Unloading Induces Osteoblastic Cell Suppression and Osteoclastic Cell Activation to Lead to Bone Loss via Sympathetic Nervous System
Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts. Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts. Osteoporosis is one of the major age-related diseases in our modern world (1Riggs B.L. Hartmann L.C. N. Engl. J. Med. 2003; 348: 618-629Crossref PubMed Scopus (829) Google Scholar, 2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Riggs B.L. J. Cell. Biochem. 2003; 88: 209-215Crossref PubMed Scopus (40) Google Scholar, 4Riggs B.L. J. Cell. Biochem. 2003; 88: 209-215Crossref PubMed Scopus (43) Google Scholar). Especially, high fracture risk in osteoporosis patients results in not only loss of quality of life but also loss of life in a certain fraction of aged patients. The number of osteoporosis patients is estimated to be close to 10% of the whole population in many advanced countries. Among this patient population, a significant number of patients have disuse osteoporosis based on bedridden conditions caused by aging-related cardiovasucular as well as cerebrovascular diseases (5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar). Bone has been known to be lost upon the removal of the mechanical stimuli, and prolonged lack of mechanical stimuli leads to disuse osteoporosis (5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar, 7Bikle D.D. Sakata T. Halloran B.P. Gravit. Space Biol. Bull. 2003; 16: 45-54PubMed Google Scholar, 8Bikle D.D. Halloran B.P. J. Bone Miner. Res. 1991; 6: 527-530PubMed Google Scholar, 9Serhan C.N. N. Engl. J. Med. 2004; 350: 1902-1903Crossref PubMed Scopus (6) Google Scholar). However, the mechanisms underlying such disuse osteoporosis are largely unknown (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). Bone mass is determined by the actions of osteoblasts, which make bone, and those of osteoclasts, which resorb bone (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Augat P. Simon U. Liedert A. Claes L. Osteoporosis Int. 2005; 16: S36-S43Crossref PubMed Scopus (220) Google Scholar, 13Strewler G.J. N. Engl. J. Med. 2004; 350: 1172-1174Crossref PubMed Scopus (38) Google Scholar, 14Mohamed A.M. N. Engl. J. Med. 2003; 349: 1671Crossref PubMed Scopus (3) Google Scholar, 15Fuller K.E. N. Engl. J. Med. 2004; 350: 189-192Crossref PubMed Scopus (18) Google Scholar). The balance between the two activities is under the control of hormones and cytokines. Usually, simultaneous changes in the two activities are considered to be coupled to compensate bone loss. However, in the case of disuse osteoporosis, bone formation activities are not enhanced even in the presence of enhanced bone resorption. Rather, bone formation is significantly suppressed in disuse osteoporosis (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, 17Harada S. Rodan G.A. Nature. 2003; 423: 349-355Crossref PubMed Scopus (1139) Google Scholar). Therefore, disuse osteoporosis is a critical pathological situation where bone mass is continuously lost without having any compensatory activity against the reduction of bone. However, how such a critical reduction in bone formation occurs in unloading-induced pathological bone loss is not yet known. Bone formation and bone resorption are under the control of the systemic hormones and local cytokines (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). However, none of these factors have been proven to be the major cause of the disuse osteoporosis. In addition to the bedridden patients, astronauts under gravity conditions also lose bone due to the loss of mechanical stress. Analysis of such astronauts returning from space indicated that sympathetic nervous tone is enhanced in their muscle (18Fu Q Levine B.D. Pawelczyk J.A. Ertl A.C. Diedrich A. Cox J.F. Zuckerman J.H. Ray C.A. Smith M.L. Iwase S. Saito M. Sugiyama Y. Mano T. Zhang R. Iwasaki K. Lane L.D. Buckey Jr., J.C. Cooke W.H. Robertson R.M. Baisch F.J. Blomqvist C.G. Eckberg D.L. Robertson D. Biaggioni I. J. Physiol. 2002; 544: 653-664Crossref PubMed Scopus (76) Google Scholar, 19Cox J.F. Tahvanainen K.U. Kuusela T.A. Levine B.D. Cooke W.H. Mano T. Iwase S. Saito M. Sugiyama Y. Ertl A.C. Biaggioni I. Diedrich A. Robertson R.M. Zuckerman J.H. Lane L.D Ray C.A White R.J. Pawelczyk J.A. Buckey Jr., J.C. Baisch F.J. Blomqvist C.G. Robertson D. Eckberg D.L. J. Physiol. 2002; 538: 309-320Crossref PubMed Scopus (73) Google Scholar). Sympathetic nervous system would regulate bone mass via bone formation by osteoblasts systemically (20Takeda S. Elefteriou F. Levasseur R. Liu X. Zhao L. Parker K.L. Armstrong D. Ducy P. Karsenty G. Cell. 2002; 111: 305-317Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). However, nothing has been known about how this system is related to pathophysiology in bone metabolism in the body. Therefore, we examined whether sympathetic nervous tone is involved in reduction of bone mass in a disuse osteoporosis model via osteoblastic and osteoclastic cells using hind limb unloading. Animals—Male 129 or C57BL/6J mice (10–14 weeks old) were used for the experiments. Mice were housed for at least 1 week prior to the study. The mice were subjected to either intraperitoneal injections of propranolol (20 μg/g of body weight/day) (21Commins S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), continuous administration