Abstract
Bone adapts to changes in the physical environment by modulating remodeling through bone resorption and formation to maintain optimal bone mass. As the most abundant connexin subtype in bone tissue, connexin 43 (Cx43)-forming hemichannels are highly responsive to mechanical stimulation by permitting the exchange of small molecules (<1.2 kDa) between bone cells and the extracellular environment. Upon mechanical stimulation, Cx43 hemichannels facilitate the release of prostaglandins E2 (PGE2), a vital bone anabolic factor from osteocytes. Although most bone cells are involved in mechanosensing, osteocytes are the principal mechanosensitive cells, and PGE2 biosynthesis is greatly enhanced by mechanical stimulation. Mechanical stimulation-induced PGE2 released from osteocytic Cx43 hemichannels acts as autocrine effects that promote β-catenin nuclear accumulation, Cx43 expression, gap junction function, and protects osteocytes against glucocorticoid-induced osteoporosis in cultured osteocytes. In vivo, Cx43 hemichannels with PGE2 release promote bone formation and anabolism in response to mechanical loading. This review summarizes current in vitro and in vivo understanding of Cx43 hemichannels and extracellular PGE2 release, and their roles in bone function and mechanical responses. Cx43 hemichannels could be a significant potential new therapeutic target for treating bone loss and osteoporosis.
Introduction
Bone is a mechanosensitive tissue that undergoes constant remodeling to adapt to the physical environment (). Enhanced mechanical stimulation has major, positive anabolic impacts on bone tissue (Warden et al., 2007; Erlandson et al., 2012), whereas disuse leads to bone loss (Lang et al., 2004). As the most abundant and long-lived cells in the adult skeleton (), osteocytes with extensive lacunar-canalicular networks are generally regarded as mechanosensory cells that help translate mechanical stimulation into biological signals by regulating the function of osteoclasts and osteoblasts on the bone surface. Prostaglandin E2 (PGE2), a member of the eicosanoid family, is an essential key factor involved in the anabolic response of bone tissue to mechanical loading. PGE2 is not stored by bone cells but is synthesized in response to mechanical stimulation (Klein-Nulend et al., 1997). PGE2 at low concentrations (0–1 nM) stimulates osteoblast proliferation and differentiation, whereas at high concentrations (≥1 nM) inhibits osteogenesis (Ozawa et al., 1990). Mechanical loading induces the expression of cyclooxygenase 2 (COX-2), a key enzyme for PGE2 synthesis (), and intracellular PGE2 is released under mechanical stimulation (; ). In vitro studies show that released PGE2 from mechanically stimulated osteocytes can reduce SOST/sclerostin expression through EP4 receptors (Galea et al., 2011) and also enhance osteoclast activity (; Matsuzaka et al., 2021). Besides osteocytes, osteoblasts also release PGE2 in response to mechanical stimulation (Duncan and Turner, 1995; Klein-Nulend et al., 1997; Saunders et al., 2001), which influences osteoblast proliferation and differentiation (Imamura et al., 1990; Miwa et al., 1991). In vivo studies also show that mechanical loading increases PGE2 levels in the tibia bone of humans (Thorsen et al., 1996) and mice (Zhao et al., 2022a; Zhao et al., 2022b). An earlier study reported that intermittent PGE2 treatment increases bone formation and bone mass (Jee et al., 1985; Tian et al., 2007), whereas, inhibition of PGE2 suppresses bone formation induced by mechanical loading (Forwood, 1996).
