Kabsun Kim

The Mechanism of Osteoclast Differentiation Induced by IL-1

IL-1 is a potent cytokine that can induce bone erosion in inflammatory sites such as rheumatoid joint regions via activation of osteoclasts. Not only is IL-1 capable of activating osteoclasts, but it is also a key cytokine involved in the differentiation, multinucleation, and survival of osteoclasts. Herein, we show that IL-1 has the potential to drive osteoclast differentiation via a receptor activator of NF-κB ligand (RANKL)/RANK-independent mechanism. Although IL-1 has a synergistic effect on RANKL-induced osteoclast formation, IL-1 alone cannot induce osteoclast differentiation from osteoclast precursors (bone marrow-derived macrophages (BMMs)) due to a lack of IL-1 signaling potential in these cells. However, we demonstrate that overexpression of the IL-1RI receptor in BMMs or induction of IL-1RI by c-Fos overexpression enables IL-1 alone to induce the formation of authentic osteoclasts by a RANKL/RANK-independent mechanism. The expression of IL-1RI is up-regulated by RANKL via c-Fos and NFATc1. Furthermore, the addition of IL-1 to IL-1RI overexpressing BMMs (IL-1/IL-1RI) strongly activates NF-κB, JNK, p38, and ERK which is a hallmark gene activation profile of osteoclastogenesis. Interestingly, IL-1/IL-1RI does not induce expression of c-Fos or NFATc1 during osteoclast differentiation, although basal levels of c-Fos and NFATc1 seem to be required. Rather, IL-1/IL-1RI strongly activates MITF, which subsequently induces osteoclast-specific genes such as osteoclast-associated receptor and tartrate-resistant acid phosphatase. Together, these results reveal that IL-1 has the potential to induce osteoclast differentiation via activation of microphthalmia transcription factor under specific microenvironmental conditions.

Akt Induces Osteoclast Differentiation through Regulating the GSK3β/NFATc1 Signaling Cascade

SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P3 and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found that overexpression of Akt enhances formation of an inactive form of GSK3β (phospho-GSK3β) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3β attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3β, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3β attenuates RANKL-induced osteoclast differentiation from SHIP-deficient BMMs. Our data suggest that the PI3K/Akt/GSK3β/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis.

Regulatory mechanism of NFATc1 in RANKL-induced osteoclast activation

NFATc1 is a master regulator of RANKL‐induced osteoclast differentiation and herein we investigate the regulatory mechanism of NFATc1 in osteoclast activation. Inactivation of NFATc1 strongly attenuates RANKL‐induced bone resorption and overexpression of a constitutively active form of NFATc1 in osteoclasts induces formation of actin rings and resorption pits on dentin slices. We demonstrate that NFATc1 binds directly to the promoter regions of its target genes and induces expression of various genes, including LTBP3, ClC7, cathepsin K, MMP9, and c‐Src, which are key players in bone resorption. Thus, NFATc1 is essential for RANKL‐induced osteoclast activation via up‐regulation of osteoclast‐activating genes.

Histone methyltransferase PRDM8 regulates mouse testis steroidogenesis.

A family of PRDM proteins are similar to histone methyltransferases (HMTases) with SET domain in that they modulate different cellular processes, including transcriptional regulation, through chromatin modifying activities. By applying a bioinformatic approach, we searched for proteins containing the SET domain and identified a double zinc-finger domain containing PRDM8 with HMTase activity. In vitro HMTase assay and immunoblot analysis revealed that PRDM8 specifically methylates H3K9 of histones which indicates transcriptional repression activity of PRDM8. Direct recruitment of PRDM8 to the promoter mediated transcriptional repression and indicated no involvement of HDAC. Tissue blot analyses identified PRDM8 transcripts from brain and testis in adult mouse. Consistent with these observations, we demonstrate that PRDM8 repressed the expression of steroidogenic markers, p450c17c and LHR, which indicates its regulatory role in mouse testis development.

Protein Inhibitor of Activated STAT 3 Modulates Osteoclastogenesis by Down-Regulation of NFATc1 and Osteoclast-Associated Receptor

Protein inhibitor of activated STAT3 (PIAS3) has been shown to regulate the activity of various transcription factors. In this study, we show that the overexpression of PIAS3 in bone marrow-derived monocyte/macrophage lineage cells attenuates osteoclast formation and down-regulates the expression of NFATc1 and osteoclast-associated receptor (OSCAR), which are important modulators in osteoclastogenesis. PIAS3 has been shown to associate with histone deacetylase 1 as well as with transcription factors, including the microphthalmia transcription factor, NFATc1, and c-Fos. Moreover, overexpression of PIAS3 inhibits the transactivation of target genes such as NFATc1 and OSCAR. This inhibitory effect of PIAS3 is possibly mediated by histone deacetylase 1 recruitment to the promoter regions of NFATc1 and OSCAR. Furthermore, silencing of PIAS3 by RNA interference in osteoclast precursors enhances osteoclast formation as well as gene expression of NFATc1 and OSCAR. Taken together, our results reveal that PIAS3 acts as a modulator in osteoclastogenesis.

