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Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts
Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Contents lists available at ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts Daisuke Kodama, Ph.D.n, Akifumi Togari, Ph.D. Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 18 January 2014 Received in revised form 4 March 2014 Accepted 22 March 2014
Background: In recent years, the role of the sympathetic nervous system in bone metabolism has been revealed. Many studies have suggested that bone loss can be induced by continuously high sympathetic tone resulting from up-regulation of osteoclastogenesis and osteoclastic activity via β2-adrenergic receptors. Although the expression of α-adrenergic receptors in osteoblasts and osteoclasts has been demonstrated, the physiological roles of these receptors in bone metabolism remain unclear. In the present review, we provide an account of the role of α1-adrenergic receptors in bone metabolism. Conclusion: Experimental studies in osteoblasts suggest that not only the suppression of β2-adrenergic receptors but also the activation of α1-adrenergic receptors could lead to a treatment for osteoporosis. Studying the signaling pathway will help elucidate the mechanism underlying the regulation of bone metabolism via the α1-adrenergic receptor and also facilitate the development of a novel therapeutic strategy for osteoporosis. & 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Alpha1-adrenergic receptor Osteoblast Sympathetic nervous system Bone metabolism
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Effects of α1-adrenergic agonists on the physiology of osteoblasts . . . . 3. Signaling pathways involved in the effects of α1-adrenergic receptors 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction In recent years, neural regulation of bone metabolism mediated by osteoblasts and osteoclasts has been demonstrated [1–4].
Abbreviations: ALP, alkaline phosphatase; BrdU, 5-bromo-20 -deoxyuridine; HOS, human osteosarcoma-derived cells; MC3T3-E1, mouse calvaria-derived osteoblastic cells; MG-63, human osteosarcoma-derived cells; OPG, osteoprotegerin; PI-PLC, phosphoinositide-phospholipase C; Pit-1, sodium-dependent inorganic phosphate transporter; PKA, protein kinase A; PLC, phospholipase C; RANK, receptor activator of NFκB; RANKL, receptor activator of NFκB ligand; RAW264.7, mouse macrophagelike cells; SaM-1, human periosteum-derived osteoblastic cells; SaOS, human osteosarcoma-derived cells; WST, water-soluble tetrazolium n Corresponding author. Tel.: þ 81 52 751 2561; fax: þ 81 52 752 5988. E-mail addresses: [email protected] (D. Kodama), [email protected] (A. Togari).
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Previous studies showed that mRNAs of α- and β-adrenergic receptors are expressed in osteoblasts and osteoclasts [5,6]. Additionally, these cells also express neurotrophins and axon guidance molecules for growing nerve fibers [7]. In immunohistochemical studies, bones were shown to be widely innervated by sympathetic nerves [8,9]. The presence of peripheral nerve axons coursing through the marrow adjacent to osteoblasts in bone tissue was shown by electron microscopy [10]. Direct nerve-osteoblastic and nerve-osteoclastic cell communication has been demonstrated using an in vitro co-culture model comprising sympathetic nerve cells derived from the mouse superior cervical ganglion and MC3T3E1 cells, mouse calvaria-derived osteoblastic cells, or osteoclasts induced from RAW264.7 cells [11,12]. These findings suggest that the sympathetic nervous system plays a direct role in the regulation of bone metabolism.
