Pathway crosstalk between Ras/Raf and PI3K in promotion of M‐CSF‐induced MEK/ERK‐mediated osteoclast survival

While M‐CSF‐mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M‐CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M‐CSF‐promoted MEK/ERK activation and osteoclast survival was investigated. M‐CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M‐CSF‐mediated Ras activation. As further support for Ras‐mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K‐selective or Raf‐selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M‐CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K‐selective Ras‐mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK‐promoted osteoclast survival induced by M‐CSF. J. Cell. Biochem. 104: 1439–1451, 2008. © 2008 Wiley‐Liss, Inc.

[1]  Michael C. Ostrowski,et al.  Regulation of CSF‐1 receptor expression , 1997, Molecular reproduction and development.

[2]  K. Guan,et al.  Positive and negative regulation of Raf kinase activity and function by phosphorylation , 2001, The EMBO journal.

[3]  W. Kolch.  Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. , 2000, The Biochemical journal.

[4]  M. Barbacid,et al.  Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation. , 1995, Oncogene.

[5]  P. Yusoff,et al.  Differences in the kinetics of activation of protein kinases and extracellular signal-related protein kinase 1 in colony-stimulating factor 1-stimulated and lipopolysaccharide-stimulated macrophages. , 1996, The Biochemical journal.

[6]  Takashi Tsuruo,et al.  Involvement of 3-Phosphoinositide-dependent Protein Kinase-1 in the MEK/MAPK Signal Transduction Pathway* , 2004, Journal of Biological Chemistry.

[7]  Angel W. Lee,et al.  Both Src-Dependent and -Independent Mechanisms Mediate Phosphatidylinositol 3-Kinase Regulation of Colony-Stimulating Factor 1-Activated Mitogen-Activated Protein Kinases in Myeloid Progenitors , 2000, Molecular and Cellular Biology.

[8]  A. Bardelli,et al.  A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor , 1993, Molecular and cellular biology.

[9]  Kozo Nakamura,et al.  Reciprocal Role of ERK and Nf-κb Pathways in Survival and Activation of Osteoclasts , 2000, The Journal of cell biology.

[10]  T. Martin,et al.  Osteoblastic cells are involved in osteoclast formation. , 1988, Endocrinology.

[11]  D. States,et al.  Colony-stimulating factor-1 requires PI3-kinase-mediated metabolism for proliferation and survival in myeloid cells , 2006, Cell Death and Differentiation.

[12]  Mengwei Zang,et al.  Microtubule Integrity Regulates Pak Leading to Ras-independent Activation of Raf-1 , 2001, The Journal of Biological Chemistry.

[13]  M. Rogers,et al.  Nitrogen‐Containing Bisphosphonates Inhibit the Mevalonate Pathway and Prevent Post‐Translational Prenylation of GTP‐Binding Proteins, Including Ras , 1998, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[14]  Kozo Nakamura,et al.  In vitro and in vivo assays for osteoclast apoptosis , 2005, Biological Procedures Online.

[15]  Aubie K. Shaw,et al.  Phosphatidylinositol 3‐kinase coordinately activates the MEK/ERK and AKT/NFκB pathways to maintain osteoclast survival , 2003, Journal of cellular biochemistry.

[16]  C. Der,et al.  Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein , 1992, Molecular and cellular biology.

[17]  A. King,et al.  Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak , 2000, Current Biology.

[18]  T. Martin,et al.  Therapeutic approaches to bone diseases. , 2000, Science.

[19]  Michael C. Ostrowski,et al.  Macrophage Colony-stimulating Factor Promotes Cell Survival through Akt/Protein Kinase B* , 1999, The Journal of Biological Chemistry.

[20]  A. Robling,et al.  Hyperactivation of p21ras and PI3K cooperate to alter murine and human neurofibromatosis type 1-haploinsufficient osteoclast functions. , 2006, The Journal of clinical investigation.

[21]  S. Ralston,et al.  Protein Geranylgeranylation Is Required for Osteoclast Formation, Function, and Survival: Inhibition by Bisphosphonates and GGTI‐298 , 2000, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[22]  Ming Zhou,et al.  Regulation of Raf-1 by direct feedback phosphorylation. , 2005, Molecular cell.

[23]  K. Moelling,et al.  Phosphorylation and regulation of Raf by Akt (protein kinase B). , 1999, Science.

[24]  C. Gatlin,et al.  TGF-beta enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M-CSF. , 1999, Biochemical and biophysical research communications.

[25]  T. Martin,et al.  Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[26]  G. Wesolowski,et al.  M-CSF, TNFα and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase , 2003, Cell Death and Differentiation.

[27]  R. Hipskind,et al.  Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages , 1995, Molecular and cellular biology.

[28]  Mengwei Zang,et al.  Interaction between Active Pak1 and Raf-1 Is Necessary for Phosphorylation and Activation of Raf-1* , 2002, The Journal of Biological Chemistry.

[29]  S. Clark,et al.  Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts , 1993, The Journal of experimental medicine.

[30]  A. Ashworth,et al.  Identification of the sites in MAP kinase kinase‐1 phosphorylated by p74raf‐1. , 1994, The EMBO journal.

[31]  T. Miyata,et al.  Commitment and Differentiation of Osteoclast Precursor Cells by the Sequential Expression of C-Fms and Receptor Activator of Nuclear Factor κb (Rank) Receptors , 1999, The Journal of experimental medicine.

[32]  J. Thiery,et al.  Ras induces NBT-II epithelial cell scattering through the coordinate activities of Rac and MAPK pathways. , 2002, Journal of cell science.

[33]  S. Volinia,et al.  Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. , 1995, Science.

[34]  C. Peyssonnaux,et al.  The Raf/MEK/ERK pathway: new concepts of activation , 2001, Biology of the cell.

[35]  J. Downward,et al.  Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor , 1999, Molecular and Cellular Biology.

[36]  M. Wigler,et al.  Multiple ras functions can contribute to mammalian cell transformation , 1995, Cell.

[37]  C. Marshall,et al.  Differential Regulation of Raf-1, A-Raf, and B-Raf by Oncogenic Ras and Tyrosine Kinases* , 1997, The Journal of Biological Chemistry.