Kazuhisa Nishishita

Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia

Abstract: Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome‐like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome‐like vacuoles and partly in the vesicles located in the trans‐Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46‐kDa polypeptide (reduced form) after a 30‐min pulse. Most of it was proteolytically processed via a 44‐kDa intermediate to a 42‐kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar‐type H+‐ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse‐labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high‐mannose oligosaccharides and processed to the active mature enzyme with complex‐type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome‐like acidic compartments.

Suppression of RANKL‐dependent heme oxygenase‐1 is required for high mobility group box 1 release and osteoclastogenesis

The differentiation of osteoclasts is regulated by several essential cytokines, such as receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony‐stimulating factor. Recently, high mobility group box 1 (HMGB1), a chromatin protein, also has been identified as one of these osteoclast differentiation cytokines. However, the molecular mechanisms that control HMGB1 release from osteoclast precursor cells are not known. Here, we report that RANKL‐induced suppression of heme oxygenase‐1 (HO‐1), a heme‐degrading enzyme, promotes HMGB1 release during osteoclastogenesis. In contrast, induction of HO‐1 with hemin or curcumin in bone marrow‐derived macrophages or RAW‐D murine osteoclast precursor cells inhibited osteoclastogenesis and suppressed HMGB1 release. Since an inhibitor for p38 mitogen‐activated protein kinase (MAPK) prevented the RANKL‐mediated HO‐1 suppression and extracellular release of HMGB1, these effects were p38 MAPK‐dependent. Moreover, suppression of HO‐1 in RAW‐D cells by RNA interference promoted the activation of caspase‐3 and HMGB1 release, whereas overexpression of HO‐1 inhibited caspase‐3 activation as well as HMGB1 release. Furthermore, these effects were regulated by redox conditions since antioxidant N‐acetylcysteine abolished the HO‐1/HMGB1/caspase‐3 axis. These results suggest that RANKL‐dependent HO‐1 suppression leads to caspase‐3 activation and HMGB1 release during osteoclastogenesis. J. Cell. Biochem. 113: 486–498, 2012.

Deltamethrin inhibits osteoclast differentiation via regulation of heme oxygenase-1 and NFATc1

Deltamethrin is a widely used pyrethroid pesticide. Although the cytotoxicity of deltamethrin has been reported, especially in neuronal cells, there is no information concerning the effects of deltamethrin on osteoclasts (OCLs). In this study, we showed that deltamethrin inhibited OCL differentiation in vitro. The effects of deltamethrin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand (RANKL) were investigated in bone marrow-derived macrophages (BMMs) or the murine monocytic cell line RAW-D. Treatment with deltamethrin inhibited OCL formation and bone resorption and up-regulated expression of heme oxygenase-1 (HO-1), an anti-oxidative stress enzyme. Deltamethrin also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator for OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of deltamethrin on intracellular signaling during the OCL differentiation of BMMs indicated that deltamethrin-treated OCLs displayed impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Jun N-terminal kinase, and Akt, and slightly delayed phosphorylation of inhibitor of nuclear factor kappa B alpha (IκBα) compared with untreated OCLs. Thus, deltamethrin possibly affects bone metabolism by inhibiting OCL differentiation.

Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice

Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H2O2) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

The Rho-specific guanine nucleotide exchange factor Plekhg5 modulates cell polarity, adhesion, migration, and podosome organization in macrophages and osteoclasts

ABSTRACT Osteoclasts are multinucleated bone‐resorbing cells that are formed by fusion of monocyte/macrophage lineage. Osteoclasts and macrophages generate podosomes that are actin‐based dynamic organelles implicated in cell adhesion, spreading, migration, and degradation. However, the detailed mechanisms of podosome organization remain unknown. Here, we identified the Rho‐specific guanine‐nucleotide exchange factor (Rho‐GEF) Plekhg5 as an up‐regulated gene during differentiation of osteoclasts from macrophages. Knockdown of Plekhg5 with small interfering RNA in both macrophages and osteoclasts induced larger cell formation with impaired cell polarity and resulted in an elongated and flattened shape. In macrophages, Plekhg5 depletion enhanced random migration, but impaired directional migration, adhesion, and matrix degradation. Plekhg5 in osteoclasts affected random migration, podosome organization, and bone resorption. Plekhg5 depletion affected signaling and localization of several Rho downstream effectors. In fact, end‐binding protein 1 (EB1), cofilin and vinculin were abnormally localized in Plekhg5‐depleted cells, and mDia1 and LIM kinase (LIMK)1 were upregulated in Plekhg5‐depleted cells compared with control cells. However, overexpression of Plekhg5 in macrophages induced an increase in its mRNA level, but failed to increase the protein level, indicating that overexpressed Plekhg5 was degraded in macrophages but not HEK293T cells. Thus, Plekhg5 affects cell polarity, migration, adhesion, degradation, and podosome organization in macrophages and osteoclasts. HIGHLIGHTSKnockdown of Plekhg5 in both macrophages and osteoclasts induced larger formation with impaired cell polarity and an elongated and flattened shape.Plekhg5 depletion in macrophages impaired directional migration, adhesion, and matrix degradation.Plekhg5 in osteoclasts affected random migration, podosome organization, and bone resorption.

