Youngkyun Lee

MicroRNA-124 regulates osteoclast differentiation.

Osteoclasts are specialized cells for bone-resorption originated from precursors of macrophage/monocyte lineage. The receptor activator of NFκB ligand (RANKL) initiates osteoclast differentiation, in which nuclear factor of activated T cell cytoplasmic 1 (NFATc1) plays a key role as a master transcription factor. In the present report, we show that microRNA-124 (miR-124) regulates osteoclastogenesis of mouse bone marrow macrophages (BMMs) by suppressing NFATc1 expression. On the other hand, synthetic inhibitor that binds specifically to miR-124 enhanced osteoclast differentiation and NFATc1 expression. The overexpression of a constitutively active form of NFATc1 prevented the inhibitory effect of miR-124 on osteoclastogenesis. Finally, miR-124 also affected the proliferation and motility of osteoclast precursors, the latter coinciding with the reduced expression of RhoA and Rac1. These findings not only reveal unprecedented role of miR-124 in osteoclastogenesis but also suggest a novel mode of regulation of NFATc1 in osteoclasts.

The role of interleukin-17 in bone metabolism and inflammatory skeletal diseases.

The balance between osteoblast-dependent bone formation and osteoclast-dependent bone resorption maintains bone homeostasis. In inflammatory conditions, this balance shifts toward bone resorption, causing osteolytic bone lesions observed in rheumatoid arthritis and periodontitis. A recently discovered family of cytokine IL-17 is widely reported to mediate diverse inflammatory processes. During the last decade, novel roles for IL-17 in skeletal homeostasis have been discovered indicating the potential importance of this cytokine in bone metabolism. This review will summarize and discuss the involvement of IL-17 during bone homeostasis in both physiologic and pathologic conditions. A better understanding of the role of IL-17 in skeletal systems warrants an advance in bone biology, as well as development of therapeutic strategies against bone-lytic diseases, such as rheumatoid arthritis and periodontitis. [BMB Reports 2013; 46(10): 479-483]

Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival

Plasma membrane calcium ATPases PMCA1 and PMCA4 regulate osteoclast differentiation and survival by regulating NFATc1 and NO.

Nrf2 is a novel regulator of bone acquisition

Nuclear factor E2 p45-related factor 2 (Nrf2) is a transcription factor involved in the expression of cytoprotective genes induced by external stresses. We investigated the role of Nrf2 in osteoclast and osteoblast differentiation. Nrf2 knockdown or deletion increased osteoclastic differentiation from bone marrow-derived macrophages (BMMs) through the upregulation of NF-κB, c-Fos, and NFATc1 transcription factors. Nrf2 also inhibited osteoblast differentiation and mineralization via suppression of key regulatory proteins, such as Runx2, osteocalcin, and osterix. Micro-computed tomography and histomorphometric analyses showed an increase in bone mass of Nrf2 knockout compared to that of wild type mice. In addition, the mineral apposition rate and the number of osteoblasts in bone were higher in Nrf2 knockout mice. However, bone resorption parameters, namely DPD and CTX levels, were not affected by Nrf2 deletion. In a coculture condition where calvarial osteoblasts and BMMs from wild type and Nrf2 knockout mice were grown, deletion of Nrf2 in osteoblasts markedly reduced osteoclast formation. This effect was due to an increase in OPG expression in Nrf2 knockout osteoblasts. Taken as a whole, these results indicate that Nrf2 is intrinsically inhibitory to both osteoblast and osteoclast differentiation but its effect on osteoblasts is dominant to its effect on osteoclasts in vivo.

MicroRNA profiling in the mouse hypothalamus reveals oxytocin-regulating microRNA.

Oxytocin (Oxt), produced in the hypothalamic paraventricular and supraoptic nuclei for transport to and release from the posterior pituitary, was originally discovered through its role in lactation and parturition. Oxt also plays important roles in the central nervous system by influencing various behaviors. MicroRNAs (miRNAs), endogenous regulators of many genes, are a class of small non‐coding RNAs that mediate post‐transcriptional gene silencing. We performed miRNA expression profiling of the mouse hypothalamus by deep sequencing. Among the sequenced and cross‐mapped small RNAs, expression of known miRNAs and unknown miRNAs candidates were analyzed. We investigated in detail one miRNA, miR‐24, and found that it is a novel regulator of Oxt and controls both transcript and peptide levels of Oxt. These results provide insights into potential neurohypophysial hormone regulation mediated by miRNAs.