http://dx.doi.org/10.1016/j.job.2014.03.004 1349-0079/& 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i
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D. Kodama, A. Togari / Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Endogenous agonists of adrenergic receptors such as noradrenaline and adrenaline stimulate both α- and β-adrenergic receptors. It is well known that α1- and β1-adrenergic receptors have a similar sensitivity to these agonists, but the β2-adrenergic receptor is less sensitive to noradrenaline than to adrenaline. In experimental studies, bone loss was induced by continuously high sympathetic tone, and this induction was reversed by β-adrenergic receptor blockade [13–15]. Many studies have suggested that up-regulation of osteoclastogenesis and osteoclastic activity via β2-adrenergic receptors enhances bone resorption [3,16,17]. Additionally, suppression of bone formation by β-adrenergic receptor activity has also been reported [15,17]. Furthermore, it has been reported that the use of β-blockers reduces the risk of bone fracture and increases bone mineral density [18–20]. The physiological role of the β-adrenergic receptor in bone metabolism has been demonstrated by many studies, whereas that of the α-adrenergic receptor has been less well studied. However, clinical studies have also reported that blockade of the α1-adrenergic receptor increased the risk of hip/femur fracture, and that hypertensive patients treated with α1-blocker showed an increased risk of osteoporosis [20,21]. These studies suggest that the α-adrenergic receptor also plays a role in sympathetic effects on bone metabolism. In the present review, we summarize the physiological role and signal transduction pathway of the α1-adrenegic receptor in osteoblasts based on our recent findings.
2. Effects of α1-adrenergic agonists on the physiology of osteoblasts In our study, mRNAs of the α1B-, α2B-, and β2-adrenergic receptors were found to be expressed in SaM-1 cells, which are human periosteum-derived osteoblasts, and in the SaOS, HOS, and MG-63 human osteosarcoma-derived cell lines [5,6]. Huang et al. [22] also demonstrated the expression of α1B- and β2-adrenergic receptors using RT-PCR and Western blotting. Additionally, on immunofluorescence microscopy, these receptors were shown to localize to the cell surface of human osteoblasts. Suzuki et al. [23] demonstrated that adrenaline and phenylephrine, an α1-adrenergic receptor agonist, increased DNA synthesis in a concentration-dependent manner in MC3T3-E1 cells as determined by 3H-thymidine incorporation. The effect of adrenaline was suppressed in the presence of α1-adrenergic receptor antagonists. Similarly, Huang et al. [22] demonstrated that DNA synthesis was enhanced by cirazoline, an α1-adrenergic receptor agonist, but inhibited by fenoterol, a β2-adrenergic receptor agonist, in human osteoblasts. We evaluated the effect of noradrenaline on cell proliferation activity according to DNA synthesis as determined by 5-bromo-20 -deoxyuridine (BrdU) incorporation, and we also evaluated the number of live cells according to dehydrogenase activity as determined by the water-soluble tetrazolium (WST) assay. In SaM-1 cells, noradrenaline increased BrdU incorporation at submicromolar concentrations and suppressed it at higher-than-micromolar concentrations. In contrast, noradrenaline only increased formazan formation in the WST assay, over the whole concentration range that we tested. In the presence of prazosin, an α1-adrenergic receptor antagonist, noradrenaline showed only suppressive effect on cell proliferation in both the BrdU assay and the WST assay. In contrast, in the presence of propranolol, a β-adrenergic receptor antagonist, the effect of noradrenaline was facilitatory in both assays [24]. These results suggest that cell proliferation is enhanced by α1-adrenergic receptors and inhibited by β-adrenergic receptors in human osteoblasts. Treatment with adrenaline also increased alkaline phosphatase (ALP) activity and sodium-dependent inorganic phosphate transporter (Pit-1) expression through the α1-adrenergic receptor in
MC3T3-E1 cells [25]. ALP reduces extracellular pyrophosphate, a potent inhibitor of calcification, and Pi is taken up by Pit-1. These molecules play important roles in the initial events of bone matrix calcification. These findings suggest that α1-adrenergic receptor activation enhances osteoblast-mediated bone formation. It is well known that the binding of receptor activator of nuclear factor kappa-B ligand (RANKL) to its receptor RANK is an essential signal for osteoclastogenesis. In MC3T3-E1 cells, the expression of RANKL and its decoy receptor, osteoprotegerin (OPG), was increased by adrenaline via the β-adrenergic receptor and α1-adrenergic receptor, respectively [26]. It has also been reported that the effects of α1-adrenergic receptor agonists on OPG expression were eliminated by the knockdown of the α1B-adrenergic receptor in human osteoblasts [22]. These results suggest that osteoclastogenesis is suppressed by α1-adrenergic receptor agonists. In summary, these studies suggest that bone metabolism is positively regulated by the α1-adrenergic receptor expressed in osteoblasts.