The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway

Osteoblasts are bone‐forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up‐regulated during osteoblast differentiation using mouse osteoblastic MC3T3‐E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down‐stream effector molecules, TFEB KD was found to cause marked up‐regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer‐binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB‐regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP‐dependent signaling pathway. J. Cell. Physiol. 231: 1321–1333, 2016.

Inhibitory effects of tert-butylhydroquinone on osteoclast differentiation via up-regulation of heme oxygenase-1 and down-regulation of HMGB1 release and NFATc1 expression

Osteoclasts (OCLs) are multinucleated bone‐resorbing cells that are differentiated by receptor activator of nuclear factor kappa‐B ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). Our recent studies have shown that heme‐oxygenase‐1 (HO‐1), a stress‐induced cytoprotective enzyme, plays an important role in OCL differentiation, although the pharmacological significance of this effect remains unknown. In this study, we investigated the effects of tert‐butylhydroquinone (tBHQ), a pharmacological HO‐1 inducer, on in vitro differentiation of bone marrow‐derived macrophages (BMMs) or murine monocytic cell line RAW‐D into OCLs. tBHQ inhibited the formation and the bone‐resorbing activity of OCLs. Moreover, tBHQ treatment decreased the expression of nuclear factor of activated T cells cytoplasmic‐1 (NFATc1), a master regulator of OCL differentiation, and of OCL markers transcriptionally regulated by NFATc1, such as Src and cathepsin K. In addition, tBHQ impaired phosphorylation of extracellular signal‐regulated kinase, p38 mitogen‐activated protein kinase (MAPK), Jun N‐terminal kinase, Akt, and inhibitor of nuclear factor kappa B alpha (IκBα). Finally, we show that tBHQ inhibited the release of high mobility group box 1 (HMGB1), a recently identified activator of OCL differentiation. Thus, tBHQ inhibits OCL differentiation through the HO‐1/HMGB1 pathways. Copyright

Cafestol has a weaker inhibitory effect on osteoclastogenesis than kahweol and promotes osteoblast differentiation

Bone homeostasis is regulated by a balance between osteoclast (OCL)-mediated bone resorption and osteoblast (OBL)-mediated bone formation. Thus, developing a compound that simultaneously inhibits OCL function and promotes OBL function would be useful as a new medical therapy for bone diseases. Here, we examined the effects of cafestol, a coffee diterpene, on the differentiation of OCLs and OBLs. Cafestol prevented OCL formation in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Interestingly, the viability of OCLs treated with 10-50 µM cafestol was significantly higher than that of untreated cells. At the molecular level, cafestol markedly decreased RANKL-induced phosphorylation of extracellular signal-regulated kinase (Erk) and inhibitor of nuclear factor kappa B alpha (IκBα). Compared to kahweol, another coffee-specific diterpene, the inhibitory effects of cafestol were milder on OCL differentiation, and cafestol and kahweol showed different characteristics in induction of the phase ΙΙ antioxidant enzymes and sensitivities in nuclear factor-erythroid 2-related factor 2 (Nrf2)-deficient BMMs. In addition to inhibiting OCLs, cafestol enhanced the differentiation of osteoblastic cells by increasing the mRNA levels of differentiation markers. Thus, cafestol inhibits OCL differentiation and promotes OBL differentiation, suggesting that cafestol may be a novel agent for bone diseases.

Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation.

Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant‐derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin‐treated murine osteoblastic MC3T3‐E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone‐resorption activity through slightly decreased the phosphorylation of extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, and inhibitor of nuclear factor kappa Bα; however, the phosphorylation of Akt and p38 slightly increased in bone marrow‐derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T‐cell cytoplasmic‐1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases. Copyright

Cobalt protoporphyrin represses osteoclastogenesis through blocking multiple signaling pathways

Cobalt protoporphyrin (CoPP) is a metallo-protoporphyrin that works as a powerful inducer of heme oxigenase-1 (HO-1) in various tissues and cells. Our recent studies have demonstrated that induction of HO-1 by several reagents inhibited differentiation and activation of osteoclasts (OCLs), which are multinucleated bone resorbing cells. However, the effects of CoPP on osteoclastogenesis remain to be elucidated. In this study, we report that CoPP inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced OCL formation in a dose dependent manner. Importantly, CoPP had little cytotoxicity, but rather enhanced cell proliferation of OCLs. CoPP suppressed the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) as well as those of OCLs markers such as Src and cathepsin K, which are transcriptionally regulated by NFATc1 in mature OCLs. Western blot analyses also showed that CoPP abolished RANKL-stimulated phosphorylation of several major signaling pathways such as IκB, Akt, ERK, JNK and p38 MAPKs in OCL precursor cells. Thus, our results show that CoPP represses osteoclastogenesis through blocking multiple signaling pathways.