IL-17 inhibits osteoblast differentiation and bone regeneration in rat

OBJECTIVE The interleukin-17 (IL-17) family is a group of pro-inflammatory cytokines that are produced by a subset of helper T cells. IL-17 family members are not only involved in the immune response of tissues but also play a role in bone metabolism. Although the role of IL-17 in osteoclast-mediated bone resorption has been extensively studied, its role during osteoblast-mediated bone formation has rarely been investigated. In this study, we examined the effect of IL-17 on osteogenesis in rats both in vitro and in vivo. DESIGN To evaluate osteogenesis in vitro, rat calvarial osteoblast precursor cells were cultured for 14 days in osteogenic medium with or without 50ng/mL IL-17. Osteogenic activity was evaluated by alkaline phosphatase and alizarin red staining. The mRNA expression of alkaline phosphatase, osteocalcin, and osterix was also measured by using real-time PCR. To test whether IL-17 affects bone formation in vivo, bone filling was examined by micro-computed tomography and histological observations at 8 weeks after critical-sized defects were made in rat calvaria. RESULTS The presence of IL-17 significantly reduced alkaline phosphatase and alizarin red staining and the expression of alkaline phosphatase, osteocalcin, and osterix in vitro. IL-17 also significantly inhibited the filling of calvarial defects in vivo. CONCLUSION IL-17 exerted a negative effect on osteogenesis in a rat model. In contrast to the previously reported beneficial effect on osteogenic differentiation of human mesenchymal stem cells, our results suggest a species or cell type-specific role for IL-17 in bone formation.

TLT-1s, Alternative Transcripts of Triggering Receptor Expressed on Myeloid Cell-like Transcript-1 (TLT-1), Inhibits the Triggering Receptor Expressed on Myeloid Cell-2 (TREM-2)-mediated Signaling Pathway during Osteoclastogenesis

Background: The triggering receptor expressed on myeloid cell (TREM)-mediated signaling is essential for osteoclastogenesis. Results: The alternative transcripts of triggering receptor expressed on myeloid cell-like transcript-1 (TLT-1s) inhibits osteoclast formation by counteracting the TREM-2 signaling pathway. Conclusion: TLT-1s is a negative regulator of osteoclastogenesis, by constitutively associating with SHP-1 and SHIP-1 phosphatases, abrogating the TREM-2 signaling upon RANKL stimulation. Significance: We discovered that an alternative transcript of TLT-1, namely TLT-1s, negatively regulates osteoclastogenesis. Triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-baring TREM family protein. In this study, we identified an alternative transcript form of TLT-1, namely TLT-1s, which has very short extracellular immunoglobulin domain consisting of only 202 amino acids. TLT-1s was mainly expressed in macrophages and osteoclast precursor cells. Upon receptor activator of nuclear factor-κB ligand stimulation, TLT-1s mRNA and protein levels were gradually decreased in BMMs. We also showed the TLT-1s is localized to the cytoplasmic membrane in osteoclast precursor cells. TLT-1s silencing strongly enhanced the formation and resorption activity of osteoclast. In addition, forced expression of TLT-1s showed reduced formation of osteoclast. Because ITIM-baring proteins inhibit immunoreceptor tyrosine-based activation motif (ITAM)-mediated receptor signaling, we tested whether TLT-1s physically interacted with TREM-2, the ITAM-associated co-stimulatory receptor essential for osteoclast differentiation. We showed that TLT-1s is associated with TREM-2 in osteoclast precursor cells. TLT-1s is also associated with tyrosine Src homology 2 domain-containing phosphatase-1 and SH2 domain-containing inositol phosphatase-1 and recruited them to the TREM2-ITAM signaling complex. In addition, knockdown of TLT-1s markedly elevated the intracellular calcium concentration and oscillation in osteoclast precursor cells. In addition, calcium-mediated induction of nuclear factor of activated T cells was also increased by TLT-1s silencing. Furthermore, TREM-2-mediated Akt activation and proliferation of osteoclast precursor cells were also enhanced in TLT-1s silenced cells. In this paper, we found the noble ITIM-baring inhibitory membrane protein; TLT-1s, which regulates ITAM-mediated signaling on osteoclastogenesis.