3. Signaling pathways involved in the effects of α1-adrenergic receptors α1-adrenergic receptors belong to the G-protein-coupled receptor family. In general, activation of α1-adrenergic receptors increases the intracellular Ca2 þ concentration ([Ca2 þ ]i) by inducing Ca2 þ release from the endoplasmic reticulum via the Gq/phosphoinositide-phospholipase C (Gq/PI-PLC) pathway. However, recent studies have demonstrated that Ca2 þ influx through Ca2 þ -permeable channels and the Na þ /Ca2 þ exchanger is involved in α1-adrenergic receptormediated Ca2 þ elevation. The molecular components underlying Ca2 þ influx and their importance in Ca2 þ signaling differ among tissues. In our study, noradrenaline induced [Ca2 þ ]i elevation via the α1-adrenergic receptor in MC3T3-E1 cells and SaM-1 cells. The effect of noradrenaline was suppressed not only by pretreatment with a PLC inhibitor, U73122, but also by removing extracellular Ca2 þ . The effect of noradrenaline was completely abolished in Ca2 þ -free extracellular solution. Therefore, Ca2 þ influx plays a predominant role in α1-adrenergic receptor-mediated Ca2 þ signaling in SaM-1 cells. Additionally, noradrenaline-induced [Ca2 þ ]i elevation was inhibited by pretreatment with either thapsigargin or storeoperated Ca2 þ channel inhibitors. These results suggest that activation of Gq-protein-coupled α1-adrenergic receptors induces [Ca2 þ ]i elevation mainly via store-operated Ca2 þ channels in human osteoblasts [27]. We have also demonstrated that noradrenaline reduces the whole-cell current in patch-clamp recordings from SaM-1 cells. The inhibitory effect of noradrenaline on the whole-cell current was eliminated by chloroethylclonidine, an α1B-adrenergic receptor-selective inhibitor, and CsCl, a potassium channel blocker. These results suggest that activation of the α1B-adrenergic receptor also suppresses potassium channels [28]. However, the inhibitory effect of noradrenaline on the whole-cell current was not affected by pretreatment with U73122 or by chelation of intracellular Ca2 þ . These results suggest that noradrenaline-induced activation of store-operated Ca2 þ channels and inhibition of potassium channels are individual effects. Then, we examined whether the Gi/o-protein is involved in the inhibitory effect of noradrenaline on the whole-cell current. Upon pretreatment with pertussis toxin, an inhibitor of the Gi/o-protein-coupled receptor, noradrenalineinduced inhibition of potassium channels was significantly suppressed. Pertussis toxin inhibits effects via the Giα-protein and also the Gβγ-protein. We next examined which pathways are involved in the inhibitory effect of noradrenaline on the wholecell current. Pretreatment with H89, a PKA inhibitor, attenuated
Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i
D. Kodama, A. Togari / Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Ca2+ NA
3
K+
α1B-AR
NA
α1B-AR
SOCCs
Gqα
AC
β γ
Giα
β γ
ATP cAMP
PLC IP3
IP3R STIM
PKA
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DG Ca2+ Cell proliferation
Fig. 1. Schematic diagram for the signaling pathway of Gq-protein-coupled and Gi/o-protein-coupled α1B-adrenergic receptors in osteoblasts. It is suggested that α1B-adrenergic receptors can be coupled to both the Gq-protein and Gi/o-protein in osteoblasts. Activation of the Gq-protein-coupled α1B-adrenergic receptor induces Ca2 þ release from the endoplasmic reticulum via the Gq/PI-PLC pathway. Reduction of the Ca2 þ concentration in endoplasmic reticulum sensed by STIM induces the activation of store-operated Ca2 þ channels. Ca2 þ influx from store-operated Ca2 þ channels appears to be the dominant pathway of noradrenaline-induced Ca2 þ elevation in human osteoblasts. In contrast, activation of the Gi/o-protein-coupled α1B-adrenergic receptor in osteoblasts suppresses potassium currents, mainly via the Gβγ-protein and partly via inhibition of the PKA pathway. This suppression of potassium channels results in membrane depolarization, which enhances cell proliferation activity. α1B AR: α1B-adrenergic receptor, AC: adenylate cyclase, DG: diacylglycerol, IP3: inositol 1,4,5-triphosphate, NA: noradrenaline, PKA: protein kinase A, PLC: phospholipase C, SOCC: store-operated Ca2 þ channel, and STIM: stromal interaction molecule.