One form of cell-cell communication is via gap junctions, the membrane-spanning channels composed of two juxtaposed hemichannels (Goodenough et al., 1996). In addition to direct gap junction intercellular communication (GJIC), halves of gap junctions, hemichannels mediate the communication between bone cells and the extracellular environment (Civitelli, 2008). Connexin-forming hemichannels exhibit relatively low substrate selectivity and permit small molecules (≤1.2 kDa) to pass through (Goodenough et al., 1996; Kumar and Gilula, 1996). Cx43 is the predominant connexin subtype expressed in osteocytes (Yellowley et al., 2000; ) and osteoblasts (). Cx43 hemichannels are highly responsive to mechanical stimulation, and their opening induced by mechanical loading mediates the release of anabolic factors such as PGE2, adenosine triphosphate (ATP) and nitric oxide (NO) from osteocytes (Jiang and Cherian, 2003; ) and osteoblasts (Thi et al., 2012). Both ATP and NO are related to the production of the bone anabolic agent PGE2 (Sugiatno et al., 2006; Genetos et al., 2007). In osteocytes, released PGE2 can act in either a feed-forward or feedback manner in regulating Cx43 expression and function. Mechanical loading-induced PGE2 release from osteocytic Cx43 hemichannels increases Cx43 expression and Cx43-forming gap junctions (). Interestingly, fluid flow-induced accumulation of extracellular PGE2 leads to the closure of Cx43 hemichannels (Riquelme et al., 2015). The Cx43 hemichannels and extracellular PGE2 play an inhibitory role in glucocorticoid-induced apoptosis (Kitase et al., 2010). Cx43 hemichannels with PGE2 release are also essential for normal bone structure (Xu et al., 2015; Zhao et al., 2022a) and the anabolic response of tibias to mechanical loading in vivo (Zhao et al., 2022a; Zhao et al., 2022b). In addition to connexins, pannexins are also capable of forming hemichannels (Plotkin et al., 2017). Pannexin1 is the most widely distributed pannexin in bone cells, and these hemichannels are involved in the release of PGE2 induced by mechanical stimulation in osteoblasts (Thi et al., 2012). However, the roles of pannexin channels in bone have not been investigated in great detail. In this review, we focus on the function of Cx43 hemichannels in releasing PGE2, and further PGE2-regulated skeletal development and cellular signals that drive bone anabolic and bone remodeling responses to mechanical stimulation.
Relationship between Cx43 hemichannels and PGE2 upon mechanical stimulation in osteocytes
Osteocytes are a rich source of PGE2 upon mechanical stimulation. Mechanical stress in the form of fluid flow causes a rapid increase in COX-2 expression (Joldersma et al., 2000) and PGE2 production in osteocytes (; ). Inhibition of COX-2 enzymatic activity with NS-398 inhibitor abolishes the stimulatory effect of fluid flow on PGE2 secretion from osteocytes (). During mechanical stimulation, Cx43 hemichannels play a crucial role in the PGE2 release from osteocytes. Low-density cultures of primary osteocytes and osteocyte-like MLO-Y4 cells with minimal cell-cell contacts, thus void of gap junctions, release more PGE2 than cells cultured at higher densities (Jiang and Cherian, 2003; ). Inhibition of Cx43 channels by chemical blocker β-glycyrrhetinic acid (Jiang and Cherian, 2003; ) or knocking down Cx43 by siRNA (Genetos et al., 2007) attenuates fluid flow-induced hemichannel activity and PGE2 production in low-density cultured osteocytes. This experimental evidence suggests that Cx43 hemichannels participate in PGE2 secretion during mechanical stimulation. To further depict the relationship between Cx43 hemichannels and PGE2 release, we develop a polyclonal antibody, Cx43 (E2), that targets the second extracellular loop domain of Cx43 and specifically blocks osteocytic Cx43 hemichannels in vitro (Siller-Jackson et al., 2008; Riquelme et al., 2013). Blocking of Cx43 hemichannels by Cx43 (E2) inhibits the opening of hemichannels and the release of PGE2 induced by flow shear stress in osteocytes (Siller-Jackson et al., 2008). Interestingly, PGE2 also has a negative feedback regulation on Cx43 hemichannels in response to mechanical stimulation. Extracellular PGE2 accumulation after the continuous opening of hemichannels by fluid flow acts on EP2/4 receptors to close Cx43 hemichannels (Riquelme et al., 2015). The negative feedback is caused by the PGE2 activation of p44/42 ERK signaling and direct Cx43 phosphorylation at S279/282 residues thereby leading to the closure of Cx43 hemichannels. (Riquelme et al., 2015). In addition, the released PGE2 from osteocytes by fluid shear stress promotes Cx43 expression and further increases Cx43 gap junctions (Jiang and Cheng, 2001; Xia et al., 2010). Consistent with the effects of fluid shear stress, direct treatment of the MLO-Y4 cells with PGE2 similarly increases Cx43 expression and gap junctions. In contrast, inhibition of PGE2 signaling by indomethacin reduced gap junction formation by fluid shear stress ().
