucine requires PDK1 and correlates with PRAS40 phosphorylation. Am. J. Physiol. Endocrinol. Metab. 298: E761–E769.

    Article  CAS  Google Scholar 

  • Sarbassov D.D., Ali S.M. & Sabatini D.M. 2005. Growing roles for the mTOR pathway. Curr. Opin. Cell Biol. 17: 596–603.

    Article  CAS  PubMed  Google Scholar 

  • Slomovitz B.M. & Coleman R.L. 2012. The PI3K/AKT/mTOR pathway as a therapeutic target in endometrial cancer. Clin. Cancer Res. 18: 5856–5864.

    Article  CAS  PubMed  Google Scholar 

  • Solursh M., Linsenmayer T.F. & Jensen K.L. 1982. Chondrogenesis from single limb mesenchyme cells. Dev. Biol. 94: 259–264.

    Article  CAS  PubMed  Google Scholar 

  • Song G., Ouyang G. & Bao S. 2005. The activation of Akt/PKB signaling pathway and cell survival. J. Cell. Mol. Med. 9: 59–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Straight A.F., Cheung A., Limouze J., Chen I., Westwood N.J., Sellers J.R. & Mitchison T.J. 2003. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299: 1743–1747.

    Article  CAS  PubMed  Google Scholar 

  • Tsakiridis T., Tong P., Matthews B., Tsiani E., Bilan P.J., Klip A. & Downey G.P. 1999. Role of the actin cytoskeleton in insulin action. Microsc. Res. Tech. 47: 79–92.

    Article  CAS  PubMed  Google Scholar 

  • Vanhaesebroeck B. & Alessi D.R. 2000. The PI3K-PDK1 connection: more than just a road to PKB. Biochem. J. 346: 561–576.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wennström S., Hawkins P., Cooke F., Hara K., Yonezawa K., Kasuga M., Jackson T., Claesson-Welsh L. & Stephens L. 1994. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr. Biol. 4: 385–393.

    Article  PubMed  Google Scholar 

  • Wezeman F.H. 1998. Morphological foundations of precartilage development in mesenchyme. Microsc. Res. Tech. 43: 91–101.

    Article  CAS  PubMed  Google Scholar 

  • Zanetti N.C. & Solursh M. 1984. Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton. J. Cell Biol. 99: 115–123.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Korea

    Hyoin Kim, Dong Hyun Kim, Bohyeon Jeong, Ju-Hee Kim & Jong Kyung Sonn

  2. Department of Biology, College of Natural Sciences, Jeju National University, Jeju-si, South Korea

    Sun-Ryung Lee

Authors
  1. Hyoin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dong Hyun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bohyeon Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ju-Hee Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sun-Ryung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jong Kyung Sonn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jong Kyung Sonn.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, H., Kim, D.H., Jeong, B. et al. Blebbistatin induces chondrogenesis of single mesenchymal cells via PI3K/PDK1/mTOR/p70S6K pathway. Biologia 72, 694–701 (2017). https://doi.org/10.1515/biolog-2017-0078

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1515/biolog-2017-0078

Key words

Search

Navigation