Jeongim Ha

A comparison of autologous and allogenic bone marrow-derived mesenchymal stem cell transplantation in canine spinal cord injury

The purpose of this study is to compare the therapeutic effects between autologous and allogenic bone-marrow-derived mesenchymal stem cell (MSC) transplantation in experimentally-induced spinal cord injury (SCI) of dogs. Thirty adult Beagle dogs (control group=10, autologous group=10, and allogenic group=10) were used in this study. Prelabeled MSCs were intrathecally transplanted through the lumbar spinal cord into the injured lesion at a density of 1 x 10(7) cells 7 days after SCI. Neurological signs of dogs in both autologous and allogenic groups were improved in their pelvic limbs after SCI compared with those in control group. Both autologous and allogenic groups showed significantly higher the Olby scores than control group (p<0.05). This finding was consistent with results of MRI and histopathological examination in both groups. Immunofluorescence analysis revealed that prelabeled autologous and allogenic MSCs were detected in the injured lesions both at 1 and 4 weeks after transplantation. However, the distribution ratio of MSCs on the injured lesion in allogenic group was significantly decreased at 4 weeks after transplantation relatively to at 1 week after transplantation. The mRNA expression for neurotrophic factors in both allogenic and autologous groups was significantly higher than that in control groups (p<0.05). Even though autologous MSC transplantation showed more beneficial effect than that of allogenic MSC transplantation, transplantation of allogenic MSCs also improved functional recovery following SCI. This study demonstrates that both autologous and allogenic MSC transplantation could be clinically useful therapeutic approaches for treating SCI.

Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-κB transactivation by RANKL

Osteoclast (Oc) differentiation is fundamentally controlled by receptor activator of nuclear factor kappaB ligand (RANKL). RANKL signalling targets include mitogen-activated protein kinases (MAPKs), nuclear factor kappaB (NF-κB), and nuclear factor of activated T cells (NFAT)c1. In this study, we found that p38 MAPK upstream components transforming growth factor-beta-activated kinase 1 (TAK1), MKK3, and MKK6 increased by RANKL in an early stage of osteoclastogenesis from primary bone marrow cells, which led to enhanced p38 activation. Retroviral transduction of dominant-negative (DN) forms of TAK1 and MKK6, but not that of MKK3, reduced Oc differentiation. Transduction of TAK1-DN and MKK6-DN and treatment with the p38 inhibitor SB203580 attenuated NFATc1 induction by RANKL. TAK1-DN, MKK6-DN, and SB203580, but not MKK3-DN, also suppressed RANKL stimulation of NF-κB transcription activity in a manner dependent on p65 phosphorylation on Ser-536. These results indicate that TAK1 and MKK6 constitute the p38 signalling pathway to participate to Oc differentiation by RANKL through p65 phosphorylation and NFATc1 induction, and that MKK6 and MKK3 have differential roles in osteoclastogenesis from bone marrow precursors.

Hyaluronan inhibits osteoclast differentiation via Toll-like receptor 4

The differentiation of osteoclasts, cells specialized for bone resorption, is governed by two key factors, macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). The extracellular matrix (ECM) is an important factor influencing cell fate. To date, little investigation on the relationship between ECM components and osteoclast differentiation has been documented. In this study, we uncovered a potent anti-osteoclastogenic effect of hyaluronan (HA), an ECM component present in bone marrow and soft connective tissues, in primary mouse and human osteoclast precursor cell cultures. The anti-osteoclastogenic function of HA was dependent on Toll-like receptor 4 (TLR4) but not on CD44. HA inhibited M-CSF-dependent signaling pathways involving Rac, reactive oxygen species and mitogen-activated protein kinases, resulting in suppression of transcription factors AP-1 and MITF that control RANK expression. Furthermore, in an in vivo mouse model of calvarial bone resorption assays HA reduced RANKL-induced bone erosion and osteoclastogenesis. Our results clearly show that HA inhibits osteoclast differentiation through TLR4 by interfering with M-CSF signaling, and point that the interaction between ECM components and innate immune receptors can play an important role in the regulation of bone metabolism.