Since the PGE2 secretion by osteocytes depends on the opening of Cx43 hemichannels in osteocytes, it is important to understand how Cx43 hemichannels are regulated by mechanical stimulation (Figure 1). We find that the dendritic processes of osteocytes transmit mechanical signals to the cell body, leading to the opening of Cx43 hemichannels in MLO-Y4 cells and primary osteocytes (). Cx43 is richly present in the cell body, not the dendritic processes of osteocytes. The integrin αVβ3, located at the dendrites of osteocytes, is an important component of the glycocalyx complex that tethers osteocytes to the canalicular wall and amplifies the magnitude of mechanical signals experienced by osteocytes (Wang et al., 2007; Riquelme et al., 2021). Upon fluid flow, the force generated by tethering elements is a magnitude higher than shear stress on the cell surface. Mechanically activated integrin αVβ3 at dendritic processes induces the activation of intracellular PI3K signaling (), which activates the downstream effector AKT (). Activated AKT directly phosphorylates Cx43 and integrin α5 () in the cell body to increase the interaction between these two proteins, opening the Cx43 hemichannels. Upon mechanical loading, activation of α5β1 through its conformational changes opens the Cx43 hemichannels in MLO-Y4 cells. Interestingly, this action is independent of integrin binding to its extracellular substrate, fibronectin (; ). Moreover, the scaffolding molecule 14-3-3θ assists in transporting both Cx43 and integrin a5 from the Golgi apparatus to the plasma membrane to form mechanosensitive Cx43 hemichannels (). Silencing 14-3-3θ prevents the accumulation of Cx43 on the cell membrane and the opening of hemichannels caused by fluid flow (). Recently, we find that Piezo1 is co-localized with Cx43 hemichannels on osteocyte cell surface. The activation of the Piezo1 leads to an increase in intracellular Ca2+ and the opening of Cx43 hemichannels through PI3K-AKT pathway in osteocytes (Zeng et al., 2022). Interestingly, the release of PGE2 is upregulated when Piezo1 is activated by either agonist or mechanical stretch (Li et al., 2019; Yang et al., 2022). The PGE2 release by Cx43 hemichannels is also regulated by extracellular ATP. Mechanical stimulation-induced ATP release through Cx43 hemichannels activates P2X receptors and promotes the Ca2+ influx to sustain the activities of Cx43 hemichannels (Zeng et al., 2022). Blockade of P2X7 purinergic receptors prevents PGE2 release from MLO-Y4 cells. However, the activation of purinergic receptors and the increase in the release of PGE2 appears to be independent of hemichannel formation (Genetos et al., 2007). Thus, the mechanism for ATP-induced PGE2 release is yet to be fully understood in osteocytes.
FIGURE 1
PGE2, Cx43 hemichannels, and mechanical stimulation in other bone cell types
Besides osteocytes, osteoblast is another mechano-responsive bone cell type (Johnson et al., 1996; Romanello et al., 2001; Liu et al., 2022). Earlier studies report that fluid flow stimulates the release of PGE2 from primary osteoblasts and MC3T3-E1 osteoblastic cells (Duncan and Turner, 1995; Klein-Nulend et al., 1997; Saunders et al., 2001). In contrast to osteocytes, osteoblasts require high magnitudes of shear stress for PGE2 production (Saunders et al., 2003; Genetos et al., 2005). Lower fluid flow levels induce greater PGE2 production in MLO-Y4 osteocyte-like cells than in 2T3 osteoblasts (Kamel et al., 2010). Moreover, Genetos and others even show that fluid flow only activates hemichannels in MLO-Y4, leading to the release of PGE2, but not osteoblastic MC3T3-E1 cells (Genetos et al., 2007). Thus, osteoblasts appear to be less mechanically sensitive to PGE2 release than osteocytes. Furthermore, increased PGE2 level (>10 nM) by hypergravity or compressive pressure promotes proliferation but suppresses differentiation of MC3T3-E1 cells (Ozawa et al., 1990; Miwa et al., 1991).
Interestingly, Cx43 hemichannels expressed in osteoblasts (Romanello and D'Andrea, 2001) can be regulated by mechanical stimulation (Romanello et al., 2003). However, PGE2 release appears not to be driven by Cx43 hemichannels in osteoblasts. Cx43-null calvarial osteoblasts still respond to mechanical stimulation, as evidenced by increased dye uptake and PGE2 release. In contrast, fluid flow-induced PGE2 release is abolished in osteoblasts deficient in pannexin1 (Thi et al., 2012). Pannexin1 is a transmembrane channel with a similar topology as connexins that only form hemichannels but not gap junctions (Penuela et al., 2013). These findings suggest that hemichannels formed by pannexin1 and not Cx43 might be responsible for fluid flow-induced PGE2 release in osteoblasts.
Recent studies indicate that osteoclasts are responsive to mechanical stress. Osteoclasts, after mechanical stretch, polarized to the M2 phenotype associated with YAP activation and nuclear translocation, which facilitates osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) (Dong et al., 2021). Jiang and others find that the extracellular PGE2, acting via EP4 receptors in osteoclasts, activates the Gαs/PI3K/AKT/MAPK signaling pathway and mediates migration and osteoclastogenesis during the progression of osteoarthritis (Jiang et al., 2022). Although it is known that osteoclasts express Cx43 on the plasma membrane and form hemichannels (Vesely et al., 1992; Ilvesaro et al., 2000; Ilvesaro and Tuukkanen, 2003), whether osteoclasts secret PGE2 through Cx43 hemichannels remains elusive.
