Inhibition of protein phosphatase 2A by okadaic acid induces translocation of nucleocytoplasmic O-GlcNAc transferase.

Post-translational modification (PTM) is crucial for many biological events, such as the modulation of bone metabolism. Phosphorylation and O-GlcNAcylation are two examples of PTMs that can occur at the same site in the protein: serine and threonine residues. This phenomenon may cause crosstalk and possible interactions between the molecules involved. Protein phosphatase 2 A (PP2A) is widely expressed throughout the body and plays a major role in dephosphorylation. At the same location where PP2A acts, O-GlcNAc transferase (OGT) can introduce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) molecules and mediates O-GlcNAc modifications. To examine the effects of PP2A inhibition on OGT localization and expression, osteoblastic MC3T3-E1 cells were treated with Okadaic Acid (OA), a potent PP2A inhibitor. In the control cells, OGT was strictly localized in the nucleus. However, OGT was observed diffusely in the cytoplasm of the OA-treated cells. This change in localization from the nucleus to the cytoplasm resulted from an increase in mitochondrial OGT expression and translocation of the nucleocytoplasmic isoform. Furthermore, knockdown of PP2A catalytic subunit α isoform (PP2A Cα) significantly affected OGT expression (p < 0.05), and there was a correlation between PP2A Cα and OGT expression (r = 0.93). These results suggested a possible interaction between PP2A and OGT, which strengthens the notion of an interaction between phosphorylation and O-GlcNAcylation.

Osteoblast Jmjd3 regulates osteoclastogenesis via EphB4 and RANKL signalling.

BACKGROUND: Osteoblasts suppress osteoclastogenesis during the reversal phase of bone remodelling and the mechanism needs to be further investigated. Here, we investigated the role of histone demethylase Jumonji domain-containing 3 (Jmjd3) in osteoblasts on regulating osteoclastogenesis. METHODS: Jmjd3 expression was silenced in osteoblasts. Osteoblasts and osteoclasts were co-cultured in direct or indirect contact ways, and osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining and Western blotting. Additionally, Ephrin receptor B4 (EphB4) and receptor activator of nuclear factor-kappa Β ligand (RANKL) expression were quantified in osteoblasts via real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. Subsequently, EphB4 was overexpressed in osteoblasts and RANKL expression and osteoclastogenesis was quantified. RESULTS: Osteoclastogenesis and marker protein expression levels was promoted when osteoclasts were co-cultured with Jmjd3-silenced osteoblasts. Silencing of Jmjd3 expression in osteoblasts decreased EphB4 expression, owing to suppression of demethylation of H3K27me3 on the promoter region of EphB4. Whereas RANKL expression was upregulated in Jmjd3-silenced osteoblasts. Overexpression of EphB4 in osteoblasts inhibited osteoclastogenesis and RANKL expression. CONCLUSION: Jmjd3 in osteoblasts is a crucial regulator of osteoblast-to-osteoclast communication through EphB4-EphrinB2, RANKL-RANK and EphB4-RANKL signalling axes, suggesting the pivotal role of Jmjd3 in bone remodelling process in bone destruction disease such as chronic apical periodontitis.

Neuropilin 1 (NRP1) Positively Regulates Adipogenic Differentiation in C3H10T1/2 Cells.

Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for several ligands, is highly expressed in many kinds of mesenchymal stem cells (MSCs), but its function is poorly understood. In this study, we explored the roles of full-length NRP1 and glycosaminoglycan (GAG)-modifiable NRP1 in adipogenesis in C3H10T1/2 cells. The expression of full-length NRP1 and GAG-modifiable NRP1 increased during adipogenic differentiation in C3H10T1/2 cells. NRP1 knockdown repressed adipogenesis while decreasing the levels of Akt and ERK1/2 phosphorylation. Moreover, the scaffold protein JIP4 was involved in adipogenesis in C3H10T1/2 cells by interacting with NRP1. Furthermore, overexpression of non-GAG-modifiable NRP1 mutant (S612A) greatly promoted adipogenic differentiation, accompanied by upregulation of the phosphorylated Akt and ERK1/2. Taken together, these results indicate that NRP1 is a key regulator that promotes adipogenesis in C3H10T1/2 cells by interacting with JIP4 and activating the Akt and ERK1/2 pathway. Non-GAG-modifiable NRP1 mutant (S612A) accelerates the process of adipogenic differentiation, suggesting that GAG glycosylation is a negative post-translational modification of NRP1 in adipogenic differentiation.

