Ras-association domain family 1 protein, RASSF1C, is an IGFBP-5 binding partner and a potential regulator of osteoblast cell proliferation.
The goal of this study was to identify downstream signaling molecules involved in mediating the IGF-independent effects of IGFBP-5 in osteoblasts. We identified RASSF1C, a member of the RASSF1 gene products, as a IGFBP-5 binding partner and as a potential mediator of IGFBP-5 effects on ERK phosphorylation and cell proliferation.


BACKGROUND
It has been predicted that the intrinsic growth factor action of insulin-like growth factor binding protein (IGFBP)-5 involves either the binding of IGFBP-5 to a putative receptor to induce downstream signaling pathways and/or intracellular translocation of IGFBP-5 to bind to potential signaling molecules involved in osteoblast cell regulation. This study reports the characterization of isoform C of the Ras association family 1 (RASSF1C) gene as an interacting partner of IGFBP-5.


METHODS
IGFBP-5 was used as bait in a yeast two-hybrid screen of a human osteosarcoma cDNA library. Expression levels of RASSF1C were measured by RT-PCR and/or Northern blot. IGFBP-5 effects on ERK phosphorylation were evaluated by immunoblot analysis. The effect of RASSF1C siRNA on cell proliferation was measured by the AlamarBlue assay.


RESULTS
One of the clones that interacted strongly with the bait under high stringency conditions corresponded to RASSF1C. The interaction between RASSF1C and IGFBP-5 was confirmed by in vitro co-immunoprecipitation studies. Northern blot and RT-PCR analysis showed that RASSF1C was expressed in a variety of osteoblast cell types that produce IGFBP-5. Addition of synthetic RASSF1C-specific small interfering (si) RNA duplex or use of a RASSF1C-specific si-hairpin plasmid caused a decrease in cell number and abolished IGFBP-5-induced extracellular signal-regulated kinase (ERK)-1/2 phosphorylation but had no effect on IGFBP-5-induced increases in alkaline phosphatase (ALP) activity.


CONCLUSIONS
We have shown a novel interaction between IGFBP-5 and RASSF1C. Our findings that silencing of RASSF1C results in the reduction of osteoblast cell proliferation and that IGFBP-5 treatment increases phosphorylation of ERK-1/2 raise the possibility that RASSF1C, a Ras effector, could, in part, contribute to mediating the effects of IGFBP-5 on ERK phosphorylation and, consequently, cell proliferation.
Amaar
Yousef G
YG
Musculoskeletal Disease Center, Jerry L. Pettis Veterans Administration Medical Center, Loma Linda, California 92357, USA.
Baylink
David J
DJ
Mohan
Subburaman
S
D000595:Amino Acid Sequence; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D020868:Gene Silencing; D006801:Humans; D047468:Immunoprecipitation; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D018975:Insulin-Like Growth Factor Binding Protein 5; D020033:Protein Isoforms; C411762:RASSF1 protein, human; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3