The signaling pathways activated by PGE2 in osteocytes upon mechanical stimulation
PGE2 can activate four subtypes of G-protein-coupled receptors (GPCRs), named EP1, EP2, EP3, and EP4 (Woodward et al., 2011). EP2 and EP4 are the most extensively studied in bone (Furuyashiki and Narumiya, 2011). EP2 is a mechanosensitive PGE2 receptor whose expression can be enhanced by fluid flow in osteocytes (). Inhibition of the EP2 receptor by antagonist AH6809 suppresses the production of PGE2 and Cx43 expression (). The fluid flow-induced PGE2 release from osteocytes exerts autocrine effects on EP2 receptors to activate the cAMP-PKA pathway in osteocytes and increase the Cx43 gap junction formation (). Further study reveals that PGE2 released from osteocytes by mechanical stimulation could lead to activation of the PI3K/Akt signaling pathway in addition to the cAMP/PKA pathway. The activation of both PI3K/Akt and cAMP/PKA pathways results in the phosphorylation and inactivation of GSK-3β (Xia et al., 2010), which is responsible for the phosphorylation of β-catenin, resulting in its ubiquitination and degradation by the 26S proteasome complex (). Consequently, the inactivated GSK-3β causes an increase in nuclear translocation and accumulation of β-catenin in osteocytes (Xia et al., 2010; Lara-Castillo et al., 2015). Increased nuclear β-catenin binds to the promoter region to promote Cx43 expression (Xia et al., 2010). In contrast, inhibition of PGE2 by a COX-2 inhibitor, Carprofen, blocks the activation of β-catenin nuclear translocation in osteocytes through the PI3K/Akt activation (Lara-Castillo et al., 2015). Canonical Wnt/β-catenin signaling is proven to stimulate anabolic actions in osteocytes (Osório, 2015; Tu et al., 2015). Deletion of β-catenin in osteocytes abolishes the bone anabolic response to mechanical loading (Javaheri et al., 2014; Kang et al., 2016). Thus, osteocytic accumulation of β-catenin may be one mechanism of mechanical-induced bone formation. In addition, the Wnt/β-catenin signaling is a well-known pathway associated with cell apoptosis (). Mechanical stimulation-induced PGE2 in osteocytes blocks glucocorticoid-induced apoptosis through activated β-catenin, a downstream effector of the PI3K/Akt pathway (Kitase et al., 2010). In addition, the cAMP/PKA signaling pathway is involved in PGE2-mediated osteocyte survival during mechanical stimulation (Kitase et al., 2010). Integrins α5β1, which requires the opening of Cx43 hemichannels in osteocytes, participate in mechanical stimulation-induced osteocyte survival through FAK/Src and the ERK pathway (Plotkin et al., 2005).
The PGE2 released upon mechanical stimulation exerts a paracrine effect on the EP2/4 receptor to suppress the sclerostin expression through the cAMP/PKA pathway (Galea et al., 2011; Genetos et al., 2011). Sclerostin is an antagonist of canonical Wnt-β-catenin signaling in osteoblasts () through binding to the Wnt co-receptor Lrp5/6 (Li et al., 2005) to suppress osteogenesis (Sawakami et al., 2006). Several in vitro studies indicate the roles of osteocyte-derived PGE2 in promoting osteoblast activity during mechanical stimulation. Increased concentration of PGE2 released into the conditioned medium by fluid flow-loaded MLO-Y4 osteocytes promotes osteoblast differentiation (Zeng et al., 2019). In a 3D trabecular bone explant co-culture model, dynamic deformational loading can significantly increase the PGE2 release from osteocytes in their native extracellular matrix environment and promote osteoblastic bone formation (). PGE2 released from osteocytes regulates osteoblast recruitment and collagen organization in the bone matrix during oscillatory fluid flow (Matsuzaka et al., 2021). Sclerostin not only inhibits bone formation but also promotes osteoclast formation. Osteocytes constitute a significant source of osteoclastogenic cytokine RANKL (; Xiong et al., 2011), which is also regulated by Wnt/β-catenin signaling (Donald et al., 2005; Nakashima et al., 2011). Sclerostin directly increases the levels of RANKL in osteocytes to regulate osteoclast activity by inhibiting β-catenin in osteoclasts (Wijenayaka et al., 2011). In contrast, sclerostin deficiency is resistant to bone resorption during mechanical unloading (Lin et al., 2009). Together, the PGE2 secreted from osteocytes upon mechanical stimulation has paracrine effects through EP2 and EP4 receptors to increase β-catenin and suppress sclerostin expression in osteocytes. Increased β-catenin in osteocytes promotes Cx43 expression, gap junction formation, mechanosensitivity, and survival of osteocytes. Suppression of sclerostin secretion promotes osteoblast activity and inhibits osteoblast activity.