Apolipoprotein E inhibits osteoclast differentiation via regulation of c-Fos, NFATc1 and NF-κB

Apolipoprotein E (ApoE) plays a major role in the transport and metabolism of lipid. Other functions of ApoE include modulation of innate and adaptive immune responses. The expression of ApoE in osteoblasts and its relevance with bone formation have also been reported. However, the effect of ApoE on osteoclasts has not yet been examined. Here, we investigated the role of ApoE in osteoclast differentiation using bone marrow-derived macrophages (BMMs) and RAW264.7 cells. We found a down-regulation of ApoE gene expression during osteoclastic differentiation of those cells. Overexpression of ApoE in BMMs and RAW264.7 cells significantly blocked the induction of c-Fos and nuclear factor of activated T cell c1 (NFATc1), transcription factors critical for expression of osteoclast marker genes, by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. ApoE inhibited osteoclast differentiation, as measured by decreased number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs). In addition, ApoE reduced the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and ATPase, H(+) transporting, lysosomal 38kDa, V0 subunit d2 (ATP6v0d2), genes involved in cell-cell fusion during osteoclastogenesis. Knock-down of ApoE using a specific siRNA promoted the RANKL-mediated induction of osteoclast differentiation. While ApoE did not affect the activation of ERK, JNK, and p38 MAPK signaling pathways by RANKL, the phosphorylation of p65 trans-activation domain on serine 536 and transcription activity of NF-κB were reduced by ApoE overexpression. These findings suggest that ApoE plays an inhibitory role in osteoclast differentiation via the suppression of RANKL-dependent activation of NF-κB and induction of c-Fos and NFATc1.

Serotonin Inhibits Osteoblast Differentiation and Bone Regeneration in Rats

BACKGROUND Although increasing evidence indicates that serotonin (SER; 5-hydroxytrypamine [5-HT]) is involved in the regulation of bone metabolism, conflicting data exist regarding whether SER promotes or inhibits osteoblast differentiation and bone formation. Regeneration of functional bone is required for proper osseointegration of dental implants. Noticeably, the use of selective SER reuptake inhibitors was recently associated with the failure of osseointegrated dental implants. The present study examines the direct role of peripheral SER on the regulation of bone regeneration. METHODS The effect of SER on osteoblast differentiation and bone regeneration was examined using rat calvarial cell cultures in vitro and a rat critical-sized calvarial defect model in vivo. RESULTS Rat calvarial cells expressed SER receptors Htr1 (5-HT1) and Htr2 (5-HT2), which are known to transmit signals in bone cells. In vitro, SER significantly reduced osteogenic differentiation and mineralization of rat calvarial cells with concomitant reduction of osteoblast marker genes including alkaline phosphatase (Alpl), osterix (Sp7), and osteocalcin (Bglap). Histologic and radiologic analyses using the rat critical-sized calvarial defect model revealed that the existence of SER significantly inhibited β-phase tricalcium phosphate-induced bone regeneration. CONCLUSION Results suggest that SER in the local bone microenvironment might play a negative role in osteoblast differentiation and bone formation in rats.

Developmental regulations of Perp in mice molar morphogenesis

Teraspanin transmembrane protein, Perp (P53 apoptosis effector related to PMP22), which is found in the plasma membrane as a component of the desmosome, is reported to be involved in the morphogenesis of the epithelium and the enamel formation of the incisor. However, its expression pattern and signaling regulation during molar development have not been elucidated in detail. We have examined the precise expression patterns of Perp in developing lower molars and employed the knock-down of Perp by antisense oligodeoxynucleotide treatment during in vitro organ cultivation at embryonic day 13 to define the precise developmental function of Perp. Perp was expressed mainly in the dental lamina and stellate reticulum regions at the bud and cap stages. After Perp knock-down, the tooth germ showed disruption of the dental lamina and stellate reticulum with altered apoptosis and proliferation. The changed expression levels of related signaling molecules from the enamel knot and desmosome were evaluated by real-time quantitative polymerase chain reaction. A renal capsule transplantation method was employed to examine the effects of Perp knock-down on molar crown development. Ultrastructural observations revealed that enamel was deposited more densely in an irregular pattern in the cusp region, and that dentin was hypo-mineralized after Perp knock-down at the cap stage. Thus, Perp might play important roles in the formation and integration of stellate reticulum, dental lamina structure and enamel formation through signaling interactions with the enamel knot and desmosome-related signaling molecules at the cap stage of lower molar development.