Sclerostin-erbB-3 interactions: modulation of erbB-3 activity by sclerostin

Abstract

To gain insights into the mechanism of action of sclerostin, a protein that regulates bone mass, we performed yeast two-hybrid analyses using human SOST (sclerostin) cDNA cloned into pGBKT7 DNA-binding domain vector as a bait, and a normalized, high-complexity, universal cDNA library in a GAL4 activating domain vector. We identified an interaction between sclerostin and the carboxyl-terminal portion of the receptor tyrosine-protein kinase erbB-3. To determine the biological relevance of this interaction, we treated MC3T3-E1 mouse osteoblast cells transfected with either a SOST expression plasmid or a control vector, with recombinant heregulin/neuregulin. Phospho-p44/42 (Thr202/Tyr204) MAPK was assessed in heregulin/neuregulin treated cells. We observed an increase in phospho-p44/42 (Thr202/Tyr204) MAPK concentrations in SOST transfected cells but not in cells transfected with a control vector, thus demonstrating a modulatory effect of sclerostin on heregulin/neuregulin signaling in osteoblasts. The data demonstrate that sclerostin functions in part, by modulating the activity of erbB-3.

Figure 1

Yeast cells containing the c-myc-sclerostin and HA-tagged "prey" protein expression are plasmids were grown for three days at 30 °C. Cells were lysed and immuno-precipitation was performed using c-myc (lanes A and D) or HA (lanes B and E) antibodies or no antibodies (lane C). The antibody complexes were captured by protein A beads, the protein precipitates were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were probed with c-myc or sclerostin monoclonal antibodies.

Figure 2

Insert of pBKT7-AD plasmid that interacted with sclerostin expressing pGBK7-DB plasmid. The sequence corresponds to nucleotides 3493–4029 (through stop codon) in GenBank NM_001982.2, human ErbB-3.

Figure 3

Transformed yeast cells containing the c-myc-sclerostin and HA-tagged erbB-3 carboxyl-terminal domain expression plasmids were grown for three days at 30 °C. Cells were lysed and immuno-precipitation was performed using c-myc (lanes A and D) or HA (lanes B and E) antibodies or no antibodies (lane C). The antibody complexes were captured by protein A beads, the protein precipitates were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were probed with c-myc or sclerostin monoclonal antibodies.

Figure 4

MC3T3 cells were transfected with a sclerostin expression plasmid (SostpcDNA3.1 (+)) or an empty vector (pcDNA3.1 (+)). The transfected cells were then treated with neuregulin/heregulin (HRGβ1) for 10 minutes or 1 hour. Phospho p44/42 was assessed by immunoblotting.