Bone Abstracts

Early-onset Paget’s disease of bone (ePDB), familial expansile osteolysis (FEO) and expansile skeletal hyperphosphatasia (ESH) are related syndromes caused by heterozygous tandem insertion duplication mutations within the signal peptide region of TNFRSF11a (encoding receptor activator of NFκB; RANK). Given that patients are always heterozygous for these mutations we have generated thirteen cell lines to investigate the molecular consequences of these mutations in vitro.

Bidirectional expression constructs (in pBI-CMV vector) were generated containing cDNAs for: myc-tagged and FLAG-tagged wildtype RANK (WTF-WTM; homozygous wildtype); myc-tagged wildtype RANK and FLAG-tagged mutant RANK (WTM-MutM; heterozygous mutant); myc-tagged and FLAG-tagged mutant RANK (MutF-MutM; homozygous mutant). The entire expression cassette was then excised and ligated into the pcDNA-FRT vector (Invitrogen). We used the Flp-In system (Invitrogen) to generate Hela cell lines expressing single copies of the bidirectional expression cassettes. Specific regions of genomic RANK DNA from each cell line sequence verified to confirm the presence of each RANK construct. qPCR confirmed that the RANK transcript is significantly more abundant in the RANK-transfected cell lines compared to the parental cell line. We performed NFκB translocation assays and IκB degradation assays to assess RANKL-dependent and RANKL-independent activation of NFκB in all cell lines over a period of up to 3 hours. WTF-WTM cells displayed a characteristic sinusoidal response to RANKL stimulation which was also apparent in the WTF-MutM cell lines, but these cell lines all had higher baseline NFκB activation before stimulation with RANKL. By contrast, all homozygous mutant cell lines displayed a very high baseline NFκB activation and this did not change in response to RANKL.

These data highlight key differences between baseline NFκB activition when the early-onset pagetic mutant RANK proteins are homozygously or heterozygously expressed and offers the opportunity to further explore the pathway specific changes that are induced by these mutations.