of guanethidine via an osmotic minipump (20 μg/g of body weight/day) (22Sherman B.E. Chole R.A. J. Bone Miner. Res. 2000; 15: 1354-1360Crossref PubMed Scopus (19) Google Scholar), or intraperitoneal injections of isoproterenol (6 μg/g of body weight/day) (23Takeda S. Elefteriou F. Levasseur R. Liu X. Zhao L. Parker K.L. Armstrong D. Ducy P. Karsenty G. Cell. 2002; 111: 305-317Abstract Full Text Full Text PDF PubMed Scopus (1373) Google Scholar). In the case of osmotic minipump implantation, the pump was implanted 12 h before the start of hind limb unloading. Osmotic minipumps were implanted into the subcutaneous tissue in the back of the animals according to the manufacturer's instruction. For dopamine β-hydroxylase (DBH) 1The abbreviations used are: DBH, dopamine β-hydroxylase; BV/TV, bone volume/tissue volume; BFR, bone formation rate; TRAP, tartrate-resistant acid phosphatase; MAR, bone mineral apposition rate; MS, mineralizing surface; CT, computerized tomography. gene deletion experiments, heterozygous knockout mice with a C57BL6/129sv F2 background and wild type litter mate mice were used (13-week-old females) (24Thomas S.A. Matsumoto A.M. Palmiter R.D. Nature. 1995; 374: 643-646Crossref PubMed Scopus (468) Google Scholar). All of the mice were injected intraperitoneally with calcein at 4 mg/kg at 4 and 2 days before sacrifice. After treatment for 10 or 14 days, mice were anesthetized with tribromoethanol at 200 mg/kg and were sacrificed by cervical dislocation. Hind Limb Unloading Model—Hind limb unloading was conducted by applying a tape to the surface of the hind limb to set a metal clip (10Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. J. Bone Miner. Res. 2002; 17: 661-667Crossref PubMed Scopus (88) Google Scholar, 16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar). The of the clip was to an The of the was to the mice at an with the hind the of the The mice were subjected to hind limb unloading for 10 or 14 control mice were also housed under the conditions for hind limb unloading for the body of the mice was the were no significant changes in body in any of the the of the study. that could be considered in our as (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Analysis of volume/tissue was determined based on using a The were by using system The bone was in an of with and at and to the of the of the The for the was set at for the (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Analysis of the of the experiments, the of were and in were as For bone formation bone in the was used to bone fraction in a of with and at and to the For the of the mice were at the of the and in and in were using a and for tartrate-resistant acid cells to bone were as osteoclasts. were an of with and at and to the of the of the was conducted to the number of and osteoclast surface as by A.M. J.A. H. P.J. J. Bone Miner. Res. PubMed Scopus Google Scholar). the cells from the bone marrow of the animals that were subjected to hind limb unloading P. Simon U. Liedert A. Claes L. Osteoporosis Int. 2005; 16: S36-S43Crossref PubMed Scopus (220) Google or in with the treatment with The cells were in the presence of acid and in with 10% After weeks in was was used to the in the cultures were the cells were in and were in for 1 The cultures were with The of was by using an levels in at the of the hind limb unloading were by L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). were from mice which were housed in a the h and were as and was based on of using a for was considered to be Hind limb unloading reduced bone in control mice as (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google a and 1 In treatment with propranolol, a at the suppressed hind limb unloading-induced reduction in bone mass as a and no significant between and to the in mice, propranolol treatment the bone loss by unloading 2 mice with propranolol significant in bone mass with mice 2 hind limb unloading suppressed the levels of bone mass and bone formation through sympathetic not only the actions of the for sympathetic which at the but also the of the the unloading-induced reduction in bone mass. Therefore, guanethidine was to at the levels in the animals that were subjected to hind limb unloading. in guanethidine treatment suppressed unloading-induced bone loss a and The of the levels in was to that in the case of propranolol was not significantly due to unloading or treatment in all of our treatment suppressed unloading-induced pathological bone loss. hind limb unloading of mice for 14 days, guanethidine treatment or was using an osmotic minipump The number of the mice used for the in is indicated as N. the hind limb unloading of 129 mice, guanethidine treatment was further whether unloading is in the of sympathetic we examined the of by isoproterenol of sympathetic tone. was in to whether isoproterenol the unloading-induced treatment reduced bone mass in mice as a and 1 and under this hind limb unloading no longer reduced bone mass a and and the levels of bone mass reduction by isoproterenol treatment were to those in mice the of these of are in with the that unloading is suppressed by for sympathetic and such is no longer in the presence of the of In to the mechanisms of actions in unloading-induced bone we conducted Hind limb unloading reduction in the levels of bone mineral apposition mineralizing