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-κB ligand in cooperation with macrophage colony–stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb−/− mice were similarly affected. In vivo studies showed that Ckb−/− mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

CXC Chemokine Ligand 2 Induced by Receptor Activator of NF-κB Ligand Enhances Osteoclastogenesis

CXCL2 has been known to regulate immune functions mainly by chemo-attracting neutrophils. In this study, we show that CXCL2 can be induced by receptor activator of NF-κB ligand, the osteoclast (OC) differentiation factor, through JNK and NF-κB signaling pathways in OC precursor cells. CXCL2 in turn enhanced the proliferation of OC precursor cells of bone marrow-derived macrophages (BMMs) through the activation of ERK. Knockdown of CXCL2 inhibited both the proliferation of and the ERK activation in BMMs. During osteoclastogenesis CXCL2 stimulated the adhesion and the migration of BMMs. Moreover, the formation of OCs from BMMs was significantly increased on treatment with CXCL2. Conversely, the CXCL2 antagonist repertaxin and a CXCL2 neutralizing Ab potently reduced receptor activator of NF-κB ligand-induced osteoclastogenesis. Furthermore, CXCL2 evoked fulminant bone erosion in the in vivo mouse experiments. Finally, prominent upregulation of CXCL2 was detected in synovial fluids and sera from rheumatoid arthritis patients, suggesting a potential involvement of CXCL2-mediated osteoclastogenesis in rheumatoid arthritis-associated bone destruction. Thus, CXCL2 is a novel therapeutic target for inflammatory bone destructive diseases.

Caffeic acid phenethyl ester inhibits osteoclastogenesis by suppressing NFκB and downregulating NFATc1 and c-Fos

Osteoclasts are multinuclear cells of myeloid lineage responsible for bone resorption. The anti-inflammatory property of caffeic acid phenethyl ester (CAPE), an active component of the propolis of honeybee hives, has been revealed. Since the regulatory mechanism of differentiation and activation of osteoclasts shares many well-known signaling pathways with that of inflammation, we investigated whether CAPE has any effect on osteoclastogenesis. CAPE potently suppressed osteoclastogenesis in cultures of bone marrow-derived precursor cells with the osteoclast differentiation factor, receptor activator of nuclear factor kappaB ligand (RANKL). While the RANKL-stimulated activation of the ERK, JNK, and p38 MAPK signaling pathways was not affected, the DNA binding and transcription activity of NF kappaB were reduced by CAPE treatment. In addition, CAPE blocked the induction of NFATc1 and c-Fos following RANKL stimulation. Forced expression of c-Fos could reverse the inhibitory effect of CAPE on osteoclastogenesis. Finally, CAPE significantly inhibited the RANKL-induced osteoclast formation in mouse calvariae in vivo. We propose that CAPE might be useful as a therapeutic agent for treatment of bone destructive diseases.

Negative Feedback Inhibition of NFATc1 by DYRK1A Regulates Bone Homeostasis

DYRK1A is a serine/threonine kinase that has been linked to mental retardation associated with Down syndrome. In the present report, we describe a previously unknown role for DYRK1A in bone homeostasis. The protein expression of DYRK1A increased during osteoclast differentiation. In vitro studies in osteoclasts revealed that DYRK1A inhibited osteoclastogenesis. Whereas DYRK1A phosphorylated and inhibited the osteoclastogenic transcription factor NFATc1, forced expression of NFATc1 induced DYRK1A expression, suggesting a negative feedback loop. Transgenic mice overexpressing DYRK1A by the extent of the increased gene dosage in Down syndrome exhibited significantly reduced bone mass despite the decreased osteoclastogenesis, which is reminiscent of osteoporotic bone phenotype in Down syndrome patients. In these mice, attenuated osteoblast differentiation and function in the presence of extra DYRK1A overrode the effect of impaired osteoclastogenesis. However, impeded osteoclastogenesis in DYRK1A transgenic mice was proven to be beneficial in protecting bone loss induced by inflammation or estrogen deficiency. These results provide novel insight into the role for DYRK1A in bone homeostasis as well as in bone destructive diseases, in which modulation of DYRK1A might be used as a strategy to treat unregulated bone resorption.

