Piezo1 opposes age-associated cortical bone loss

Abstract

As we age, our bones undergo a process of loss, often accompanied by muscle weakness and reduced physical activity. This is exacerbated by decreased responsiveness to mechanical stimulation in aged skeleton, leading to the hypothesis that decreased mechanical stimulation plays an important role in age-related bone loss. Piezo1, a mechanosensitive ion channel, is critical for bone homeostasis and mechanotransduction. Here, we observed a decrease in Piezo1 expression with age in both murine and human cortical bone. Furthermore, loss of Piezo1 in osteoblasts and osteocytes resulted in an increase in age-associated cortical bone loss compared to control mice. The loss of cortical bone was due to an expansion of the endosteal perimeter resulting from increased endocortical resorption. In addition, expression of Tnfrsf11b, encoding anti-osteoclastogenic protein OPG, decreases with Piezo1 in vitro and in vivo in bone cells, suggesting that Piezo1 suppresses osteoclast formation by promoting Tnfrsf11b expression. Our results highlight the importance of Piezo1-mediated mechanical signaling in protecting against age-associated cortical bone loss by inhibiting bone resorption in mice.

Keywords: Piezo1; age-associated bone loss; endocortical bone resorption; mechanical stimulation.

FIGURE 1

Piezo1 expression decreases with age. (a) mRNA levels of Piezo1 in 6‐ and 24‐month‐old C57BL/6J and DBA mice (n = 9–11, here and throughout, values are mean ± SD). (b) qPCR of Piezo1 mRNA in human cortical bone isolated from human femoral neck removed during hip replacement surgeries from young (n = 9 with mean age of 47.1 ± 7.01 years old) and aged patients (n = 13 with mean age of 73.8 ± 3.2 years old). (c) Quantification and representative images of Piezo1 mRNA in situ hybridization using RNAscope on femoral cortical bone of 6‐ and 24‐month‐old C57BL/6J mice. Red dots indicate positive Piezo1 signals. Scale bar, 100 μm. (d) Quantification and representative images of Piezo1 mRNA in situ hybridization on human cortical bone of femoral neck from young and old individuals. Red dots indicate positive Piezo1 signals. *p < 0.05 using Student's t test.

FIGURE 2

Deletion of Piezo1 in osteoblasts and osteocytes exacerbates the cortical bone loss with age. (a) Cancellous bone volume per tissue volume (BV/TV) and the representative μCT images measured in the L4 vertebra of 6‐ and 24‐month‐old female Piezo1^f/f (n = 8, 10) and Dmp1‐Cre; Piezo1^f/f (n = 8, 12) mice. (b) Cortical thickness and the representative μCT images (scale bar, 1 mm) measured in the femoral diaphysis of 6‐ and 24‐month‐old female Piezo1^f/f (n = 8, 10) and Dmp1‐Cre; Piezo1^f/f (n = 8, 12) mice. (c) X‐ray images of tibia from 24‐month‐old Piezo1^f/f and Dmp1‐Cre; Piezo1^f/f littermate. Fractions represent 0 and 4 mice displayed tibia facture out of 10 and 12 mice, respectively. Arrow indicates the location of fracture. (d–f) Periosteal circumference (d), endocortical circumference (e), and moment of inertia (f) analysis of the femoral diaphysis by micro‐CT from 6‐ and 24‐month‐old female Piezo1^f/f (n = 8, 10) and Dmp1‐Cre; Piezo1^f/f (n = 8, 12) mice. (g) Representative histological longitudinal section images of cortical bone showing cortical porosity in 6‐ and 24‐month‐old female Piezo1^f/f and Dmp1‐Cre; Piezo1^f/f mice. Red arrow heads indicate pores in cortical bone. Scale bar, 50 μm. (h) Cortical porosity measured by μCT in femur from 6‐ and 24‐month‐old female Piezo1^f/f (n = 8, 10) and Dmp1‐Cre; Piezo1^f/f (n = 8, 12) mice. *p < 0.05 with the comparisons indicated by the brackets using 2‐way ANOVA. #, p < 0.05 for interaction using 2‐way ANOVA. ns, nonsignificant.

