Michitsugu Arai

Expression of mRNAs for neuropeptide receptors and β-adrenergic receptors in human osteoblasts and human osteogenic sarcoma cells

In human periosteum-derived osteoblastic cells (SaM-1) and human osteosarcoma-derived cells (SaOS-2, HOS, MG-63), the mRNA expressions of calcitonin gene-related peptide receptor (CGRP-R), substance P receptor (SP-R), neuropeptide Y receptor (NPY-R), beta-adrenergic receptors (beta1-R, beta2-R, beta3-R), vasoactive intestinal polypeptide type 1 and type 2 receptors (VIP-1R, VIP-2R) and pituitary adenylate cyclase activating polypeptide receptor (PACAP-R) were examined by reverse transcription-polymerase chain reaction (RT-PCR). According to the magnitude of the mRNA expression of alkaline phosphatase (ALP), the relative state of commitment of these osteoblastic cell lines to the osteoblast lineage was SaM-1 > SaOS-2 > HOS > MG-63. CGRP-R, NPY-R, VIP-1R and beta2-R, but not SP-R, VIP-2R, PACAP-R, beta1-R and beta3-R, were expressed in osteoblasts as well as osteosarcoma cells. Expression of these receptors seems to be a common feature in osteoblastic cells, but the magnitude of expression was not dependent upon the relative state of commitment of the osteoblastic cells to the osteoblast lineage. In addition, VIP mRNA was not expressed in osteoblastic cells, suggesting the absence of an autocrine system of VIP in osteoblasts. These observations suggest that these neuropeptides and norepinephrine are involved in local regulation of human bone metabolism.

Adrenergic stimulation of osteoclastogenesis mediated by expression of osteoclast differentiation factor in MC3T3-E1 osteoblast-like cells

It is well known that adrenergic agonists efficiently activate beta-adrenoceptors on osteoblastic cells and can stimulate bone resorption in intact mouse calvaria. Recently, an osteoclastogenic factor of osteoblastic origin was found to be a novel tumor necrosis factor ligand family member and was termed osteoclast differentiation factor (ODF). Using a reverse transcription-polymerase chain reaction approach, we investigated the effect of epinephrine on mRNA levels of ODF and its decoy receptor, osteoclastogenesis inhibitory factor (OCIF), in MC3T3-E1 cells. Treatment with epinephrine (1 microM) rapidly increased ODF and OCIF mRNA levels, which peaked after 0.5 hr of treatment. Epinephrine (1 microM) also increased interleukin (IL)-6, IL-11, and cyclooxygenase (COX)-II mRNA levels, as well as increased prostaglandin E(2) (PGE(2)) accumulation in the culture medium. Treatment of the cells with IL-11 (10 ng/mL) or PGE(2) (1 microM) increased ODF and OCIF mRNA levels as observed with epinephrine. However, increases in ODF and OCIF mRNA levels by epinephrine were more rapid than those by IL-11, and were not influenced by NS-398 (100 microM; an inhibitor of COX-II), suggesting a direct effect of epinephrine on ODF and OCIF mRNA expressions as well as an indirect effect mediated by IL-11 and PGE(2) production. Epinephrine-induced increases in ODF and OCIF mRNA levels were inhibited by pretreatment with timolol (1 microM; beta-antagonist) and phentolamine (1 microM; alpha-antagonist), respectively. Furthermore, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells from mouse bone marrow cells was stimulated by isoproterenol (0.1 to 10 microM) or epinephrine (0.1 to 10 microM). The action of isoproterenol, a beta-agonist, was clearly stronger than that of epinephrine, suggesting the importance of the physiological balance between ODF and OCIF productions for osteoclastogenesis. These findings suggest that beta-adrenergic stimulation induces not only IL-6, IL-11, and PGE(2) but also ODF expression in osteoblastic cells, leading to a stimulation of osteoclastogenesis.

Effects of β-adrenergic agonists on bone-resorbing activity in human osteoclast-like cells

In the present study, we demonstrate for the first time that beta-adrenergic agonists stimulate bone-resorbing activity in human osteoclast-like multinucleated cells (MNCs). Osteoclast-like MNCs constitutively expressed mRNA for alpha1B-, alpha2B- and beta2-adrenergic receptor (AR) in addition to characteristic markers of mature osteoclast, such as calcitonin receptor (CT-R), tartrate-resistant acid phosphatase (TRAP), alphaV-chain of integrin (Int alphaV), carbonic anhydrase II (CA-II) and cathepsin K (Cathe K). Epinephrine (1 microM; alpha,beta-adrenergic agonist) up-regulated expression of Int alphaV, CA-II and Cathe K in the osteoclast-like MNCs. Osteoclastic resorbing activity was markedly increased by isoprenaline (1 microM; beta-adrenergic agonist), moderately by epinephrine, but poorly by phenylephrine (1 microM; alpha1-adrenergic agonist). The actin ring, which was suggested to be correlated with bone-resorbing activity, was clearly observed in osteoclast-like MNCs treated with isoprenaline and epinephrine, but faintly in those treated with phenylephrine. These findings suggest that beta-adrenergic agonists directly stimulate bone-resorbing activity in matured osteoclasts.