surface and 10 days of hind limb unloading and propranolol treatment suppressed the unloading-induced in MAR, MS, and The of the were to those in the case of bone mass treatment also suppressed the hind limb unloading-induced reduction in MAR, MS, and in the mice subjected to hind limb unloading for 2 the of the were to those in the case of bone mass of sympathetic has to the of with to bone formation activity in vivo. treatment suppressed the levels of mineral apposition rate, mineralizing surface, and bone formation rate, in the presence of isoproterenol 10 days of hind limb unloading to all of these parameters is that the of the in were to those in the of isoproterenol on bone mass These of based on indicated that bone formation is the of sympathetic tone in mice subjected to hind limb treatment suppressed unloading-induced reduction in bone formation in vivo. Mice were with guanethidine as in the to The number of mice in is indicated as N. calcein of the at the of the hind limb unloading or in or The the of calcein used to in in the of the of the and were in all at to the in the as under and The mice were injected intraperitoneally with calcein at 4 mg/kg 4 and 2 days before at 2 are as and for from of the and mice significant significant between the and or significant between and treatment suppressed the of bone formation and unloading not further these parameters in vivo. Mice were with isoproterenol as in the to The number of mice in is indicated as N. calcein of the at the of the hind limb unloading or in or The the of calcein used to in in the of the of the and were in all at to the in the as under and The mice were injected intraperitoneally with calcein at 4 mg/kg 4 and 2 days before at 10 are as and for from of the and significant significant between the and or further examined whether our observations be at cell levels in For this a nodule formation was conducted by using bone marrow cells from the bone of the mice were subjected to hind limb unloading or control in the presence or the of the treatment with Hind limb unloading reduced nodule formation in the cultures of cells from the animals a and 1 treatment in suppressed the unloading-induced reduction in the mineralized nodule formation in a and treatment suppressed the levels of nodule formation in loaded control mice a and 1 and in the presence of isoproterenol treatment in hind limb unloading to further the levels of nodule formation in bone marrow cells in a and these in indicated that sympathetic tone mediates unloading-induced reduction in of bone marrow cell treatment in suppressed the levels of nodule and unloading conditions to further the levels in Mice were with isoproterenol as in to The number of mice in is indicated as N. The cells from the bone marrow of the animals subjected to hind limb unloading with the isoproterenol or treatment were in the presence of acid and After was and the of the nodule was significant significant between the and or In to the of the sympathetic nervous tone on unloading-induced reduction in bone mass and bone formation in a model knockout mice were subjected to hind limb unloading. Hind limb unloading reduced bone mass by about in wild type mice 1 loss for the dopamine β-hydroxylase gene the reduction in bone loss by about hind limb unloading The of bone loss due to unloading as was significantly reduced from in to in The nodule formation in cultures of bone marrow cells of the wild type was reduced by unloading 1 The marrow cells from gene heterozygous knockout mice indicated suppression of hind limb unloading-induced reduction in nodule formation the of unloading-induced bone bone resorption also occurs as critical to bone mass. Unloading in mice caused an increase in osteoclast number and osteoclast surface based on in as 1 In of sympathetic tone by treatment with propranolol or suppressed such unloading-induced increase in osteoclast number a and for propranolol, for and osteoclast surface for propranolol, for The levels of and were enhanced by either unloading or isoproterenol treatment to levels and 2 The simultaneous presence of unloading conditions and isoproterenol treatment in levels in the increase in osteoclast number and surface to those in mice subjected to either one of the two conditions and 4 2 and unloading-induced increase in deoxypyridinoline into 1 was also suppressed by guanethidine treatment of sympathetic tone unloading-induced systemic bone loss by the levels of urinary deoxypyridinoline Mice were subjected to hind limb unloading and guanethidine treatment as under and After 14 days, of the mice were and the of deoxypyridinoline in the was as under and significant The number of mice in is indicated as N. significant between the and or significant between and that sympathetic nervous tone is unloading-induced bone loss via reduction in osteoblastic cell activity as well as enhancement in osteoclastic cell is the that sympathetic control of the bone mass is involved in the unloading-induced bone loss by Unloading-induced bone loss was suppressed by the treatment of the animals with propranolol, a that in the sympathetic nervous system are the of unloading-induced bone loss. The sympathetic nervous from the nervous which as a into the that unloading-induced bone loss was suppressed by the treatment of the animals with These that the of in the of