the effect of noradrenaline on the whole-cell current, but not significantly. In contrast, pretreatment with gallein, a Gβγ-protein inhibitor, significantly suppressed the inhibitory effect of noradrenaline on the whole-cell current. Therefore, it is suggested that noradrenaline-induced inhibition of potassium channels occurs mainly via the Gβγ protein pathway of the Gi/o-coupled α1B-adrenergic receptor [24]. Additionally, the positive effects of noradrenaline on cell proliferation were significantly inhibited by CsCl, pertussis toxin, gallein, and H89, but not by U73122, in both the BrdU assay and the WST assay. Several studies have demonstrated that membrane potential and cell proliferation capacity are regulated by the activity of potassium channels in several cell types including osteoblasts. These results suggest that noradrenaline enhances cell proliferation by inhibiting Cs-sensitive potassium channels via the Gi/o-protein-coupled α1Badrenergic receptor in human osteoblasts [24].
α1-adrenergic receptors could lead to a treatment for osteoporosis. We have demonstrated that noradrenaline increases cell proliferation activity via the α1-adrenergic receptor at low concentrations and suppresses cell proliferations via the β-adrenergic receptor at high concentrations [24]. This biphasic effect is thought to result from the difference in the affinity of noradrenaline for these receptors. These results suggest that β-blockers may be effective against bone loss with a pathologically high sympathetic tone, whereas α1-adrenergic receptor agonists may be effective against dysregulation of bone metabolism with low sympathetic tone. In general, agonists of α1-adrenergic receptors also affect blood vessels. It is likely that reduction of the blood flow to bone downregulates bone metabolism. Therefore, osteoblast-selective effects may be preferable for the treatment of osteoporosis. Studying the associated signaling pathway will help elucidate the mechanism underlying the regulation of bone metabolism via the α1-adrenergic receptor and also facilitate the development of a novel therapeutic strategy for osteoporosis.
4. Conclusion Recent studies have demonstrated that a single subtype of receptor can be associated with different types of heterotrimeric G-proteins [29–31]. It is also suggested that coupling with a particular type of G-protein can be regulated by the location, phosphorylation, and expression of G-protein-coupled receptors [32–34]. Our studies suggest that α1B-adrenergic receptors can be coupled to both the Gq-protein and Gi/o-protein in human osteoblasts. By activating the Gq-protein-coupled α1B-adrenergic receptor, noradrenaline induces Ca2 þ influx through store-operated Ca2 þ channels via the Gq/PI-PLC pathway. In contrast, activation of the Gi/o-protein-coupled α1B-adrenergic receptor suppresses potassium channels, and cell proliferation activity is enhanced mainly by the inhibition of potassium channels (Fig. 1). Although further studies are needed to identify the channels activated and suppressed by noradrenaline, these studies suggest that the α1B-adrenergic receptor plays multiple roles in the physiology of osteoblasts. Experimental studies in osteoblasts suggest that not only the suppression of β2-adrenergic receptors but also the activation of
Ethical approval This review did not require ethical approval.
Conflicts of interest All authors have no conflicts of interest.
Acknowledgment Our studies mentioned in this review were partly supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 21890276 to D.K. and by a Grant-in-Aid from Strategic Research AGU-Platform Formation (2008-2012).
Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i
D. Kodama, A. Togari / Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i
Contents lists available at ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts Daisuke Kodama, Ph.D.n, Akifumi Togari, Ph.D. Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 18 January 2014 Received in revised form 4 March 2014 Accepted 22 March 2014
Background: In recent years, the role of the sympathetic nervous system in bone metabolism has been revealed. Many studies have suggested that bone loss can be induced by continuously high sympathetic tone resulting from up-regulation of osteoclastogenesis and osteoclastic activity via β2-adrenergic receptors. Although the expression of α-adrenergic receptors in osteoblasts and osteoclasts has been demonstrated, the physiological roles of these receptors in bone metabolism remain unclear. In the present review, we provide an account of the role of α1-adrenergic receptors in bone metabolism. Conclusion: Experimental studies in osteoblasts suggest that not only the suppression of β2-adrenergic receptors but also the activation of α1-adrenergic receptors could lead to a treatment for osteoporosis. Studying the signaling pathway will help elucidate the mechanism underlying the regulation of bone metabolism via the α1-adrenergic receptor and also facilitate the development of a novel therapeutic strategy for osteoporosis. & 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Alpha1-adrenergic receptor Osteoblast Sympathetic nervous system Bone metabolism
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Effects of α1-adrenergic agonists on the physiology of osteoblasts . . . . 3. Signaling pathways involved in the effects of α1-adrenergic receptors 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction In recent years, neural regulation of bone metabolism mediated by osteoblasts and osteoclasts has been demonstrated [1–4].
Abbreviations: ALP, alkaline phosphatase; BrdU, 5-bromo-20 -deoxyuridine; HOS, human osteosarcoma-derived cells; MC3T3-E1, mouse calvaria-derived osteoblastic cells; MG-63, human osteosarcoma-derived cells; OPG, osteoprotegerin; PI-PLC, phosphoinositide-phospholipase C; Pit-1, sodium-dependent inorganic phosphate transporter; PKA, protein kinase A; PLC, phospholipase C; RANK, receptor activator of NFκB; RANKL, receptor activator of NFκB ligand; RAW264.7, mouse macrophagelike cells; SaM-1, human periosteum-derived osteoblastic cells; SaOS, human osteosarcoma-derived cells; WST, water-soluble tetrazolium n Corresponding author. Tel.: þ 81 52 751 2561; fax: þ 81 52 752 5988. E-mail addresses: [email protected] (D. Kodama), [email protected] (A. Togari).
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Previous studies showed that mRNAs of α- and β-adrenergic receptors are expressed in osteoblasts and osteoclasts [5,6]. Additionally, these cells also express neurotrophins and axon guidance molecules for growing nerve fibers [7]. In immunohistochemical studies, bones were shown to be widely innervated by sympathetic nerves [8,9]. The presence of peripheral nerve axons coursing through the marrow adjacent to osteoblasts in bone tissue was shown by electron microscopy [10]. Direct nerve-osteoblastic and nerve-osteoclastic cell communication has been demonstrated using an in vitro co-culture model comprising sympathetic nerve cells derived from the mouse superior cervical ganglion and MC3T3E1 cells, mouse calvaria-derived osteoblastic cells, or osteoclasts induced from RAW264.7 cells [11,12]. These findings suggest that the sympathetic nervous system plays a direct role in the regulation of bone metabolism.
http://dx.doi.org/10.1016/j.job.2014.03.004 1349-0079/& 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i
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D. Kodama, A. Togari / Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Endogenous agonists of adrenergic receptors such as noradrenaline and adrenaline stimulate both α- and β-adrenergic receptors. It is well known that α1- and β1-adrenergic receptors have a similar sensitivity to these agonists, but the β2-adrenergic receptor is less sensitive to noradrenaline than to adrenaline. In experimental studies, bone loss was induced by continuously high sympathetic tone, and this induction was reversed by β-adrenergic receptor blockade [13–15]. Many studies have suggested that up-regulation of osteoclastogenesis and osteoclastic activity via β2-adrenergic receptors enhances bone resorption [3,16,17]. Additionally, suppression of bone formation by β-adrenergic receptor activity has also been reported [15,17]. Furthermore, it has been reported that the use of β-blockers reduces the risk of bone fracture and increases bone mineral density [18–20]. The physiological role of the β-adrenergic receptor in bone metabolism has been demonstrated by many studies, whereas that of the α-adrenergic receptor has been less well studied. However, clinical studies have also reported that blockade of the α1-adrenergic receptor increased the risk of hip/femur fracture, and that hypertensive patients treated with α1-blocker showed an increased risk of osteoporosis [20,21]. These studies suggest that the α-adrenergic receptor also plays a role in sympathetic effects on bone metabolism. In the present review, we summarize the physiological role and signal transduction pathway of the α1-adrenegic receptor in osteoblasts based on our recent findings.