The JNK-dependent CaMK pathway restrains the reversion of committed cells during osteoclast differentiation

Osteoclastogenesis involves the commitment of macrophage-lineage precursors to tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear pre-osteoclasts (pOCs) and subsequent fusion of pOCs to form multinuclear mature osteoclasts. Despite many studies on osteoclast differentiation, little is known about the signaling mechanisms that specifically mediate the osteoclastic commitment. In this study, we found that inhibition of JNK at the pOC stage provoked reversion of TRAP+ cells to TRAP– cells. The conversion to TRAP– cells occurred with concomitant return to the state with higher expression of macrophage antigens, and greater activity of phagocytosis and dendritic-differentiation potential. JNK inhibition at the pOC stage reduced NFATc1 and CaMK levels, and addition of active NFATc1 partially rescued the effect of JNK inhibition. In addition, the level of NFATc1 was decreased by knockdown of CaMK by RNAi and by catalytic inhibition of CaMK, which both caused the reversion of pOCs to macrophages. These data suggest that JNK activity is specifically required for maintaining the committed status during osteoclastogenesis and that the CaMK-NFATc1 pathway is the key element in that specific role of JNK.

CXCL2 mediates lipopolysaccharide-induced osteoclastogenesis in RANKL-primed precursors.

The strong inflammatory agent lipopolysaccharide (LPS) has been shown to cause bone lysis in vivo. However, the etiology of LPS-induced bone destruction is still elusive. Here, we show that LPS stimulates the induction of CXCL2 in bone marrow macrophages (BMMs), osteoclast precursors, at the transcription level even in the absence of the synthesis of new proteins including interferon β. Reactive oxygen species were involved in the secretion of CXCL2 but not in the mRNA expression. CXCL2 mRNA induction by LPS was mediated by p38, JNK, and NFκB signaling pathways. Moreover, c-Fos and p65 were directly recruited to CXCL2 promoter. The conditioned medium from LPS-treated BMMs could enhance migration of osteoclast precursors, which was blocked by treatment with CXCL2-neutralizing antibody or CXCR2 receptor antagonist. The blockade of CXCL2 also reduced LPS-induced osteoclastogenesis. More significantly, CXCL2-neutralization prevented bone destruction in mice treated with LPS. Therefore, CXCL2 might be a useful therapeutic target for inflammatory bone destructive diseases.

AWP1 binds to tumor necrosis factor receptor-associated factor 2 (TRAF2) and is involved in TRAF2-mediated nuclear factor-kappaB signaling.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is an adaptor protein which involves in the activation of the transcription factor, nuclear factor kappaB (NF-κB), in the tumor necrosis factor (TNF) receptor pathway. This signaling is modulated by proteins that interact with tumor necrosis factor receptor-associated factor 2. In this study, we identified the zinc-finger protein AWP1 as a tumor necrosis factor receptor-associated factor 2-interacting protein through yeast two-hybrid screening. We found that AWP1 directly interacted with the C-terminal tumor necrosis factor receptor-associated factor (TRAF) domain of tumor necrosis factor receptor-associated factor 2. Knockdown of AWP1 using small hairpin RNA significantly decreased nuclear factor kappaB activity but increased tumor necrosis factor alpha (TNFα)-induced apoptosis, presumably by decreasing the induction of nuclear factor kappaB-responsive anti-apoptotic molecules, including FICE-like inhibitory protein (FLIP), X-linked inhibitor of apoptosis protein (XIAP), Bcl-2, and Bcl-xL. In contrast, overexpression of wild-type AWP1 inhibited nuclear factor kappaB activation. Detailed domain mapping experiments showed that the AN1 domain of AWP1 mediated the functional interaction with tumor necrosis factor receptor-associated factor 2, and the A20 domain was responsible for the negative regulation of nuclear factor kappaB activation. Importantly, ectopic AWP1 overexpression led to an A20 domain-dependent increase in K-48 ubiquitination of tumor necrosis factor receptor-associated factor 2. These data indicate that AWP1 binds to tumor necrosis factor receptor-associated factor 2 and that regulates tumor necrosis factor alpha-induced nuclear factor kappaB activation through two distinct domains.