Cx43 hemichannels in bone development under physiological level of mechanical stress
Previous studies using Cx43 knockout and transgenic mouse models provide insightful information regarding the importance of Cx43 hemichannels in bone development under physiological mechanical conditions. Although global deleting Cx43 (Cx43−/−) caused early postnatal death due to an obstruction of the right ventricular outflow tract, they showed retarded intramembranous, endochondral ossification, craniofacial abnormalities, and osteoblast dysfunction (Lecanda et al., 2000; Thi et al., 2010; ; Ishikawa et al., 2016). Moreover, mice with specific deletion of Cx43 by expressing the Cre recombinases in osteoprogenitors (DM1-Cre;Cx43-/flx) (Watkins et al., 2011), preosteoblasts (Osx1-Cre;Cx43flx/flx) (Hashida et al., 2014), early osteoblasts/osteocytes (Col1α1-Cre;Cx43-/flx) (; ), mature osteoblasts/osteocytes (OCN-Cre;Cx43-/flx) (), and osteocytes (8-kb DMP1-Cre;Cx43flx/flx) (; ) all showed thinner cortical thickness, larger marrow area, and total cross-sectional area. The change of cortical bone structure in these cKO mice was due to both increased periosteal osteoblastic bone formation (Watkins et al., 2011; ; Watkins et al., 2012; Pacheco-Costa et al., 2015) and an even greater unbalanced increase in endosteal osteoclastic bone resorption (Watkins et al., 2011; ; Watkins et al., 2012; Lloyd et al., 2013). In addition, Cx43 overexpressing in osteocytes (Cx43OT) preserves osteocyte viability and bone formation to ameliorate age-induced cortical bone loss (Davis et al., 2018). It is worth noting that Cx43 deficiency affects the production and release of PGE2 in osteoblasts and osteocytes. A lower amount of PGE2 is found in the primary calvaria cells of Cx43−/− mice than in wild-type (WT) mice (Grimston et al., 2006). The absence of Cx43 in osteocytes by Cx43 shRNA attenuates PGE2 synthesis by COX-2 (). Although Cx43 knockout during bone developmental stages from the early stage (DM1-Cre) to the late stage (DMP1-Cre) of osteoblast differentiation shows the similar bone structure, Cx43 deficiency abolishes both Cx43 gap junctions and hemichannels. Thus, it is impossible to determine whether Cx43 gap junctions or/and hemichannels are responsible for the observed phenotypes. It is worth noting that other cell types may play indirect roles in osteoblast differentiation after knocking out Cx43. For example, Cx43 in osteoblasts/osteocytes indirectly modulates skeletal muscle growth and function (Shen et al., 2015; Li et al., 2021). In turn, skeletal muscle can also influence bone growth by releasing osteogenic myokines (Hamrick et al., 2010). Thus, the roles of Cx43 hemichannel in bone and other tissue crosstalk need to be further studied.
Besides Cx43-deficient mice, several Cx43 gene mutations can lead to a skeletal disease called oculodentodigital dysplasia (ODDD), with phenotypic presentations of syndactyly, craniofacial abnormalities, and long broad bones (Paznekas et al., 2003). To date, four mouse strains (Cx43I130T/+ (Kalcheva et al., 2007), Cx43Jrt(G60S)/+ (Flenniken et al., 2005), Cx43G138R/+ (Dobrowolski et al., 2008) and Cx43K258Stop/- (Pacheco-Costa et al., 2015; Moorer et al., 2017)) with missense point mutations in one allele of the Cx43 gene are generated to mimic the phenotypes of ODDD. The mutations alter Cx43 protein conformation, thus leading to changed hemichannel activities (Dobrowolski et al., 2007). Increased (Dobrowolski et al., 2008) or decreased (Kalcheva et al., 2007) Cx43 hemichannel functions are found in these Cx43 mutants, indicating that normal Cx43 hemichannel function is crucial in maintaining bone structure.