2. Effects of α1-adrenergic agonists on the physiology of osteoblasts In our study, mRNAs of the α1B-, α2B-, and β2-adrenergic receptors were found to be expressed in SaM-1 cells, which are human periosteum-derived osteoblasts, and in the SaOS, HOS, and MG-63 human osteosarcoma-derived cell lines [5,6]. Huang et al. [22] also demonstrated the expression of α1B- and β2-adrenergic receptors using RT-PCR and Western blotting. Additionally, on immunofluorescence microscopy, these receptors were shown to localize to the cell surface of human osteoblasts. Suzuki et al. [23] demonstrated that adrenaline and phenylephrine, an α1-adrenergic receptor agonist, increased DNA synthesis in a concentration-dependent manner in MC3T3-E1 cells as determined by 3H-thymidine incorporation. The effect of adrenaline was suppressed in the presence of α1-adrenergic receptor antagonists. Similarly, Huang et al. [22] demonstrated that DNA synthesis was enhanced by cirazoline, an α1-adrenergic receptor agonist, but inhibited by fenoterol, a β2-adrenergic receptor agonist, in human osteoblasts. We evaluated the effect of noradrenaline on cell proliferation activity according to DNA synthesis as determined by 5-bromo-20 -deoxyuridine (BrdU) incorporation, and we also evaluated the number of live cells according to dehydrogenase activity as determined by the water-soluble tetrazolium (WST) assay. In SaM-1 cells, noradrenaline increased BrdU incorporation at submicromolar concentrations and suppressed it at higher-than-micromolar concentrations. In contrast, noradrenaline only increased formazan formation in the WST assay, over the whole concentration range that we tested. In the presence of prazosin, an α1-adrenergic receptor antagonist, noradrenaline showed only suppressive effect on cell proliferation in both the BrdU assay and the WST assay. In contrast, in the presence of propranolol, a β-adrenergic receptor antagonist, the effect of noradrenaline was facilitatory in both assays [24]. These results suggest that cell proliferation is enhanced by α1-adrenergic receptors and inhibited by β-adrenergic receptors in human osteoblasts. Treatment with adrenaline also increased alkaline phosphatase (ALP) activity and sodium-dependent inorganic phosphate transporter (Pit-1) expression through the α1-adrenergic receptor in
MC3T3-E1 cells [25]. ALP reduces extracellular pyrophosphate, a potent inhibitor of calcification, and Pi is taken up by Pit-1. These molecules play important roles in the initial events of bone matrix calcification. These findings suggest that α1-adrenergic receptor activation enhances osteoblast-mediated bone formation. It is well known that the binding of receptor activator of nuclear factor kappa-B ligand (RANKL) to its receptor RANK is an essential signal for osteoclastogenesis. In MC3T3-E1 cells, the expression of RANKL and its decoy receptor, osteoprotegerin (OPG), was increased by adrenaline via the β-adrenergic receptor and α1-adrenergic receptor, respectively [26]. It has also been reported that the effects of α1-adrenergic receptor agonists on OPG expression were eliminated by the knockdown of the α1B-adrenergic receptor in human osteoblasts [22]. These results suggest that osteoclastogenesis is suppressed by α1-adrenergic receptor agonists. In summary, these studies suggest that bone metabolism is positively regulated by the α1-adrenergic receptor expressed in osteoblasts.
