Role of pannexin 1 channels in load-induced skeletal response

Abstract

The pannexin 1 (Panx1) channel is a mechanosensitive channel that interacts with P2X7 receptors (P2X7R) to form a functional complex that has been shown in vitro to play an essential role in osteocyte mechanosignaling. While the participation of P2X7R in skeletal responses to mechanical loading has been demonstrated, the role of Panx1 and its interplay with P2X7R still remain to be determined. In this study, we use a global Panx1^-/- mouse model and in vivo mechanical loading to demonstrate that Panx1 channels play an essential role in load-induced skeletal responses. We found that absence of Panx1 not only disrupts the P2X7R-Panx1 signaling complex, but also alters load-induced regulation of P2X7R expression. Moreover, lack of Panx1 completely abolished load-induced periosteal bone formation. Load-induced regulation of β-catenin and sclerostin expression was dysregulated in Panx1^-/- , compared to wild-type, bone. This finding suggests that Panx1 deficiency disrupts Wnt/β-catenin signaling by lowering β-catenin while favoring inhibition of bone formation by increasing load-induced sclerostin expression. This study demonstrates the existence of a Panx1-dependent mechanosensitive mechanism that not only modulates ATP signaling but also coordinates Wnt/β-catenin signaling that is essential for proper skeletal response to mechanical loading.

Keywords: Panx1; mechanical loading; osteocytes; sclerostin; β-catenin.

Figure 1

The P2X7R-Panx1 mechanosignaling complex response to mechanical loading in wildtype mice is time dependent. Expression of Panx1 and P2X7R in loaded wildtype compared to age-matched non-loaded wildtype after 2 or 4 weeks of loading (treadmill running). Representative western blots for Panx1 and P2X7R from 2 weeks and 4 weeks loaded (positive symbol) and non-loaded controls (negative symbol) are shown below the graph. Protein levels were first normalized with the respective β-actin loading protein control, and then with the age-matched non-loaded wildtype levels. **P<0.01, ***P<0.001, non-loaded wildtype (dotted line) vs. age-matched loaded wildtype, nonparametric t-test followed by Mann-Whitney comparison test. Loading duration group: n= 5 (2 weeks), n = 6 (4 weeks). Data presented as means ± SD.

Figure 2

P2X7R expression in response to mechanical loading in Panx1^−/− mice. (A) Expression of P2X7R in non-loaded and loaded Panx1^−/− compared to age-matched non-loaded wildtype (dotted line) after 2 or 4 weeks of loading (treadmill running). (B) Expression of P2X7R in loaded Panx1^−/− compared to age-matched non-loaded Panx1^−/− (dotted line) after 2 or 4 weeks of loading. (C) Representative western blots for P2X7R from 2 weeks and 4 weeks loaded Panx1^−/− mice (positive symbol) and non-loaded age match controls (negative symbol). Protein levels were first normalized with the respective β-actin loading control, and then with the age-matched non-loaded wildtype levels. Comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparison test. For the comparison between Panx1^−/− non-loaded vs. age-matched loaded, an unpaired t-test was used followed by Mann-Whitney comparison test. Loading duration group: n = 5 (2 weeks), n = 6 (4 weeks). Data presented as means ± SD.

Figure 3

Load-induced changes in the periosteal and endocortical surfaces of femoral midshaft after 2 weeks of loading in wildtype and Panx1^−/− mice. Top panels: Representative cross-section images showing double calcein labels for age-matched non-loaded and loaded wildtype (A) and Panx1^−/− (B) bones. Scale bar = 200 µm. Middle panels: Representative images of double calcein labeling on lateral endocortical (Ec.) surfaces (indicated with arrows in the top panels) of non-loaded and loaded wildtype and Panx1^−/− bones. Scale bar = 20 µm. Bottom panels: Representative images of double calcein labeling on medial periosteal (Ps.) surfaces (indicated with arrowheads in the top panels) of non-loaded and loaded wildtype and Panx1^−/− bones. Scale bar = 20 µm.

Figure 4

mRNA expression of β-catenin and sclerostin after 4 weeks loading in wildtype and Panx1^−/− mice. q-PCR analysis of (A) β-catenin (Ctnnb1) and (B) sclerostin (Sost) mRNA expression in loaded wildtype, loaded Panx1^−/−, and non-loaded Panx1^−/− relative to age-matched non-loaded wildtype, and loaded Panx1^−/− relative to age-matched non-loaded Panx1^−/−. The delta-delta CT method was used for data analysis, where the value obtained for each gene of interest is first normalized to the reference gene (18S rRNA gene) and then to either age-matched non-loaded wildtype or age-matched non-loaded Panx1^−/−. All data are presented as mean ± SD, n = 5; **P < 0.01, ***P < 0.001. P values were obtained using either one-way ANOVA followed by Tukey’s multiple comparison test or unpaired t-test followed by Mann-Whitney test.