Vestigial-Like 3 Plays an Important Role in Osteoblast Differentiation by Regulating the Expression of Osteogenic Transcription Factors and BMP Signaling.

Our previous gene profiling analysis showed that the transcription cofactor vestigial-like 3 (VGLL3) gene expression was upregulated by mechanical tension in the mouse cranial suture, coinciding with accelerated osteoblast differentiation. Therefore, we hypothesized that VGLL3 plays a significant role in osteogenic differentiation. To clarify the function of VGLL3 in osteoblasts, we examined its expression characteristics in mouse bone tissue and the osteoblastic cell line MC3T3-E1. We further examined the effects of Vgll3 knockdown on osteoblast differentiation and bone morphogenetic protein (BMP) signaling. In the mouse cranial suture, where membranous ossification occurs, VGLL3 was immunohistochemically detected mostly in the nucleus of osteoblasts, preosteoblasts, and fibroblastic cells. VGLL3 expression in MC3T3-E1 cells was transient and peaked at a relatively early stage of differentiation. RNA sequencing revealed that downregulated genes in Vgll3-knockdown cells were enriched in gene ontology terms associated with osteoblast differentiation. Interestingly, most of the upregulated genes were related to cell division. Targeted Vgll3 knockdown markedly suppressed the expression of major osteogenic transcription factors (Runx2, Sp7/osterix, and Dlx5) and osteoblast differentiation. It also attenuated BMP signaling; moreover, exogenous BMP2 partially restore osteogenic transcription factors' expression in Vgll3-knockdown cells. Furthermore, overexpression of Vgll3 increased the expression of osteogenic transcription factors. These results suggest that VGLL3 plays a critical role in promoting osteoblast differentiation and that part of the process is mediated by BMP signaling. Further elucidation of VGLL3 function will increase our understanding of osteogenesis and skeletal disease etiology.

Histone Demethylase Jmjd3 Regulates the Osteogenic Differentiation and Cytokine Expressions of Periodontal Ligament Cells.

Periodontal ligament (PDL) cells are critical for the bone remodeling process in periapical lesions since they can differentiate into osteoblasts and secrete osteoclastogenesis-promoting cytokines. Post-translational histone modifications including alterations of the methylation status of H3K27 are involved in cell differentiation and inflammatory reaction. The histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes methylation of H3K27. We investigated whether Jmjd3 is involved in the osteogenic differentiation and secretion of PDL cells' inflammatory factors. Jmjd3 expression in periapical lesions was examined by immunostaining. Using siRNA specific for Jmjd3 or the specific Jmjd3 inhibitor GSK-J4, we determined Jmjd3's roles in osteogenic differentiation and cytokine production by real-time RT-PCR. The locations of Jmjd3 and NF-κB were analyzed by immunocytochemistry. Compared to healthy PDLs, the periapical lesion samples showed higher Jmjd3 expression. Treatment with GSK-J4 or Jmjd3 siRNA suppressed PDL cells' osteogenic differentiation by suppressing the expressions of bone-related genes (Runx2, Osterix, and osteocalcin) and mineralization. Jmjd3 knockdown decreased the expressions of cytokines (TNF-α, IL-1ß, and IL-6) induced by lipopolysaccharide extracted from Porphyromonas endodontalis (Pe-LPS). Pe-LPS induced the nuclear translocations of Jmjd3 and NF-κB; the latter was inhibited by GSK-J4 treatment. Jmjd3 appears to regulate PDL cells' osteogenic differentiation and proinflammatory cytokine expressions.

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors.

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

Upregulated osterix expression elicited by Runx2 and Dlx5 is required for the accelerated osteoblast differentiation in PP2A Cα-knockdown cells.

Serine/threonine protein phosphatase 2A (PP2A) is involved in regulating various physiological processes including cell cycle, growth, apoptosis, and signal transduction. Osteoblast differentiation is controlled by main bone specific transcription factors including Osterix, distal-less homeobox 5 (Dlx5), and Runt-related transcription factor 2 (Runx2). We previously reported that knockdown of PP2A Cα increases the expression of Osterix, leading to the accelerated osteoblast differentiation through the upregulation of bone-related genes. In this study, we examined whether Dlx5 and Runx2 are involved in the upregulated Osterix expression in PP2A Cα-knockdown osteoblasts (shPP2A cells). The expression of Dlx5 as well as Osterix was significantly higher in shPP2A cells in the initial stage of osteoblast differentiation compared with the control cells (shCont). The expression of Runx2 protein was also higher in shPP2A cells compared with shCont cells although its mRNA level was comparable. Reduction of Dlx5 or Runx2 decreased Osterix expression and alkaline phosphatase activity in shPP2A cells. Luciferase assay showed that Osterix promoter activity was drastically elevated in shPP2A cells compared with that in shCont cells. The deletion or mutation of the Dlx5 and Runx2 binding sites significantly suppressed Osterix promoter activity in shPP2A cells. These results indicate that Dlx5 and Runx2 are critical factors for the upregulated Osterix expression in shPP2A cells, which is considered to be important for the accelerated osteoblast differentiation in these cells.

PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts.

The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-κB activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-κB and also induced NLRP3 expression in osteoblasts. Inhibition of NF-κB attenuated SNAP-P. g.-induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-κB and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-κB. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-κB pathway in periodontal diseases.

Histone demethylase Jmjd3 regulates osteoblast apoptosis through targeting anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bim.

Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.

Protein phosphatase 2A Cα regulates proliferation, migration, and metastasis of osteosarcoma cells.

Osteosarcoma is the most frequent primary bone tumor. Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes, such as cell cycle, growth, apoptosis, and signal transduction. In this study, we examined the expression and function of PP2A Cα in osteosarcoma cells. PP2A Cα expression was expected to be higher in malignant osteosarcoma tissues. PP2A Cα expression level and PP2A activity was higher in malignant osteosarcoma LM8 cells compared with that in primary osteoblasts and in the osteoblast-like cell line MC3T3-E1. Okadaic acid, an inhibitor of PP2A, reduced cell viability and induced apoptosis in LM8 cells. PP2A Cα-knockdown LM8 cells (shPP2A) exhibited less striking filopodial and lamellipodial structures than that in original LM8 cells. Focal adhesion kinase phosphorylation and NF-κB activity decreased in shPP2A-treated cells. Sensitivity to serum deprivation-induced apoptosis increased in shPP2A-treated cells, accompanied by a lower expression level of anti-apoptotic BCL-2 in these cells. Reduction of PP2A Cα resulted in a decrease in the migration ability of LM8 cells in vitro. Reduction in PP2A Cα levels in vivo suppressed proliferation and metastasis in LM8 cells. PP2A Cα expression was also higher in human osteosarcoma MG63 and SaOS-2 cells than that in primary osteoblasts and MC3T3-E1 cells, and reduction in PP2A Cα levels suppressed the cell proliferation rate and migration ability of MG63 cells. These results indicate that PP2A Cα has a critical role in the proliferation and metastasis of osteosarcoma cells; therefore, its inhibition could potentially suppress the malignancy of osteosarcoma cells.

Reduction of PP2A Cα stimulates adipogenesis by regulating the Wnt/GSK-3ß/ß-catenin pathway and PPARγ expression.

Serine/threonine protein phosphatase 2A (PP2A) regulates several physiological processes such as the cell cycle, cell growth, apoptosis, and signal transduction. In this study, we examined the expression and role of PP2A Cα in adipocyte differentiation. PP2A Cα expression and PP2A activity decreased during adipocyte differentiation in C3H10T1/2 and 3T3-L1 cells and the expression of adipocyte marker genes such as PPARγ and adiponectin increased. To further clarify the role of PP2A Cα in adipocyte differentiation, we constructed PP2A knockdown cells by infecting C3H10T1/2 cells with a lentivirus expressing a shRNA specific for the PP2A Cα (shPP2A cells). Silencing of PP2A Cα in C3H10T1/2 cells dramatically stimulated adipocyte differentiation and lipid accumulation, which were accompanied by expression of adipocyte marker genes. Silencing of PP2A Cα suppressed Wnt10b expression and reduced the levels of the inactivated form of GSK-3ß (phospho-GSK-3ß), leading to the reduction of ß-catenin levels in the nucleus and its transcriptional activity. Treatment with LiCl, a GSK-3ß inhibitor, and inhibition of PPARγ expression suppressed the accelerated adipogenesis of shPP2A cells. Our data indicate that PP2A Cα plays an important role in the regulation of adipocyte differentiation by regulating the Wnt/GSK-3ß/ß-catenin pathway and PPARγ expression.

Histone demethylase Utx regulates differentiation and mineralization in osteoblasts.