RANKL induces NFATc1 acetylation and stability via histone acetyltransferases during osteoclast differentiation

NFATc1 (nuclear factor of activated T-cells c1), a key transcription factor, plays a role in regulating expression of osteoclast-specific downstream target genes such as TRAP (tartrate-resistant acid phosphatase) and OSCAR (osteoclast-associated receptor). It has been shown that RANKL [receptor activator of NF-κB (nuclear factor κB) ligand] induces NFATc1 expression during osteoclastogenesis at a transcriptional level. In the present study, we demonstrate that RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via HATs (histone acetyltransferases), such as p300 and PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor], thereby stabilizing NFATc1 proteins. PCAF physically interacts with NFATc1 and directly induces NFATc1 acetylation and stability, subsequently increasing the transcriptional activity of NFATc1. In addition, RANKL-mediated NFATc1 acetylation is increased by the HDAC (histone deacetylase) inhibitors sodium butyrate and scriptaid. Overexpression of HDAC5 reduces RANKL- or PCAF-mediated NFATc1 acetylation, stability and transactivation activity, suggesting that the balance between HAT and HDAC activities might play a role in the regulation of NFATc1 levels. Furthermore, RANKL and p300 induce PCAF acetylation and stability, thereby enhancing the transcriptional activity of NFATc1. Down-regulation of PCAF by siRNA (small interfering RNA) decreases NFATc1 acetylation and stability, as well as RANKL-induced osteoclastogenesis. Taken together, the results of the present study demonstrate that RANKL induces HAT-mediated NFATc1 acetylation and stability, and subsequently increases the transcriptional activity of NFATc1 during osteoclast differentiation.

MicroRNA-26a regulates RANKL-induced osteoclast formation.

Osteoclasts are unique cells responsible for the resorption of bone matrix. MicroRNAs (miRNAs) are involved in the regulation of a wide range of physiological processes. Here, we examined the role of miR-26a in RANKL-induced osteoclastogenesis. The expression of miR-26a was up-regulated by RANKL at the late stage of osteoclastogenesis. Ectopic expression of an miR-26a mimic in osteoclast precursor cells attenuated osteoclast formation, actin-ring formation, and bone resorption by suppressing the expression of connective tissue growth factor/CCN family 2 (CTGF/CCN2), which can promote osteoclast formation via up-regulation of dendritic cell-specific transmembrane protein (DC-STAMP). On the other hand, overexpression of miR-26a inhibitor enhanced RANKL-induced osteoclast formation and function as well as CTGF expression. In addition, the inhibitory effect of miR-26a on osteoclast formation and function was prevented by treatment with recombinant CTGF. Collectively, our results suggest that miR-26a modulates osteoclast formation and function through the regulation of CTGF.

Negative Feedback Control of Osteoclast Formation through Ubiquitin-mediated Down-regulation of NFATc1

The regulation of NFATc1 expression is important for osteoclast differentiation and function. Herein, we demonstrate that macrophage-colony-stimulating factor induces NFATc1 degradation via Cbl proteins in a Src kinase-dependent manner. NFATc1 proteins are ubiquitinated and rapidly degraded during late stage osteoclastogenesis, and this degradation is mediated by Cbl-b and c-Cbl ubiquitin ligases in a Src-dependent manner. In addition, NFATc1 interacts endogenously with c-Src, c-Cbl, and Cbl-b in osteoclasts. Overexpression of c-Src induces down-regulation of NFATc1, and depletion of Cbl proteins blocks NFATc1 degradation during late stage osteoclastogenesis. Taken together, our data provide a negative regulatory mechanism by which macrophage-colony-stimulating factor activates Src family kinases and Cbl proteins, and subsequently, induces NFATc1 degradation during osteoclast differentiation.

MHC class II transactivator negatively regulates RANKL-mediated osteoclast differentiation by downregulating NFATc1 and OSCAR

Nuclear factor of activated T cells (NFAT) c1 plays a key role in receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation and function via induction of osteoclast-specific target genes including osteoclast-associated receptor (OSCAR), cathepsin K, and tartrate-resistant acid phosphatase. To elucidate which downstream target genes are regulated by NFATc1 during osteoclastogenesis, we used microarray analyses to examine gene expression profiles in the context of bone marrow-derived macrophages overexpressing a constitutively active form of NFATc1. Herein, we demonstrate that MHC class II transactivator (CIITA) is up-regulated downstream of NFATc1. Overexpression of CIITA in osteoclast precursors attenuates RANKL-induced osteoclast formation through down-regulation of NFATc1 and OSCAR. Epigenetic overexpression of CIITA regulates NFATc1 and OSCAR by competing with c-Fos and NFATc1 for CBP/p300 binding sites. Furthermore, silencing of CIITA by RNA interference in osteoclast precursors enhances osteoclast formation as well as NFATc1 and OSCAR expression. Taken together, our data reveal that CIITA can act as a modulator of RANKL-induced osteoclastogenesis.

Silibinin inhibits osteoclast differentiation mediated by TNF family members

Silibinin is a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), with known hepatoprotective, anticarcinogenic, and antioxidant effects. Herein, we show that silibinin inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 cells as well as from bone marrow-derived monocyte/macrophage cells in a dose-dependent manner. Silibinin has no effect on the expression of RANKL or the soluble RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts. However, we demonstrate that silibinin can block the activation of NF-κB, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK) in osteoclast precursors in response to RANKL. Furthermore, silibinin attenuates the induction of nuclear factor of activated T cells (NFAT) c1 and osteoclast-associated receptor (OSCAR) expression during RANKL-induced osteoclastogenesis. We demonstrate that silibinin can inhibit TNF-α-induced osteoclastogenesis as well as the expression of NFATc1 and OSCAR. Taken together, our results indicate that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL and TNF-α.