[Show abstract][Hide abstract] ABSTRACT: Claudin 18 (Cldn-18) belongs to a large family of transmembrane proteins that are important components of tight junction strands. Although several claudin members are expressed in bone, the functional role for any claudin member in bone is unknown. Here we demonstrate that disruption of Cldn-18 in mice markedly decreased total body bone mineral density, trabecular bone volume, and cortical thickness in Cldn-18(-/-) mice. Histomorphometric studies revealed that bone resorption parameters were increased significantly in Cldn-18(-/-) mice without changes in bone formation. Serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b) and mRNA expression levels of osteoclast specific markers and signaling molecules were also increased. Loss of Cldn-18 further exacerbated calcium deficiency induced bone loss by influencing bone resorption, thereby resulting in mechanically weaker bone. In vitro studies with bone marrow macrophages revealed Cldn-18 disruption markedly enhanced receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation but not macrophage colony-stimulating factor (MCSF)-induced bone marrow macrophage (BMM) proliferation. Consistent with a direct role for Cldn-18 in regulating osteoclast differentiation, overexpression of wild type but not PDZ binding motif deleted Cldn-18 inhibited RANKL-induced osteoclast differentiation. Furthermore, our findings indicate that Cldn-18 interacts with Zonula occludens 2 (ZO-2) to modulate RANKL signaling in osteoclasts. In conclusion, we demonstrate that Cldn-18 is a novel negative regulator of bone resorption and osteoclast differentiation.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 07/2012; 27(7):1553-65. DOI:10.1002/jbmr.1600 · 6.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men.
[Show abstract][Hide abstract] ABSTRACT: There is now increasing evidence which suggests an important role for reactive oxygen species (ROS) in the pathogenesis of osteoporosis. However, little is known on the molecular components of the oxidative stress pathway or their functions in bone. In this study, we evaluated the role and mechanism of action of glutaredoxin (Grx) 5, a protein that is highly expressed in bone. Osteoblasts were transfected with Grx5 siRNA and treated with hydrogen peroxide (H(2)O(2)). Grx5 siRNA treatment increased apoptosis while Grx5 overexpression protected MC3T3-E1 cells against H(2)O(2) induced apoptosis and ROS formation. Grx5 deficiency results in impaired biogenesis of Fe-S cluster in yeast. Accordingly, activity of mitochondrial aconitase, whose activity is dependent on Fe-S cluster, decreased in Grx5 siRNA treated cells. Since reduced formation of Fe-S cluster would lead to increased level of free iron, a competitive inhibitor of manganese superoxide dismutase (MnSOD), we measured MnSOD activity in Grx5 deficient osteoblasts and found MnSOD activity was significantly reduced. The consequence of long term inhibition of Grx5 on osteoblast apoptosis was evaluated using lentiviral shRNA technology. Grx5 shRNA cells exhibited higher caspase activity and cardiolipin oxidation in the presence of H(2)O(2). MnSOD activity was rescued by the addition of MnCl(2) to Grx5 shRNA osteoblasts in the presence of H(2)O(2). Our findings are consistent with the hypothesis that Grx5 is an important determinant of osteoblast apoptosis and acts via a molecular pathway that involves regulation of ROS production, cardiolipin oxidation, caspase activity, Fe-S cluster formation, and MnSOD activity.
Bone 06/2009; 44(5):795-804. DOI:10.1016/j.bone.2009.01.003 · 4.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T-box (Tbx)3, a known transcriptional repressor, is a member of a family of transcription factors, which contain a highly homologous DNA binding domain known as the Tbx domain. Based on the knowledge that mutation of the Tbx3 gene results in limb malformation, Tbx3 regulates osteoblast proliferation and its expression increases during osteoblast differentiation, we predicted that Tbx3 is an important regulator of osteoblast cell functions. In this study, we evaluated the consequence of transgenic overexpression of Tbx3 on osteoblast differentiation. Retroviral overexpression increased Tbx3 expression >100-fold at the mRNA and protein level. Overexpression of Tbx3 blocked mineralized nodule formation (28 +/- 8 vs. 7 +/- 1%) in MC3T3-E1 cells. In support of these data, alkaline phosphatase (ALP) activity was reduced 33-70% (P < 0.05) in both MC3T3-E1 cells and primary calvaria osteoblasts overexpressing Tbx3. In contrast, Tbx3 overexpression did not alter ALP activity in bone marrow stromal cells. Tbx3 overexpression blocked the increase in expression of key osteoblast marker genes, ALP, bone sialoprotein, and osteocalcin that occurs during normal osteoblast differentiation, but had little or no effect on expression of proliferation genes p53 and Myc. In addition, Tbx3 overexpression abolished increased osterix and runx2 expression observed during normal osteoblast differentiation, but the change in Msx1 and Msx2 expression over time was similar between control and Tbx3 overexpressing cells. Interestingly, osterix and runx2, but not Msx1 and Msx2, contain Tbx binding site in the regulatory region. Based on these data and our previous findings, we conclude that Tbx3 promotes proliferation and suppresses differentiation of osteoblasts and may be involved in regulating expression of key transcription factors involved in osteoblast differentiation.