Alteration of methylation status of lysine 27 on histone H3 (H3K27) associates with dramatic changes in gene expression in response to various differentiation signals. Demethylation of H3K27 is controlled by specific histone demethylases including ubiquitously transcribed tetratricopeptide repeat X chromosome (Utx). However, the role of Utx in osteoblast differentiation remains unknown. In this study, we examined whether Utx should be involved in osteoblast differentiation. Expression of Utx increased during osteoblast differentiation in MC3T3-E1 cells and primary osteoblasts. GSK-J1, a potent inhibitor of H3K27 demethylase, increased the levels of trimethylated H3K27 (H3K27me3) and decreased the expressions of Runx2 and Osterix and ALP activity in MC3T3-E1 cells. Stable knockdown of Utx by shRNA attenuated osteoblast differentiation and decreased ALP activity, calcium content, and bone-related gene expressions. Silencing of Utx increased the level of H3K27me3 on the promoter regions of Runx2 and Osterix and decreased the promoter activities of Runx2 and Osterix. Taken together, our present results propose that Utx plays important roles in osteoblast differentiation by controlling the expressions of Runx2 and Osterix.

Double Stranded RNA-Dependent Protein Kinase is Necessary for TNF-α-Induced Osteoclast Formation In Vitro and In Vivo.

Double-stranded RNA-dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF-α plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF-α-induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF-α-induced osteoclastogenesis. The TNF-α-induced osteoclast differentiation in bone marrow-derived macrophages and RAW264.7 cells was markedly suppressed by the pretreatment of PKR inhibitor, 2-aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K, and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF-α-induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF-α-induced activation of NF-κB and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF-κB and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF-α-induced expression of NFATc1 and c-fos, master transcription factors in osteoclastogenesis. TNF-α-induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF-α-induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF-α-induced osteoclast differentiation in vitro and in vivo.

Histone demethylase Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and osterix.

Post-translational modifications of histones including methylation play important roles in cell differentiation. Jumonji domain-containing 3 (Jmjd3) is a histone demethylase, which specifically catalyzes the removal of trimethylation of histone H3 at lysine 27 (H3K27me3). In this study, we examined the expression of Jmjd3 in osteoblasts and its roles in osteoblast differentiation. Jmjd3 expression in the nucleus was induced in response to the stimulation of osteoblast differentiation as well as treatment of bone morphogenetic protein-2 (BMP-2). Either treatment with Noggin, an inhibitor of BMP-2, or silencing of Smad1/5 suppressed Jmjd3 expression during osteoblast differentiation. Silencing of Jmjd3 expression suppressed osteoblast differentiation through the expression of bone-related genes including Runx2, osterix, osteopontin, bone sialoprotein (BSP), and osteocalcin (OCN). Silencing of Jmjd3 decreased the promoter activities of Runx2 and osterix and increased the level of H3K27me3 on the promoter regions of Runx2 and osterix. Introduction of the exogenous Runx2 and osterix partly rescued osteoblast differentiation in the shJmjd3 cells. The present results indicate that Jmjd3 plays important roles in osteoblast differentiation and regulates the expressions of BSP and OCN via transcription factors Runx2 and osterix.

Oral Porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3ß signaling pathway.

Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3ß induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis.

Protein phosphatase 2A Cα regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor.

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation.

Mechanical stretching determines the orientation of osteoblast migration and cell division.

Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical stretching controls osteoblast morphology and alignment. However, little is known about its effects on osteoblast migration. Here, we investigated changes in the morphology and migration of preosteoblastic MC3T3-E1 cells after the removal of continuous or cyclic stretching. Actin staining and time-lapse recording were performed after stretching removal. The continuous and cyclic groups showed parallel and perpendicular alignment to the stretch direction, respectively. A more elongated cell morphology was observed in the cyclic group than in the continuous group. In both stretch groups, the cells migrated in a direction roughly consistent with the cell alignment. Compared to the other groups, the cells in the cyclic group showed an increased migration velocity and were almost divided in the same direction as the alignment. To summarize, our study showed that mechanical stretching changed cell alignment and morphology in osteoblasts, which affected the direction of migration and cell division, and velocity of migration. These results suggest that mechanical stimulation may modulate the direction of bone tissue formation by inducing the directional migration and cell division of osteoblasts.

Interaction between PKR and PACT mediated by LPS-inducible NF-κB in human gingival cells.

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 µg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-κB to the nucleus after 30 min, and inhibition of NF-κB decreased the PACT-PKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT-PKR association in Sa3 cells. Our results also suggest that NF-κB is involved in the PACT-PKR interaction and the production of pro-inflammatory cytokines in periodontitis.

Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction.

To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses' whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta.

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.