Effects of retinoic acid on bone formation and resorption in cultured mouse calvaria.

1. The effects of retinoids on bone metabolism were examined in newborn mouse calvaria. 2. Incubation of calvaria with 0.01-1 microM retinoic acid for 4 days decreased their alkaline phosphatase (ALP) activity, mineral content and collagen content in a concentration-dependent fashion. 3. With treatment for 2 days, retinoic acid (1 microM) decreased the ALP activity and collagen content, but not the mineral content. 4. All these inhibitory effects were observed in calvaria from 0-day-old mice, but no inhibition of ALP activity was observed in calvaria from 14-day-old mice. 5. 1-Hydroxyethylidene-1,1-bisphosphonate (HEBP, 1 mM), which inhibits bone resorption, prevented the effect of retinoic acid (1 microM) on the bone mineral content, but not the effects on ALP and collagen (synthesized by osteoblasts). HEBP (1 mM) alone had no effect on the calvarial mineral and collagen contents. 6. These findings indicate that retinoic acid both stimulates bone resorption and inhibits osteoblastic activity by different mechanisms, and that stimulation of bone resorption by retinoic acid is inhibited by HEBP.

The role of the sympathetic nervous system in controlling bone metabolism

Experimental studies have generally shown that increased sympathetic nervous activity causes bone loss via an increase in bone resorption and a decrease in bone formation. Increased bone resorption is based on the stimulation of both osteoclast formation and osteoclast activity. These effects are associated with β2-adrenergic activity towards both osteoblastic and osteoclastic cells. Decreased bone formation is based on the inhibition of osteoblastic activity through β2-adrenergic receptors on osteoblasts. Such findings indicate that β-blockers may be effective against osteoporosis, in which case there is increased sympathetic activity. In fact, in a population-based, case-control study, the current use of β-blockers has been demonstrated to be associated with a reduced risk of fractures. These clinical studies suggest that pharmacological blockade of the β-adrenergic system is beneficial to the human skeleton. In another prospective study, however, no association between β-blocker use and fracture risk was shown in perimenopausal and older women. To confirm this important new therapeutic avenue to prevent bone loss, the relationship between the pharmacological effectiveness of β-blockers and the pathogenesis of osteoporosis must be explored in detail.

Expression of mRNA for axon guidance molecules, such as semaphorin-III, netrins and neurotrophins, in human osteoblasts and osteoclasts

In the present study, we demonstrated the constitutive expression of diffusible axon guidance molecules such as neurotrophins, semaphorin-III, netrin-1, and netrin-2-like protein, which are known to function as a chemoattractant and/or chemorepellent for growing nerve fibers, in human osteoblastic and osteoclastic cells. The findings, obtained by RT-PCR, ELISA, and Western blot analysis suggest the extension of axons of peripheral sensory and sympathetic neurons to osteoblastic and osteoclastic cells and the possible neural regulation of bone metabolism in these osteogenic cells.

Effects of Propranolol on Bone Metabolism in Spontaneously Hypertensive Rats

The effects of propranolol (PRO), a nonselective β-adrenergic receptor (β-AR) antagonist with membrane-stabilizing action on bone metabolism, were examined in spontaneously hypertensive rats (SHR) showing osteoporosis with hyperactivity of the sympathetic nervous system. Treatment of SHR with PRO at 1 and 5 mg/kg p.o. for 12 weeks increased bone mass of the lumbar vertebra and proximal tibia without affecting blood pressure, but PRO at 50 and 100 mg/kg with hypotensive action did not increase bone mass. Next, the effects of PRO at 0.1, 1, and 10 mg/kg on bone status were examined in more detail. Compared with the SHR control, not only bone mass but also biomechanical parameters of strength and toughness of the lumbar vertebrae were increased in SHR treated with PRO at 0.1 and 1 mg/kg, suggesting antiosteoporotic action. PRO at 1 mg/kg statistically increased histomorphometry indices of bone formation, whereas PRO at doses of 0.1, 1, and 10 mg/kg decreased those of bone resorption. Antiosteoporotic effect of PRO is attenuated at 10 mg/kg compared with 0.1 and 1 mg/kg. In addition, treatment with timolol, a nonselective β-AR antagonist without membrane-stabilizing action, or butoxamine, a selective β2-AR antagonist, at 1 mg/kg increased bone mass in SHR. These results suggested that treatment of SHR with β-blockers at low dose improved bone loss and bone fragility. This antiosteoporotic effect of β-blockers seems to be caused by the blocking action of β2-AR, regardless of the membrane-stabilizing action.

