Atsuhiko Hikita

Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only Bcl-2 family member Bim

Osteoclasts (OCs) undergo rapid apoptosis without trophic factors, such as macrophage colony‐stimulating factor (M‐CSF). Their apoptosis was associated with a rapid and sustained increase in the pro‐apoptotic BH3‐only Bcl‐2 family member Bim. This was caused by the reduced ubiquitylation and proteasomal degradation of Bim that is mediated by c‐Cbl. Although the number of OCs was increased in the skeletal tissues of bim−/− mice, the mice exhibited mild osteosclerosis due to reduced bone resorption. OCs differentiated from bone marrow cells of bim−/− animals showed a marked prolongation of survival in the absence of M‐CSF, compared with bim+/+ OCs, but the bone‐resorbing activity of bim−/− OCs was significantly reduced. Overexpression of a degradation‐resistant lysine‐free Bim mutant in bim−/− cells abrogated the anti‐apoptotic effect of M‐CSF, while wild‐type Bim did not. These results demonstrate that ubiquitylation‐dependent regulation of Bim levels is critical for controlling apoptosis and activation of OCs.

Negative Regulation of Osteoclastogenesis by Ectodomain Shedding of Receptor Activator of NF-κB Ligand

Receptor activator of NF-κB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.

The roles of TGF-β signaling in carcinogenesis and breast cancer metastasis

Transforming growth factor-β (TGF-β) ligand is a multifunctional growth factor that regulates various cell behavior, such as cell proliferation, differentiation, migration, and apoptosis. Because TGF-β is a potent growth inhibitor, abnormalities in TGF-β signaling result in carcinogenesis. In addition to tumor suppressor function, TGF-β acts as an oncogenic factor. In particular, TGF-β signaling plays an important role during metastasis of breast cancer. Recently, epithelial-mesenchymal transition (EMT) has been shown to confer malignant properties such as cell motility and invasiveness to cancer cells and plays crucial roles during cancer metastasis. Moreover, breast stem-like cells exhibit EMT properties. Because TGF-β is a potent regulator of EMT as well as cell stemness, TGF-β signaling might play a crucial role in the regulation of breast cancer stem cells.

Regulation of osteoclast apoptosis and motility by small GTPase binding protein Rac1.

The role of Rac1 in osteoclast survival and bone‐resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M‐CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts.

Jun Dimerization Protein 2 (JDP2), a Member of the AP-1 Family of Transcription Factor, Mediates Osteoclast Differentiation Induced by RANKL

Osteoclasts are multinucleated cells that resorb bones, and are derived from hematopoietic cells of the monocyte/macrophage lineage. The receptor activator of NF-κB ligand (RANKL, also called ODF/TRANCE/OPGL) stimulates both osteoclast differentiation from osteoclast progenitors and activation of mature osteoclasts. To identify genes responsible for osteoclast differentiation, we used a molecular indexing technique. Here, we report a clone of one of these genes whose transcription is induced by soluble RANKL (sRANKL) in both the RAW264.7 cells of the mouse macrophage cell line and the mouse primary bone marrow cells. The predicted protein was found to be a mouse homologue of Jun dimerization protein 2 (JDP2), a member of the AP-1 family of transcription factors, containing a basic region-leucine zipper motif. Transient transfection experiments revealed that overexpression of JDP2 leads to activation of both tartrate-resistant acid phosphatase (TRAP) and cathepsin K gene promoters in RAW264.7 cells. Infection of mouse primary bone marrow cells with retroviruses expressing JDP2-facilitated sRANKL-mediated formation of TRAP-positive multinuclear osteoclasts. Importantly, antisense oligonucleotide to JDP2 strongly suppressed sRANKL-induced osteoclast formation of RAW264.7 cells. Our findings suggest that JDP2 may play an important role in the RANK-mediated signal transduction system, especially in osteoclast differentiation.

Pro-inflammatory Cytokine Tumor Necrosis Factor-α Induces Bone Morphogenetic Protein-2 in Chondrocytes via mRNA Stabilization and Transcriptional Up-regulation

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-α (TNF-α) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-α induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3′-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3′-untranslated regions, and transient transfection experiments demonstrated that TNF-α increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-α modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-κB.

