Noradrenaline stimulates cell proliferation by suppressing potassium channels via G_i/o‐protein‐coupled α_1B‐adrenoceptors in human osteoblasts

Abstract

BACKGROUND AND PURPOSERecent studies demonstrated that the sympathetic nervous system regulates bone metabolism via b2-adrenoceptors. Although a-adrenoceptors are also expressed in osteogenic cells, their functions in bone metabolism have been less studied. We previously demonstrated that noradrenaline suppressed potassium currents via a1B-adrenoceptors in the human osteoblast SaM-1 cell line. The aim of this study was to investigate the signal transduction pathway and the physiological role of noradrenaline in human osteoblasts in more detail. EXPERIMENTAL APPROACHTo investigate signal transduction through a1B-adrenoceptors, we used whole-cell patch clamp recording and Ca fluorescence imaging. Potassium channels regulate membrane potential and cell proliferation activity in non-excitable cells, so we evaluated cell proliferation activity by BrdU incorporation and WST assay. KEY RESULTSIn SaM-1 cells, bath-applied noradrenaline elevated intracellular Ca 2+ concentration and this effect was abolished by both chloroethylclonidine, an a1B-adrenoceptor antagonist, and U73122, a PLC inhibitor. However, the inhibitory effect of noradrenaline on whole-cell current was unaffected by U73122. In contrast, in cells pretreated with either Pertussis toxin, a Gi/o-protein-coupled receptor inhibitor, or gallein, a Gbg-protein inhibitor, the inhibitory effect of noradrenaline on whole-cell current was significantly suppressed. Noradrenaline-induced enhancement of cell proliferation was inhibited by CsCl, a non-selective potassium channel blocker, gallein and H89, a PKA inhibitor, but not by U73122. CONCLUSIONS AND IMPLICATIONSNoradrenaline facilitated cell proliferation by regulation of potassium currents in human osteoblasts via Gi/o-protein-coupled a1B-adrenoceptors, not via coupling to Gq-proteins. Abbreviationsa-MEM, a-modified minimum essential medium; BK channel, large-conductance calcium-activated potassium channel; BrdU, 5-bromo-2′

α1B‐Adrenoceptor signalling regulates bone formation through the up‐regulation of CCAAT/enhancer‐binding protein δ expression in osteoblasts

“…The physiological action of adrenaline (epinephrine) was previously demonstrated to be mediated by α 1 ‐ as well as ß‐ adrenoceptor signalling in osteoblasts. Our previous studies also showed that cellular proliferation was facilitated via α 1B ‐ adrenoceptors and inhibited via ß‐ adrenoceptors in human osteoblastic SaM‐1 cells (Kodama & Togari, ). In the present study, phenylephrine stimulated chloroethylclonidine, cellular proliferation on BMSCs (Supporting Information Fig.…”

“…α 1 ‐Adrenoceptors were previously shown to have an important role in regulating the functionality of osteoblasts (Suzuki et al, ; Huang et al, ; Hirai et al, ). We also reported direct nerve‐osteoblast communication in vitro and demonstrated that NA increased cell proliferation by suppressing K + channels via Gi/o‐coupled α 1B ‐adrenoceptors in human osteoblasts (Obata et al, ; Kodama & Togari, ); however, the physiological role of α 1 ‐adrenoceptor signalling in bone in vivo remains unclear. Thus, to obtain a deeper understanding of bone remodelling by the sympathetic nervous system, we investigated the role of α 1 ‐adrenoceptor signalling in bone metabolism.…”

“…To investigate the physiological function of α 1B ‐adrenoceptor signalling in osteoblasts, we determined whether Cebpd contributed to the regulation of cellular function in osteoblasts. Based on previous findings in which α 1 ‐adrenoceptor signalling in osteoblasts mediated cellular proliferation (Suzuki et al, ; Kodama & Togari, ), we investigated whether Cebpd in osteoblasts contributed to cellular proliferation. Cebpd was silenced in MC3T3‐E1 cells, and cell proliferation activity was evaluated as DNA synthesis by the BrdU incorporation assay.…”

“…Cell proliferation activity was assessed by BrdU incorporation using the Cell Proliferation ELISA BrdU kit (Roche Applied Science, http://www.google.com.ph/search?hl=en-PH&gbv=2&q=penzberg+germany&stick=H4sIAAAAAAAAAOPgE-LSz9U3MC5MTjMxVuIEsU0sS9LStLSyk63084vSE_MyqxJLMvPzUDhWGamJKYWliUUlqUXFALJNBAVFAAAA&sa=X&ved=0ahUKEwiQ-dTH56XKAhXBGZQKHVTkAj8QmxMIcigAMA4) according to the manufacturer's instructions (Kodama & Togari, ). MC3T3‐E1 cells were grown in α‐MEM supplemented with 10% FBS and 1% penicillin/streptomycin to ~70% confluency, followed by transfection with either siRNA targeting Cebpd or non‐silencing RNA diluted in Opti‐MEM using Lipofectamine RNAiMAX ( Invitrogen ) according to the manufacturer's protocol.…”

Sympathetic Denervation-Induced MSC Mobilization in Distraction Osteogenesis Associates with Inhibition of MSC Migration and Osteogenesis by Norepinephrine/adrb3

“…It has been shown that norepinephrine (NE), a major mediator of the sympathetic nervous system, influences osteoblasts both in culture and in vivo [19]–[22]. NE has also been demonstrated to stimulate osteoclast formation, thereby enhancing bone resorption [19].…”