Genetic interactions between Polycystin-1 and TAZ in osteoblasts define a novel mechanosensing mechanism regulating bone formation in mice

Abstract

Molecular mechanisms transducing physical forces in the bone microenvironment to regulate bone mass are poorly understood. Here, we used mouse genetics, mechanical loading, and pharmacological approaches to test the possibility that polycystin-1 and TAZ have interdependent mechanosensing functions in osteoblasts. We created and compared the skeletal phenotypes of control Pkd1^flox/+;TAZ^flox/+, single Pkd1^Oc-cKO, single TAZ^Oc-cKO, and double Pkd1/TAZ^Oc-cKO mice to investigate genetic interactions. Consistent with an interaction between polycystins and TAZ in bone in vivo, double Pkd1/TAZ^Oc-cKO mice exhibited greater reductions of BMD and periosteal MAR than either single TAZ^Oc-cKO or Pkd1^Oc-cKO mice. Micro-CT 3D image analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone volume and cortical bone thickness in double Pkd1/TAZ^Oc-cKO mice compared to either single Pkd1^Oc-cKO or TAZ^Oc-cKO mice. Double Pkd1/TAZ^Oc-cKO mice also displayed additive reductions in mechanosensing and osteogenic gene expression profiles in bone compared to single Pkd1^Oc-cKO or TAZ^Oc-cKO mice. Moreover, we found that double Pkd1/TAZ^Oc-cKO mice exhibited impaired responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing gene expression compared to control mice. Finally, control mice treated with a small molecule mechanomimetic MS2 had marked increases in femoral BMD and periosteal MAR compared to vehicle control. In contrast, double Pkd1/TAZ^Oc-cKO mice were resistant to the anabolic effects of MS2 that activates the polycystin signaling complex. These findings suggest that PC1 and TAZ form an anabolic mechanotransduction signaling complex that responds to mechanical loading and serve as a potential novel therapeutic target for treating osteoporosis.

Fig 1.. Conditional deletion of TAZ in mature osteoblasts on postnatal bone homeostasis.

a & b Bone mineral density by DEXA scan in male and female mice. c Bone structure by micro-CT 3D images analysis from both male and female mice. Data are expressed as the mean ± S.D. from serum samples of individual mice (n=6). *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type control mice. P values were determined by 1-way ANOVA with Tukey’s multiple-comparisons test.

Fig 2.. Histological analysis of conditional TAZ deletion in bone.

a & b Goldner staining of non-decalcified bone. Representative images of distal femur sections displayed a gene-dosage dependent reduction in trabecular bone volume and cortical thickness from 8-week-old conditional TAZ deleted mice compared with age-matched control mice. c Periosteal mineral apposition rate (MAR) by calcein double labeling. There was a significant reduction in periosteal MAR in single TAZ^Oc-Het heterozygous mice compared with age-matched control mice and an even greater decrement in TAZ^Oc-cKO null mice, indicating a gene-dosage effect of TAZ on osteoblast-mediated bone formation. d TRAP staining (red color) for osteoclast activity. Data are expressed as the mean ± S.D. from 6 individual mice (n=6). *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type control mice. P values were determined by 1-way ANOVA with Tukey’s multiple-comparisons test.

Fig 3.. Conditional deletion of Pkd1 and TAZ in mature osteoblasts on postnatal bone homeostasis.

a & b Bone mineral density by DEXA scan in male and female mice. c Bone structure by micro-CT 3D images analysis from male mice. Data are expressed as the mean ± S.D. from serum samples of individual mice (n=6). *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type mice, ^##P < 0.01 compared with, TAZ^Oc-cKO mice, and ^&&P < 0.01 compared with Pkd1^Oc-cKO mice, respectively. P values were determined by 1-way ANOVA with Tukey’s multiple-comparisons test.

Fig 4.. Histological analysis of conditional Pkd1 and TAZ deletion in bone.

a & b Goldner staining of non-decalcified bone. Representative images of distal femur sections displayed significant reductions in trabecular bone volume and cortical thickness from 8-week-old conditional Pkd1 and/or TAZ deleted mice compared with age-matched control mice. c Periosteal mineral apposition rate (MAR) by Calcein double labeling. There was a significant reduction in periosteal MAR in single Pkd1^Oc-cKO or TAZ^Oc-cKO mice compared with age-matched control mice and an even greater decrement in double Pkd1/TAZ^Oc-cKO null mice, indicating a synergic effect of PC1 and TAZ on osteoblast-mediated bone formation. d TRAP staining (red color) for osteoclast activity. Data are expressed as the mean ± S.D. from 6 individual mice (n=6). *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type mice. P values were determined by 1-way ANOVA with Tukey’s multiple-comparisons test.

Fig 5.. An impairment of anabolic response to mechanical loading in conditional Pkd1 and TAZ deletion in bone.

Representative images of midshaft tibia cross sections from no-load and loaded ulnae of wild-type control and compound Pkd1/TAZ^Oc-cKO null mice after loading. Data are mean ± S.D. from 6 tibias of wild-type control and Pkd1/TAZ^Oc-cKO mice. ***P < 0.001 compared with wild-type mice. P values were determined by 1-way ANOVA with Tukey’s multiple-comparisons test.

Fig 6.. Cell-based BRET assays for MS2-target engagement assays.

a Chemical structure of MS2 and an example of predicted 3D binding mode for MS2 (ball-and-sticks rendering in CPK colors) in PC2 (light blue). PC1 (red) as bound to PC2 in the homology structure is superimposed. b An example of calculated 2D binding mode and residues for MS2 in PC1/PC2 C-tails. c A diagram of BRET constructs and reactions in the presence of DeepBlue C with or without MS2 stimulation. d BRET signal changes from wild-type and mutant constructs with or without MS2 incubation. e & f Time-dependent changes of PC1 and PC2 proteins with or without MS2 incubation during osteogenic differentiation cultures in MC3T3-E1 cell line. Incubation Data are presented as the mean ± SD from 3 independent experiments (n = 3). **P < 0.01 compared with vehicle control.

Fig 7.. Effects of MS2 on bone formation in wild-type and compound Pkd1/TAZ^Oc-cKO null mice.

a Bone mineral density by DEXA scan. b Bone structure by micro-CT 3D images analysis. c Periosteal mineral apposition rate (MAR) by Calcein double labeling. d Osteoclast activities by TRAP immunostaining after MS2 (50 mg/kg) treatment for 4 weeks compared to vehicle control. Data are mean ± S.D. from 6 tibias of wild-type control and compound Pkd1/TAZ^Oc-cKO null mice. *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type control mice. P values were determined by 1-way ANOVA with Tukey’s multiple-comparisons test.