Regional differences in chondrocyte metabolism in osteoarthritis: A detailed analysis by laser capture microdissection

OBJECTIVE To determine the change in metabolic activity of chondrocytes in osteoarthritic (OA) cartilage, considering regional difference and degree of cartilage degeneration. METHODS OA cartilage was obtained from knee joints with end-stage OA, at both macroscopically intact areas and areas with various degrees of cartilage degeneration. Control cartilage was obtained from age-matched donors. Using laser capture microdissection, cartilage samples were separated into superficial, middle, and deep zones, and gene expression was compared quantitatively in the respective zones between OA and control cartilage. RESULTS In OA cartilage, gene expression changed markedly with the site. The expression of cartilage matrix genes was highly enhanced in macroscopically intact areas, but the enhancement was less obvious in the degenerated areas, especially in the upper regions. In contrast, in those regions, the expression of type III collagen and fibronectin was most enhanced, suggesting that chondrocytes underwent a phenotypic change there. Within OA cartilage, the expression of cartilage matrix genes was significantly correlated with SOX9 expression, but not with SOX5 or SOX6 expression. In OA cartilage, the strongest correlation was observed between the expression of type III collagen and fibronectin, suggesting the presence of a certain link(s) between their expression. CONCLUSION The results of this study revealed a comprehensive view of the metabolic change of the chondrocytes in OA cartilage. The change of gene expression profile was most obvious in the upper region of the degenerated cartilage. The altered gene expression at that region may be responsible for the loss of cartilage matrix associated with OA.

Addition of an arch support improves the biomechanical effect of a laterally wedged insole

In order to examine if the addition of an arch support could improve the biomechanical effect of the laterally wedged insole, three-dimensional gait analysis was performed on 20 healthy volunteers. Kinetic and kinematic parameters at the knee and subtalar joints were compared among the following four types of insoles; a 5-mm thick flat insole, a flat insole with an arch support (AS), a 6 degrees inclined laterally wedged insole (LW), and a laterally wedged insole with an arch support (LWAS). The knee adduction moment averaged for the entire stance phase was reduced by the use of LW and LWAS by 7.7% and 13.3%, respectively, from that with FLAT. The difference in knee adduction moment between LW and LWAS was most obvious in the late stance, which was ascribed to the difference in the progression angle between those insoles. The analyses also revealed that LW tended to increase step width, and that such an increase was completely eliminated by the addition of an arch support to LW. This reduction of step width could be another mechanism for the further reduction of the moment with LWAS. The analyses of biomechanical parameters at the subtalar joints suggested that LWAS allowed the subject to walk in a more natural manner, while exerting greater biomechanical effects than LW. Thus, the addition of an arch support to the laterally wedged insole reduced knee adduction moment more efficiently, possibly through the elimination of potential negative effects of the laterally wedged insole.

Zonal Gene Expression of Chondrocytes in Osteoarthritic Cartilage

OBJECTIVE To determine the chondrocyte metabolism in respective zones of osteoarthritic (OA) cartilage. METHODS OA cartilage was obtained from macroscopically intact areas of 4 knee joints with end-stage OA. The cartilage was divided into 3 zones, and gene expression profiles were determined in the respective zones by a custom-designed microarray that focused on chondrocyte-related genes. For the genes whose expression was significantly different among the zones, the expression was compared between OA and control cartilage in the respective zones by an analysis using laser capture microdissection and real-time polymerase chain reaction (PCR). For some genes, the correlation of expression was investigated in specific cartilage zones. RESULTS A total of 198 genes (approximately 40% of those investigated) were found to be expressed at significantly different levels among the zones. Expression of 26 of those genes was evaluated by laser capture microdissection and real-time PCR, which confirmed the validity of microarray analysis. The expression of cartilage matrix genes was mostly enhanced in OA cartilage, at similar levels across the zones but at different magnitudes among the genes. The expression of bone-related genes was induced either in the superficial zone or in the deep zone, and positive correlations were found among their expression in the respective zones. The expression of 5 proteinase genes was most enhanced in the superficial zone, where their expression was correlated, suggesting the presence of a common regulatory mechanism(s) for their expression. CONCLUSION In OA cartilage, the metabolic activity of chondrocytes differed considerably among zones. Characteristic changes were observed in the superficial and deep zones.

Molecular mechanism of the life and death of the osteoclast.

Abstract:  The life span of osteoclasts is critically regulated by various cytokines, and therapeutics such as bisphosphonates act directly on osteoclasts and induce apoptosis of the cells. This article will focus on the molecular mechanism of osteoclast apoptosis and summarize the recent advances in this field with an emphasis on the role of intracellular signaling pathways.