Recent studies have shown that adiponectin and its receptors were detected not only in adipocytes but also in bone-forming cells [17–20]. However, little is known about the physiological action of adiponectin on osteoblasts. In this study, we demonstrated that osteoblastic MC3T3-E1 cells expressed AdipoR1, but not AdipoR2. We found that the suppression of AdipoR1 expression by its siRNA inhibited the differentiation and mineralization of the cells, and we suggested that adiponectin promoted these processes through the action of AdipoR1 on the osteoblasts. We also found that adiponectin activated AMP kinase in the cells, and that AICAR, a pharmacological AMP kinase activator [21, 22], promoted the differentiation and mineralization of the cells. Both adiponectin and AICAR also stimulated the proliferation of the cells. Taken together, adiponectin seemed to promote the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells possibly via the AdipoR1 and AMP kinase signaling pathway. Recent studies have also shown that adiponectin promotes osteoblast proliferation [17, 19] and exerts an enhancing action on human osteoblast differentiation and mineralization . Although we did not carry out experiments using the inhibitor or siRNA of AMP kinase, our results seem to be consistent with those reported in these studies. Further, we disclosed the involvement of the AMP kinase signaling pathway in the stimulatory actions of adiponectin on osteoblasts.
Luo et al. have reported that the adiponectin concentration in fetal bovine serum (FBS) was approximately 60 μg/ml, which was about six-fold higher than that in human serum. They have shown that the addition of FBS into the medium actually enhances the ALP activity of primary human osteoblasts; however, the addition of adiponectin-free FBS does not enhance the activity . Therefore, the influence of adiponectin, which is present in high concentrations in FBS, must be ignored when the differentiation and mineralization of osteoblasts in long cultures are examined. In this study, we knocked down the expression of AdipoR1 by transfecting its siRNA and investigated how the lack of adiponectin action in the microenvironment of MC3T3-E1 cells would affect the cell functions. There seem to be two advantages of using MC3T3-E1 cells as a model system to study the role of adiponectin signaling on osteoblast growth and differentiation. First, since the cells produce adiponectin endogenously , there is no need to add adiponectin to the cultures. Second, since the present study and a previous one  showed that the cells do not express AdipoR2, data interpretation will not be complicated by the compensatory effect of AdipoR2 after the siRNA knockdown of AdipoR1. By reducing AdipoR1 expression through its siRNA transfection, we were able to demonstrate that endogenously produced adiponectin was important in osteoblast proliferation and differentiation as well as in matrix mineralization.
Shinoda et al. have shown that there were no abnormalities in the bone mass or turnover of either adiponectin-deficient mice or adiponectin-transgenic mice that overexpressed adiponectin in the liver . They have reported that circulating adiponectin suppresses osteoblastogenesis through the endocrine pathway, while local adiponectin stimulates it through the autocrine/paracrine pathway . Additionally, they have suggested that these contradictory effects of adiponectin might explain no apparent bone changes in its gene-modified mice. In this study, we found that the blockage of the AdipoR1 expression by transfecting its siRNA inhibited the differentiation and mineralization of MC3T3-E1 cells. Since this siRNA transfection obstructed not only the autocrine/paracrine pathway of adiponectin but also its endocrine pathway, cytokine might play an important role in the activation of osteoblasts in the microenvironment of bone marrow.
Yokota et al. have shown that adiponectin is also found in bone marrow fat cells , and Berner et al. have shown that the level of adiponectin is higher in diluted bone marrow fluid than in the serum of rats . These facts suggest that fat cells as well as bone cells might contribute to the production and high concentration of adiponectin found in bone marrow. Because adiponectin receptors are known to be expressed ubiquitously , it is possible that adiponectin can influence not only osteoblasts but also other lineages of bone marrow cells. In fact, adiponectin has been shown to exert an inhibitory effect on B lymphopoiesis and a stimulatory effect on myelopoiesis in bone marrow [24, 25]. Thus, further studies seem to be necessary to clarify the interplay among osteoblasts and other cells in the biological actions of adiponectin in the microenvironment of bone marrow.
Runx-2 has been identified as an important transcription factor that is involved in osteoblast differentiation and mineralization . Hence, it is possible that the stimulatory actions of adiponectin and AICAR on MC3T3-E1 cells in this study might be mediated by this transcription factor. However, neither of these agents affected mRNA or protein expressions of Runx-2, suggesting that the transcription factor might be not involved in the stimulatory effects of adiponectin on MC3T3-E1 cells. On the other hand, there remains a possibility that the Runx-2 activity can be altered by the adiponectin signaling pathway. It has been demonstrated that Runx-2 was activated by Erk via phosphorylation [26, 27]. Although the Erk activity was reported to be not altered by adiponectin , it is not clear from our current experiments whether the adiponectin downstream effector AMP kinase can phosphorylate Runx-2 either directly or indirectly. Alternatively, we indicated that Osx mRNA expression was enhanced by adiponectin and AICAR. Although Osx is generally regarded as the downstream effector of Runx-2, the upregulation of Osx mRNA has been reported without the alteration of Runx-2 mRNA levels or in the absence of Runx-2 [28, 29], suggesting that Osx and Runx-2 can be differently regulated and Osx can modulate osteoblastic function independent of Runx-2. Moreover, Shinoda et al. have recently shown that adiponectin activates the intracellular signaling of insulin in bone marrow cells through the phosphorylation of IRS-1 and Akt, the main downstream molecules of insulin , thereby suggesting that insulin/IGF-I signaling pathways might be alternative candidates for mediating the stimulatory effects of adiponectin on osteoblasts.
The biological effects of adiponectin are known to be mediated through the two adiponectin receptor subtypes AdipoR1 and R2 , which subsequently activate AMP kinase, PPARα ligand, and mitogen-activated protein kinase (MAPK), and regulate fatty-acid oxidation and glucose uptake . AMP kinase is a highly conserved heterotrimeric signaling kinase responsive to hypoxia, exercise, and cellular stress, and it is associated in a variety of cellular responses, including suppression of gluconeogenesis in the liver, promotion of glucose uptake in skeletal muscle, inhibition of fatty acid and sterol synthesis, increase in fatty acid oxidation, and inhibition of lipolysis [30–34]. In contrast, there exists little or no information about the potential roles of this signaling molecule in bone metabolism. In this study, we found that AMP kinase activation promoted the proliferation, differentiation, and mineralization of MC3T3-E1 cells. AMP kinase is known to have the ability to phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, a key regulatory enzyme in the synthesis of cholesterol and other isoprenoid compounds . Statins, which also inactivate HMG-CoA reductase, have been shown to promote osteoblastogenesis via cellular BMP-2 production [36, 37]. Thus, further studies are necessary to investigate whether or not the activation of AMP kinase would inactivate HMG-CoA reductase in MC3T3-E1 cells and enhance their differentiation and mineralization.
A couple of studies have revealed that there are interplays between the AMP kinase pathway and the MAPK pathway in the cellular responses of osteoblasts as well as the islet cells in the pancreas. Kefas et al. have previously shown that the prolonged activation of AMP kinase by AICAR promotes apoptosis of the beta cells in the islets through a sequential activation of JNK [38, 39]. Luo et al. have reported that the adiponectin-induced proliferative response of human osteoblasts was mediated by the AdipoR/JNK pathway, while the differentiation response was mediated via the AdipoR/p38 MAPK pathway . Although we found that the stimulatory effects of adiponectin on MC3T3-E1 cells might be mediated by AMP kinase, these previous results suggest that the involvement of MAPK or other pathways must also be investigated in future.