the could the unloading-induced osteoporosis. unloading to further the bone that was reduced by a These of observations further that sympathetic nervous tone is involved in unloading-induced pathological bone loss. also examined the of heterozygous deletion of the is for the sympathetic nervous tone. Therefore, we subjected the heterozygous knockout mice to hind limb unloading. Unloading-induced bone loss was by the of the half-dosage of dopamine β-hydroxylase that the presence of a of gene in the animals is for the of the unloading-induced bone loss. the that be by due to the systemic and of sympathetic the that the sympathetic nervous system pathological loss of bone in unloading-induced Since bone formation is the critical activity to the levels of bone loss due to is the major to the mechanisms for unloading-induced loss of bone mass. on osteoblastic cell activity in that the reduction in bone formation activity in due to unloading was suppressed by a of including propranolol and bone cell using the bone marrow cells from the animals subjected to either or of the sympathetic nervous tone that these suppressed unloading-induced reduction in mineralized nodule formation. The of the reduction in the formation of osteoblastic bone weeks in would be that cell for osteoblastic cell could be reduced at the of the cells from animals at the of unloading. suppression was by the treatment with propranolol. also to bone in the presence or of isoproterenol or propranolol and guanethidine in using bone marrow cells that were from wild type was no of these in to the nodule formation in the bone marrow cells from mice not These the that the population in bone marrow in would be reduced by the unloading or by treatment with such as propranolol, and isoproterenol the of the experiments. Therefore, the of sympathetic nervous tone unloading-induced bone loss cell on osteoblastic cell is known that propranolol at of 10 μg/g body could in is also known that such could be enhanced by the treatment with However, has yet been no in or in of a between and bone mass. However, at this we fully the that propranolol or isoproterenol used in our have bone mass via of and these have to be in the is to the in the of the has been known to in days, isoproterenol and guanethidine is not known whether such in their on also their of the bone mass. However, propranolol and guanethidine unloading-induced bone loss in our experiments. be due to the of bone where the bone could be in a with these of could be proven to be in the treatment of unloading-induced osteoporosis, has to be considered that in the treatment of the patients. In the we have to certain of the by which bone be without the or these be used only for those patients could be to be based on the indicated that sympathetic regulate unloading-induced enhancement in bone resorption. was by the observations that of sympathetic tone by propranolol and suppressed unloading-induced bone and this leads to suppression in bone loss. indicated that unloading bone resorption through the increase in osteoclast number and osteoclast These in the bone resorption parameters due to unloading were suppressed by treatment with propranolol and observations the for sympathetic tone on unloading-induced bone resorption were not to bone, suppression of sympathetic tone by the treatment with guanethidine also suppressed unloading-induced increase in deoxypyridinoline into which is a systemic bone resorption These that sympathetic tone unloading-induced bone resorption as of sympathetic tone in the of bone resorption unloading was further by the on the mice subjected to simultaneous unloading and isoproterenol isoproterenol treatment or unloading could cause an increase in the levels of bone loss as well as an increase in the levels of bone resorption parameters number and osteoclast The simultaneous presence of unloading conditions and isoproterenol treatment in an increase in bone loss as well as an increase in osteoclast number and osteoclast surface to levels to those in the of either one of the two conditions These further the that sympathetic nervous tone and the unloading would Unloading bone loss and fracture risk in bedridden patients. In deoxypyridinoline into was to increase in astronauts a are to conditions in bone loss is caused by an of osteoclastic activity the body is subjected to unloading this is in patients and and the for such a of bone activity has not been Since the nervous system could at a our of the sympathetic tone as for the unloading-induced increase in bone resorption and loss of bone mass would the of to unloading. we have used knockout mice to the of such a deletion of the gene on the bone loss due to this is also to in the as well as The to from has been in to be subjected to by stress. a the heterozygous mice results not of a for the sympathetic nervous system However, our on the of unloading on bone in knockout mice is at least in in with the that sympathetic tone is involved in the bone loss due to unloading. In our that sympathetic tone is in of the pathological reduction in bone mass upon unloading by osteoblastic cell actions and osteoclastic These that of the of systemic in the bone loss in unloading conditions could a to to patients with disuse osteoporosis C.N. N. Engl. J. Med. 2004; 350: 1902-1903Crossref PubMed Scopus (6) Google Scholar).