3. Signaling pathways involved in the effects of α1-adrenergic receptors α1-adrenergic receptors belong to the G-protein-coupled receptor family. In general, activation of α1-adrenergic receptors increases the intracellular Ca2 þ concentration ([Ca2 þ ]i) by inducing Ca2 þ release from the endoplasmic reticulum via the Gq/phosphoinositide-phospholipase C (Gq/PI-PLC) pathway. However, recent studies have demonstrated that Ca2 þ influx through Ca2 þ -permeable channels and the Na þ /Ca2 þ exchanger is involved in α1-adrenergic receptormediated Ca2 þ elevation. The molecular components underlying Ca2 þ influx and their importance in Ca2 þ signaling differ among tissues. In our study, noradrenaline induced [Ca2 þ ]i elevation via the α1-adrenergic receptor in MC3T3-E1 cells and SaM-1 cells. The effect of noradrenaline was suppressed not only by pretreatment with a PLC inhibitor, U73122, but also by removing extracellular Ca2 þ . The effect of noradrenaline was completely abolished in Ca2 þ -free extracellular solution. Therefore, Ca2 þ influx plays a predominant role in α1-adrenergic receptor-mediated Ca2 þ signaling in SaM-1 cells. Additionally, noradrenaline-induced [Ca2 þ ]i elevation was inhibited by pretreatment with either thapsigargin or storeoperated Ca2 þ channel inhibitors. These results suggest that activation of Gq-protein-coupled α1-adrenergic receptors induces [Ca2 þ ]i elevation mainly via store-operated Ca2 þ channels in human osteoblasts [27]. We have also demonstrated that noradrenaline reduces the whole-cell current in patch-clamp recordings from SaM-1 cells. The inhibitory effect of noradrenaline on the whole-cell current was eliminated by chloroethylclonidine, an α1B-adrenergic receptor-selective inhibitor, and CsCl, a potassium channel blocker. These results suggest that activation of the α1B-adrenergic receptor also suppresses potassium channels [28]. However, the inhibitory effect of noradrenaline on the whole-cell current was not affected by pretreatment with U73122 or by chelation of intracellular Ca2 þ . These results suggest that noradrenaline-induced activation of store-operated Ca2 þ channels and inhibition of potassium channels are individual effects. Then, we examined whether the Gi/o-protein is involved in the inhibitory effect of noradrenaline on the whole-cell current. Upon pretreatment with pertussis toxin, an inhibitor of the Gi/o-protein-coupled receptor, noradrenalineinduced inhibition of potassium channels was significantly suppressed. Pertussis toxin inhibits effects via the Giα-protein and also the Gβγ-protein. We next examined which pathways are involved in the inhibitory effect of noradrenaline on the wholecell current. Pretreatment with H89, a PKA inhibitor, attenuated
Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i
D. Kodama, A. Togari / Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Ca2+ NA
3
K+
α1B-AR
NA
α1B-AR
SOCCs
Gqα
AC
β γ
Giα
β γ
ATP cAMP
PLC IP3
IP3R STIM
PKA
membrane depolarization
DG Ca2+ Cell proliferation
Fig. 1. Schematic diagram for the signaling pathway of Gq-protein-coupled and Gi/o-protein-coupled α1B-adrenergic receptors in osteoblasts. It is suggested that α1B-adrenergic receptors can be coupled to both the Gq-protein and Gi/o-protein in osteoblasts. Activation of the Gq-protein-coupled α1B-adrenergic receptor induces Ca2 þ release from the endoplasmic reticulum via the Gq/PI-PLC pathway. Reduction of the Ca2 þ concentration in endoplasmic reticulum sensed by STIM induces the activation of store-operated Ca2 þ channels. Ca2 þ influx from store-operated Ca2 þ channels appears to be the dominant pathway of noradrenaline-induced Ca2 þ elevation in human osteoblasts. In contrast, activation of the Gi/o-protein-coupled α1B-adrenergic receptor in osteoblasts suppresses potassium currents, mainly via the Gβγ-protein and partly via inhibition of the PKA pathway. This suppression of potassium channels results in membrane depolarization, which enhances cell proliferation activity. α1B AR: α1B-adrenergic receptor, AC: adenylate cyclase, DG: diacylglycerol, IP3: inositol 1,4,5-triphosphate, NA: noradrenaline, PKA: protein kinase A, PLC: phospholipase C, SOCC: store-operated Ca2 þ channel, and STIM: stromal interaction molecule.
