Among the targets that were identified by the genome-wide association studies, we were particularly interested by WNT16, a major regulator of cortical bone mass (Moverare-Sktric, Nature Med, 2014). WNTs are secreted ligands that bind to its receptor Frizzled, thereby activating a signaling pathway that promotes bone formation by osteoblasts. However, in order to be secreted out of the cell, and to activate the signaling pathway, the palmitoleoylation (addition of lipid called palmitoleic acid) of WNTs ligands is required. Palmitoleoylation is performed by Porcupine in the endoplasmic retinaculum, whereas the removal of the lipid is performed by NOTUM, a secreted lipase. As we observed in preliminary studies that NOTUM inhibition also leads to an increase in cortical bone mass and strength in mice, we hypothesized that the NOTUM’s effect on cortical bone was dependent on WNT16 or vice versa.
Effect of Porcupine inhibition on bone mass. (Funck-Brentano T et al, J Endocrinol, 2018)
During this first part of the scientific project, we focused on the role of palmitolyeolation of WNTs by Porcupine on bone density and bone strength. Using a murine pharmacological approach, we inhibited either Porcupine (that adds palmitoleate to WNTs) or NOTUM (that removes palmitoleate from WNTs). We found that Porcupine inhibitors, currently in Phase I trials for cancer therapy, dramatically reduced bone density and bone strength. Both cortical and trabecular bone was affected due to a decrease in bone formation and an increase in bone resorption. On the other hand, NOTUM inhibition increased cortical bone mass and strength, mainly by enhancing endocortical bone formation. Altogether, these results demonstrate that palmitolyeolation of WNTs is a key factor for the site-specific regulation of bone mass and strength. This mechanism could be further tested as pharmacologic targets for the prevention of osteoporosis, or as biomarkers to identify patients at risk of fractures.
Methods
Twelve-week-old female mice were treated for three weeks by oral gavage with two different Porcupine inhibitors (PORCN_I : LGK974 at 3 [LGK_Lo] or 6 mg/kg [LGK_Hi] or C59 at 10 mg/kg, Selleckchem), a NOTUM inhibitor (30 mg/kg, Lexicon) or vehicle (n= 10 per group). Bone density was evaluated by DXA (PIXIMUS). Cortical bone was evaluated at the femur by µCT and trabecular bone was evaluated at the spine. Static and dynamic histomorphometry was also performed at the femur and spine. Strength was evaluated at the tibia by the 3-point bending test.
Results
Total body BMD was decreased by PORCN_I while it was increased by NOTUM inhibition. Cortical thickness at the femur was also decreased by PORCN_I and increased by NOTUM inhibition. Vertebral trabecular bone volume fraction was substantially decreased by PORCN_I but unchanged by NOTUM inhibition.
In conclusion, palmitoleoylation of WNTs by Porcupine is a major determinant of both trabecular and cortical bone mass. In addition, our findings suggest that Porcupine inhibitors, under development for cancer treatment, may have deleterious skeletal side-effects.
This study was published in Journal of Endocrinology in May 2018
Studies in progress:
Based on the results of this first part of the project, we are currently studying the effect of co-treatment by Porcupine and NOTUM inhibitors. The aim of this study is to determine whether NOTUM inhibitors may affect cortical bone in a WNT-independent manner. If in this setting NOTUM inhibition exerts no effect on cortical bone mass and density, we will conclude that the effect of NOTUM is solely driven by the Wnt signaling pathway. On the contrary, if NOTUM inhibition still increases cortical bone mass despite the absence of functional WNTs, we will conclude that NOTUM inhibition is partly independent of the Wnt signaling pathway.
Ageing and senescence: In a second set of experiments, we aimed to determine if the age-dependent bone loss is mediated by NOTUM. We treated young and old female mice by our NOTUM inhibitor according to the same methods presented above. The goal of this experiment is to assess the skeletal effects of NOTUM inhibition in aging mice and to determine if NOTUM can affect senescent markers.
