Ras-association domain family 1 protein, RASSF1C, is an IGFBP-5 binding partner and a potential regulator of osteoblast cell proliferation.

Abstract

UNLABELLED:

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.

INTRODUCTION:

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.

MATERIALS AND 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.

• Authors: Yousef G Amaar, David J Baylink, Subburaman Mohan
• Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research (J Bone Miner Res) Vol. 20 Issue 8 Pg. 1430-9 (Aug 2005) ISSN: 0884-0431 [Print] United States
• PMID: 16007340 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Insulin-Like Growth Factor Binding Protein 5
• Protein Isoforms
• RASSF1 protein, human
• RNA, Small Interfering
• Tumor Suppressor Proteins
• Mitogen-Activated Protein Kinase 1
• Mitogen-Activated Protein Kinase 3

Topics

• Amino Acid Sequence
• Bone and Bones (cytology, metabolism)
• Cell Proliferation
• Cells, Cultured
• Gene Silencing
• Humans
• Immunoprecipitation
• Insulin-Like Growth Factor Binding Protein 5 (genetics, metabolism)
• Mitogen-Activated Protein Kinase 1 (metabolism)
• Mitogen-Activated Protein Kinase 3 (metabolism)
• Molecular Sequence Data
• Osteoblasts (chemistry, metabolism, physiology)
• Phosphorylation
• Protein Isoforms (genetics, metabolism)
• RNA, Small Interfering (genetics)
• Tumor Suppressor Proteins (analysis, genetics, metabolism)