the effect of noradrenaline on the whole-cell current, but not significantly. In contrast, pretreatment with gallein, a Gβγ-protein inhibitor, significantly suppressed the inhibitory effect of noradrenaline on the whole-cell current. Therefore, it is suggested that noradrenaline-induced inhibition of potassium channels occurs mainly via the Gβγ protein pathway of the Gi/o-coupled α1B-adrenergic receptor [24]. Additionally, the positive effects of noradrenaline on cell proliferation were significantly inhibited by CsCl, pertussis toxin, gallein, and H89, but not by U73122, in both the BrdU assay and the WST assay. Several studies have demonstrated that membrane potential and cell proliferation capacity are regulated by the activity of potassium channels in several cell types including osteoblasts. These results suggest that noradrenaline enhances cell proliferation by inhibiting Cs-sensitive potassium channels via the Gi/o-protein-coupled α1Badrenergic receptor in human osteoblasts [24].
α1-adrenergic receptors could lead to a treatment for osteoporosis. We have demonstrated that noradrenaline increases cell proliferation activity via the α1-adrenergic receptor at low concentrations and suppresses cell proliferations via the β-adrenergic receptor at high concentrations [24]. This biphasic effect is thought to result from the difference in the affinity of noradrenaline for these receptors. These results suggest that β-blockers may be effective against bone loss with a pathologically high sympathetic tone, whereas α1-adrenergic receptor agonists may be effective against dysregulation of bone metabolism with low sympathetic tone. In general, agonists of α1-adrenergic receptors also affect blood vessels. It is likely that reduction of the blood flow to bone downregulates bone metabolism. Therefore, osteoblast-selective effects may be preferable for the treatment of osteoporosis. Studying the associated signaling pathway will help elucidate the mechanism underlying the regulation of bone metabolism via the α1-adrenergic receptor and also facilitate the development of a novel therapeutic strategy for osteoporosis.
4. Conclusion Recent studies have demonstrated that a single subtype of receptor can be associated with different types of heterotrimeric G-proteins [29–31]. It is also suggested that coupling with a particular type of G-protein can be regulated by the location, phosphorylation, and expression of G-protein-coupled receptors [32–34]. Our studies suggest that α1B-adrenergic receptors can be coupled to both the Gq-protein and Gi/o-protein in human osteoblasts. By activating the Gq-protein-coupled α1B-adrenergic receptor, noradrenaline induces Ca2 þ influx through store-operated Ca2 þ channels via the Gq/PI-PLC pathway. In contrast, activation of the Gi/o-protein-coupled α1B-adrenergic receptor suppresses potassium channels, and cell proliferation activity is enhanced mainly by the inhibition of potassium channels (Fig. 1). Although further studies are needed to identify the channels activated and suppressed by noradrenaline, these studies suggest that the α1B-adrenergic receptor plays multiple roles in the physiology of osteoblasts. Experimental studies in osteoblasts suggest that not only the suppression of β2-adrenergic receptors but also the activation of
Ethical approval This review did not require ethical approval.
Conflicts of interest All authors have no conflicts of interest.
Acknowledgment Our studies mentioned in this review were partly supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 21890276 to D.K. and by a Grant-in-Aid from Strategic Research AGU-Platform Formation (2008-2012).
Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i
D. Kodama, A. Togari / Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Please cite this article as: Kodama D, Togari A. Signaling pathway and physiological role of the alpha-1 adrenergic receptor in human osteoblasts. J Oral Biosci (2014), http://dx.doi.org/10.1016/j.job.2014.03.004i