Our group has generated two transgenic mouse models to dissect the function of Cx43 gap junctions and hemichannels in osteocytes, respectively. A 10 kb-DMP1 promoter drives the two transgenic mouse models R76W and ∆130–136 with the overexpression of dominant negative Cx43 mutants in osteocytes (Xu et al., 2015). The R76W site mutant, of which Cx43 amino acid residue arginine-76 (R) is replaced by tyrosine, inhibits gap junctions. The ∆130–136 mutant with the deletion of seven residues in the cytoplasmic loop of Cx43 protein at amino acids 130–136 inhibits both gap junctions and hemichannels. The bone phenotype in R76W mice is mostly like WT mice, except for increased endosteal osteoclast activity and bone remodeling markers in serum. In contrast, the ∆130–136 mice exhibit increased osteocyte apoptosis, endosteal resorption, and periosteal bone formation, resulting in higher tissue, cortical, and marrow cavity area of femoral midshaft at the femoral mid-diaphysis. The bone phenotypes in ∆130–136 mice are similar, but even more severe, than osteocyte-specific Cx43 cKO mice driven by 8-kb DMP1 promoter (), indicating Cx43 deficiency in osteocytes impairs the functions of Cx43 hemichannel in bone development. Compared to WT and R76W, the ∆130–136 mice show lower PGE2 levels in tibia diaphysis (Zhao et al., 2022a). As discussed above, PGE2 released by osteocytes mediated by Cx43 hemichannels is proven to maintain osteocyte survival and prevent their apoptosis (Kitase et al., 2010). Consistently, both ∆130–136 and 8-kb DMP1-Cre;Cx43 cKO mice show increased osteocyte apoptosis in cortical bone. Impaired Cx43 hemichannels of osteocytes are also found in osteocyte-specific integrin a5 cKO mice driven by 10-kb DMP1 promoter, which expresses lower serum PGE2 levels and increased osteocytes apoptosis and cortical thickness in tibias (Zhao et al., 2022b). Thus, under a physiological mechanical environment, Cx43 hemichannels and PGE2 in osteocytes likely play a predominant role in osteocyte vitality and bone structure.
PGE2 release through osteocytic Cx43 hemichannels promotes bone anabolism upon mechanical loading
PGE2 stimulates bone resorption and formation (Jee and Ma, 1997; ). Continuous PGE2 treatment decreases cancellous bone mass due to bone resorption exceeding bone formation (Tian et al., 2007). Whereas moderate PGE2 treatment by intraperitoneal injection (Jee et al., 1985; Ueno et al., 1985) and local metaphyseal injection (Welch et al., 1993; Yang et al., 1993) increases both trabecular and cortical bone mass in growing rats. In addition, PGE2 prevents bone loss induced by ovariectomy (Mori et al., 1990; Harada et al., 1995), disuse (Jee et al., 1992), and orchidectomy (Li et al., 1995) in rats. Recent studies show that PGE2 activates EP4 in sensory nerves to promote bone formation and inhibit adipogenesis by inhibiting sympathetic activity through the central nervous system (; Hu et al., 2020). The release of PGE2, a known direct product of bone mechanical stimulation, has important anabolic effects on the skeleton. In healthy women, a rapid and significant increase of PGE2 levels in the proximal tibial metaphysis is observed using the microdialysis technique in response to weight-bearing mechanical loading (Thorsen et al., 1996). Inhibition of PGE2 by a COX-2 inhibitor, NS-398, completely blocks tibial bone formation induced by four-point bending loading in rats (Forwood, 1996; Li et al., 2002). On the contrary, activation of the PGE2 receptor using ONO-4819 (agonist for prostaglandin E receptor subtype EP4) has an additive effect on bone formation in response to mechanical loading (Hagino et al., 2005). The involvement of Cx43 in the anabolic function of mechanical loading and PGE2 has been reported in earlier studies. Mice with Cx43 deficiency show altered bone anabolic response to mechanical loading. Col1α1-Cre;Cx43 cKO mice show attenuated tibial endosteal bone formation during non-physiological four-point (Grimston et al., 2006) or three-point tibial bending (Grimston et al., 2008). A lower level of PGE2 is found in the primary calvaria cells from Cx43−/− mice than in WT mice during mechanical stretching (Grimston et al., 2006). In DM1-Cre;Cx43 cKO mice, axial tibia loading results in a greater decrease of endosteal bone formation compared to WT mice (Grimston et al., 2012). However, Cx43 deficiency has a positive effect on periosteal bone formation. Deletion of Cx43 in osteoblasts/osteocytes (DM1-Cre;Cx43 cKO and OCN-Cre;Cx43 cKO mice) showed an enhanced tibial periosteal response to tibial axial compression (Grimston et al., 2012) or tibial cantilever bending (Zhang et al., 2011). Similarly, deletion of Cx43 in osteocytes (DMP1-Cre; Cx43 cKO mice) showed enhanced periosteal bone formation in response to ulna compression (). Nevertheless, these cKO mice have both impaired gap junctions and hemichannels, as well as potential channel-independent roles of Cx43. Thus, the specific role of gap junctions and hemichannels formed by Cx43 hemichannels in response to mechanical loading in the bone cannot be resolved with Cx43 deletion models.