Coexpression of GTP cyclohydrolase I and inducible nitric oxide synthase mRNAs in mouse osteoblastic cells activated by proinflammatory cytokines.

Proinflammatory cytokines, a combination of IL‐1β, TNF‐α, and IFN‐γ, caused mRNA expression of GTP cyclohydrolase I (GTP‐CH), the rate‐limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis, and of inducible nitric oxide synthase (iNOS) in a well‐characterized osteoblastic clone MC3T3‐E1 cell line. We found the expression of the GTP‐CH gene in osteoblasts for the first time. The expression of GTP‐CH and iNOS mRNAs was found to be maximal at 3 and 9 h, respectively. The expression of both genes elicited increases in BH4 and NO levels. Pharmacological studies using 2,4‐diamino‐6‐hydroxypyrimidine, an inhibitor of GTP‐CH activity, showed that BH4 is involved in the activity of iNOS, but not in the induction of iNOS mRNA. The results using an inhibitor of nuclear factor (NF)‐κB and activating protein‐1 (AP‐1) activation suggested that coinduction of the two genes in response to cytokines occurred via activation of NF‐κB and AP‐1. In MC3T3‐E1 cells BH4 and sepiapterin, producing BH4, could protect against apoptosis, i.e. the degradation of nuclear DNA in the cells, induced by NO derived from S‐nitroso‐N‐acetyl‐d,l‐penicillamine. These results suggest that the induction of BH4 together with NO by proinflammatory cytokines could protect against NO‐induced apoptosis in MC3T3‐E1 cells.

Effects of capsaicin-induced sensory denervation on bone metabolism in adult rats

Bone metabolism has recently been revealed to be under nerve regulation. In this study, the integrity of the sensory innervation contributing to bone metabolism was examined by capsaicin-induced sensory neuron lesions. Eight-week-old male Wistar strain rats in a modeling phase of skeletal growth were divided into four groups (8 rats per group) and treated with capsaicin at one of three different doses (37.5, 75, 150 mg/kg) or vehicle, subcutaneously. Five weeks later, high-dose (150 mg/kg) capsaicin treatment had reduced trabecular bone volume (BV/TV) due to increased trabecular separation (Tb.Sp) in the proximal tibia and the modification of mechanical properties such as strength, ductility, and toughness toward increasing bone fragility in the trunk of the sixth lumbar vertebrae (L6). Moderate-dose (75 mg/kg) capsaicin treatment had no significant effect on trabecular BV/TV or bone mechanical properties but increased Tb.Sp as seen high-dose capsaicin treatment. Bone histomorphometry showed osteoclast number (Oc.N/BS) and surface (Oc.S/BS) were increased in both the moderate-dose and high-dose capsaicin treatment groups. High-dose capsaicin significantly increased the level of tartrate-resistant acid phosphatase form 5b (TRAP 5b) in plasma, a systemic bone resorption marker, but had no influence on plasma osteocalcin concentration, a bone formation marker, suggesting that capsaicin-induced sensory nerve denervation increased bone resorption but had no influence on bone formation. Low-dose (37.5mg/kg) capsaicin had no influence on bone remodeling. These results suggest that sensory nerve innervation contributes to the maintenance of trabecular bone mass and its mechanical properties by inhibiting bone resorption.

Alteration of in vitro bone metabolism and tooth formation by zinc

1. The effects of zinc on bone metabolism and tooth formation was examined in organ cultures of calvaria and tooth germ, and in cell cultures of osteoblast-like cells, MC3T3-E1. 2. Treatment of calvaria with zinc (10, 100 microM) for 4 days both increased alkaline phosphatase (ALP) activity in bone and reduced the secretion of N-acetyl beta-glucosaminidase from bone, without affecting bone mineral or collagen content. The increase in ALP activity produced by zinc (10 microM) was inhibited neither by actinomycin D (5 micrograms/ml) nor by cycloheximide (0.5 micrograms/ml). 3. Treatment of MC3T3-E1 cells with zinc (50, 100 microM) for 25 days also increased ALP activity, but reduced calcium content in cells and in the matrix layer. 4. These results indicate that zinc increases ALP activity in osteoblasts without affecting de novo enzyme synthesis, and that it inhibits bone mineralization, in accordance with the inhibition of osteoclastic activity. 5. Treatment of tooth germ with zinc (100 microM) for 7 days also produced an increase in ALP activity and inhibition of mineralization. These results indicate that the increased ALP activity produced by zinc is a common phenomenon in hard tissues, and, further, that zinc inhibits mineralization during tissue formation.