FIGURE 3

Deletion of Piezo1 in osteoblasts and osteocytes increases osteoclast formation at endocortical surface. (a) Representative images of TRAP staining at the endocortical surface of 6‐ and 24‐month‐old female Piezo1^f/f and Dmp1‐Cre; Piezo1^f/f mice. Scale bar, 50 μm. (b) Osteoclast number (N.Oc/B.Pm, left) and osteoclast surface (Oc.S/BS, right) measured at femoral endocortical surface of 6‐ and 24‐month‐old female Piezo1^f/f (n = 5, 5) and Dmp1‐Cre; Piezo1^f/f (n = 5, 5) mice. *p < 0.05 with the comparisons indicated by the brackets using 2‐way ANOVA. #, p < 0.05 for interaction using 2‐way ANOVA. (c) Representative images of TRAP staining and quantification of TRAP⁺ osteoclasts in cocultures of bone marrow derived macrophages with MLO‐Y4 cells with or without Piezo1 knocked down. (d) mRNA of osteoclastic genes Ctsk and Acp5 in cocultures of bone marrow derived macrophages with MLO‐Y4 cells with or without Piezo1 knocked down. (e) mRNA of Acp5 in cocultures of bone marrow derived macrophages with MLO‐Y4 cells in the presence of Piezo1 agonist Yoda1. (f) Acp5 expression in femoral cortical bone cultured ex vivo in the presence of Piezo1 agonist Yoda1. *p < 0.05 using Student's t test.

FIGURE 4

Deletion of Piezo1 in osteoblasts and osteocytes suppresses Tnfrsf11b expression. (a) qPCR of Piezo1 and Tnfrsf11b mRNA in control or Piezo1 knock‐down MLO‐Y4 cells. *p < 0.05 using Student's t test. (b) Relative mRNA levels of Piezo1 and Tnfrsf11b in MLO‐Y4 cells treated with 10 μM Yoda1 for 2 h. *p < 0.05 using Student's t test. (c) Piezo1 and Tnfrsf11b mRNA levels measured in tibia of 3‐month‐old female C57BL/6J (n = 7) mice loaded with one bout of compressive loading. Mice were harvested 4 h after loading. *p < 0.05 using Student's t test. (d) Tnfrsf11b mRNA levels in femoral cortical bone of 6‐ and 24‐month‐old female C57BL/6J mice (n = 6). *p < 0.05 using Student's t test. (e) Tnfrsf11b mRNA levels in Veh or Yoda1 treated bone marrow stromal cells isolated from 6‐ and 24‐month‐old female C57BL/6J mice. *p < 0.05 with the comparisons indicated by the brackets using 2‐way ANOVA. #, p < 0.05 for interaction using 2‐way ANOVA. (f) Representative images and quantification of Tnfrsf11b mRNA in situ hybridization using RNAscope on femoral cortical bone of 3‐month‐old female Piezo1^f/f and Dmp1‐Cre; Piezo1^f/f mice (n = 3). Red dots indicate positive Tnfrsf11b signals. Scale bar, 100 μm. (g) Tnfrsf11b mRNA levels in osteocyte‐enriched femoral cortical bone of 4‐month‐old male Piezo1^f/f and Dmp1‐Cre; Piezo1^f/f mice (n = 5). *p < 0.05 using Student's t test.

FIGURE 5

Piezo1 controls Tnfrsf11b expression via Ca²⁺/CaM/mTOR signaling pathway. (a) qPCR of Tnfrsf11b mRNA in MLO‐Y4 cells treated with Veh or 10 μM Yoda1 for 2 h. Cells were cultured in αMEM either with or without calcium. (b) Relative mRNA levels of Tnfrsf11b in MLO‐Y4 cells treated with Veh or 10 μM Yoda1 for 2 h in the presence of 5 μM intracellular calcium chelator BAPTA. (c) Relative mRNA levels of Tnfrsf11b in MLO‐Y4 cells treated with 10 μM Yoda1 for 2 h in the presence of 1.5 μM calmidazolium (CMZ), a membrane‐permeable CaM antagonist. (d) Relative mRNA levels of Tnfrsf11b in MLO‐Y4 cells treated with Veh or 10 μM Yoda1 for 2 h in the presence of 2.5 μM mTOR inhibitor PP242. (e) Relative mRNA levels of Tnfrsf11b in MLO‐Y4 cells treated with Veh or 10 μM Yoda1 for 2 h in the presence of 10 μM SB203590 (p38/MAPK inhibitor), 50 μM PD98059 (MEK/ERK inhibitor), and 200 μM L‐NAME (eNOS inhibitor). (f) Schematic illustration of the pathway by which Piezo1 controls Tnfrsf11b expression. n = 3 per group. Each experiment was repeated at least two times. *p < 0.05 with the comparisons indicated by the brackets using 2‐way ANOVA. #, p < 0.05 for interaction using 2‐way ANOVA.