Recently, our group find a close relationship between Cx43 hemichannels and PGE2 release in skeletal response to mechanical loading in vivo. In this study, PGE2 levels in the diaphysis are significantly increased in WT and gap junction impaired R76W upon tibial cyclic compression loading. Increased PGE2 level suppresses the sclerostin expression in osteocytes and bone formation on the endosteal surface. However, Δ130-136 mice with impaired gap junctions and hemichannels show unchanged PGE2 levels and sclerostin expression in osteocytes. As a result, the increased bone mass caused by mechanical loading is not seen in Δ130-136 mice. Attenuated bone formation and increased resorption on the endosteal surface lead to the enlargement of the bone marrow cavity and inhibited bone mass gain (Zhao et al., 2022a). The data points to the role of Cx43 hemichannels in mediating PGE2 release and the anabolic action of mechanical loading. To further investigate whether the changed mechanical response in Δ130-136 mice is due to Cx43 hemichannels, a specific mouse monoclonal blocking antibody Cx43 (M1) was developed and used to investigate the role of Cx43 hemichannels in vivo. Administration of this particular antibody exhibits similar effects as Δ130-136 mice, including the attenuated PGE2 level and inhibited anabolic response to mechanical loading on the endosteal surface. PGE2 administration, however, can rescue the attenuated endosteal osteogenic response to mechanical loading impeded by the Cx43 (M1) antibody. PGE2 acts in a paracrine manner to suppress sclerostin expression in vitro (Galea et al., 2011; Genetos et al., 2011). These in vivo studies demonstrate that Cx43 hemichannels activated by mechanical stimulation release PGE2 from osteocytes to suppress sclerostin expression in osteocytes and enhance osteoblast activity and bone formation on endosteal surfaces. We further demonstrate the important role of Cx43 hemichannels and PGE2 release in bone anabolic response to mechanical loading through the use of osteocyte-specific integrin a5 cKO mice driven by a 10-kb DMP1 promoter (Zhao et al., 2022b). Since the interaction between integrin α5 and Cx43 is essential for the hemichannel opening by mechanical loading (), integrin α5 deficiency impedes load-induced Cx43 hemichannel opening and PGE2 release (; Zhao et al., 2022b). Integrin α5 cKO mice in osteocytes exhibit attenuated loading effects on catabolic sclerostin reduction and anabolic β-catenin increase, contributing to decreased endosteal osteoblasts and bone formation (Figure 1). Our studies show that the anabolic effect of Cx43 hemichannel-released PGE2 is on the endosteal surface (Zhao et al., 2022a; Zhao et al., 2022b). Consistently, previous studies also report that the effect of PGE2 on bone anabolic response to mechanical loading appears to be more on the endosteal surface than the periosteal surface (Forwood, 1996; Li et al., 2002; Hagino et al., 2005).
Interestingly, inhibited Cx43 hemichannels in Δ130-136 mice, Cx43 (M1) group, and integrin α5 cKO mice showed an increase of osteoclast activity on the endosteal surface during mechanical loading. Whether this catabolic response is related to PGE2 requires further investigation. The mechanosensitivity of bone is gradually diminished during aging (Holguin et al., 2014; Holguin et al., 2016), accompanied by impaired mechanotransduction () and decreased Cx43 expression in osteocytes (Kar et al., 2013). We speculate that attenuated Cx43 expression in osteocytes is related to abolished Cx43 hemichannel activity and PGE2 release in aged bone. Indeed, significantly reduced PGE2 EP4 receptors in the sensory nerve of aged bone attenuate the sensibility to changes in bone metabolism (Lv et al., 2022).
Cx43 hemichannels play a protective role against bone loss during mechanical unloading
Reduced or no mechanical loading, such as long bed rest (Spector et al., 2009), and astronauts in space missions (Keyak et al., 2009), harms skeletal health, which is associated with an imbalanced bone turnover. Suppressed osteoblastic bone formation and activated osteoclastic bone resorption (; Kondo et al., 2005) increase bone loss and fracture risk. Previous in vitro studies show that Cx43 hemichannels participate in response to mechanical unloading. Zero-gravity by parabolic flight decreases Cx43 expression in osteocytes (Di et al., 2011). Simulated microgravity by a random position machine (RPM) increases the activity of Cx43 hemichannels and the release of PGE2 (Xu et al., 2017). Dominant negative integrin β1 mutants driven by an OCN promoter show a lower cancellous bone mass in the distal femoral metaphysis caused by increased bone resorption and decreased bone formation during short-term hindlimb unloading, a model mimicking unloading/disuse (Iwaniec et al., 2005). Given that integrin a5β1 regulates the opening of the Cx43 hemichannels, this study implies the vital role of Cx43 hemichannels in bone response to unloading. High extracellular PGE2 due to sustained opening of hemichannels during unloading (Xu et al., 2017) resulted in osteoclast resorption and bone loss (Coon et al., 2007; Knippenberg et al., 2007; Tian et al., 2007). Consistently, deletion of Cx43 in osteoblast/osteocyte-specific Cx43 cKO mice driven by the 2.3-kb Col1α1 promoter (Grimston et al., 2011) and OCN promoter (Lloyd et al., 2012; Lloyd et al., 2013) show protection against unloading-induced bone loss. In contrast, our previous study shows that enhanced Cx43 hemichannels in R76W mice protect from osteocyte apoptosis in cortical bone during mechanical unloading (Zhao et al., 2020). PGE2 is an inhibitor of sclerostin expression, and both sclerostin and PGE2 inhibitors are known to be associated with cell apoptosis (). It is assumed that PGE2 from Cx43 hemichannels has a protective role against osteocyte apoptosis. Multifaced roles of Cx43 could cause the difference seen between Cx43 deletion and hemichannel-impaired models. Further studies are needed to establish the underlying mechanisms of Cx43 hemichannels and PGE2 in response to mechanical unloading.
Future perspectives
With research advances and the development of new transgenic mouse models, the inter-relationship between Cx43 hemichannels and extracellular PGE2 in mediating osteogenic response to mechanical loading is becoming more evident. However, several questions remain, including 1) if extracellular PGE2 released by hemichannels upon mechanical loading has any direct action on osteoclasts; 2) if the loading-induced bone anabolic response is partly regulated through the influence of osteocyte-released PGE2 on the nerve system. Early studies reported that temporarily blocking peripheral neurons by bupivacaine reduces bone formation in compressed ulna (Sample et al., 2008). Recently, PGE2 has been found to act on sensory neurons and affect sympathetic nerve activity, and regulate bone homeostasis (; Lv et al., 2021). It is speculated that the PGE2 released from Cx43 hemichannels in osteocytes may not only have autocrine and paracrine effects on osteocytes and the other types of bone cells, respectively, but may also systematically influence sensory nerves. The interactions of bone cells and sensory neurons regulated by PGE2 remain largely unknown. These could all be potential directions for future research. Moreover, further research on unveiling the mechanism of action for hemichannels and PGE2 may help in the discovery and development of potential therapeutics that aid in treating bone loss, in particular, in the elderly population with lost sensitivity to anabolic responses to mechanical stimulation.
Statements
Author contributions
DZ, JW and JJ drafted the manuscript, FA and JJ conceived the idea and revised the manuscript. All authors contributed to manuscript revision read and approved the submitted version.
Funding
This work was supported by the National Institutes of Health (NIH) Grants: AR072020 (to JJ) and F32DK134051 (to FA), and Welch Foundation grant: AQ-1507 (to JJ).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
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Summary
Keywords
connexin 43, hemichannel, prostaglandin E2, bone, mechanical stimulation
Citation
Zhao D, Wu J, Acosta FM, Xu H and Jiang JX (2023) Connexin 43 hemichannels and prostaglandin E2 release in anabolic function of the skeletal tissue to mechanical stimulation. Front. Cell Dev. Biol. 11:1151838. doi: 10.3389/fcell.2023.1151838
Received
26 January 2023
Accepted
05 April 2023
Published
13 April 2023
Volume
11 - 2023
Edited by
Catherine Wright, Glasgow Caledonian University, United Kingdom
Reviewed by
José Luis Vega, University of Antofagasta, Chile
Xiaolin Tu, Chongqing Medical University, China
Updates
Copyright
© 2023 Zhao, Wu, Acosta, Xu and Jiang.
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*Correspondence: Jean X. Jiang, jiangj@uthscsa.edu
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