Lmo7 recruits myosin II heavy chain to regulate actomyosin contractility and apical domain size in Xenopus ectoderm.

Apical constriction, or a reduction in size of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction; however, the detailed analysis of molecular mechanisms is still pending. Here, we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is primarily localized at apical junctions and promotes the formation of the dense circumferential actomyosin belt. Strikingly, Lmo7 binds non-muscle myosin II (NMII) and recruits it to apical junctions and the apical cortex. This NMII recruitment is essential for Lmo7-mediated apical constriction. Lmo7 knockdown decreases NMIIA localization at apical junctions and delays neural tube closure in Xenopus embryos. Our findings suggest that Lmo7 serves as a scaffold that regulates actomyosin contractility and apical domain size.

Adipocyte-specific GPRC6A ablation promotes diet-induced obesity by inhibiting lipolysis.

The G protein-coupled receptor GPRC6A regulates various physiological processes in response to its interaction with multiple ligands, such as extracellular basic amino acids, divalent cations, testosterone, and the uncarboxylated form of osteocalcin (GluOC). Global ablation of GPRC6A increases the susceptibility of mice to diet-induced obesity and related metabolic disorders. However, given that GPRC6A is expressed in many tissues and responds to a variety of hormonal and nutritional signals, the cellular and molecular mechanisms underlying the development of metabolic disorders in conventional knockout mice have remained unclear. On the basis of our previous observation that long-term oral administration of GluOC markedly reduced adipocyte size and improved glucose tolerance in WT mice, we examined whether GPRC6A signaling in adipose tissue might be responsible for prevention of metabolic disorders. We thus generated adipocyte-specific GPRC6A knockout mice, and we found that these animals manifested increased adipose tissue weight, adipocyte hypertrophy, and adipose tissue inflammation when fed a high-fat and high-sucrose diet compared with control mice. These effects were associated with reduced lipolytic activity because of downregulation of lipolytic enzymes such as adipose triglyceride lipase and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of adipose triglyceride lipase in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.

Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos.

Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein beneath the plasma membrane that functions to control epithelial morphogenesis. Here we report a previously uncharacterized role of EPB41L5 in controlling ciliary function. We found that EPB41L5 forms a complex with IQCB1 (previously known as NPHP5), a ciliopathy protein. Overexpression of EPB41L5 reduced IQCB1 localization at the ciliary base in cultured mammalian epithelial cells. Conversely, epb41l5 knockdown increased IQCB1 localization at the ciliary base. epb41l5-deficient zebrafish embryos or embryos expressing C-terminally modified forms of Epb41l5 developed cilia with reduced motility and exhibited left-right patterning defects, an outcome of abnormal ciliary function. We observed genetic synergy between epb41l5 and iqcb1. Moreover, EPB41L5 decreased IQCB1 interaction with CEP290, another ciliopathy protein and a component of the ciliary base and centrosome. Together, these observations suggest that EPB41L5 regulates the composition of the ciliary base and centrosome through IQCB1 and CEP290.

NFAT5 promotes oral squamous cell carcinoma progression in a hyperosmotic environment.

Epidermal growth factor receptor (EGFR) is highly expressed in several types of cancer cells including oral squamous cell carcinoma (OSCC). EGF/EGFR signaling is recognized as an important molecular target in cancer therapy. However, cancer cells often become tolerant to EGF/EGFR signaling-targeted therapies. In the tumor microenvironment, the tumor incites inflammation and the inflammation-derived cytokines make a considerable impact on cancer development. In addition, hyperosmolarity is also induced, but the role of osmotic stress in cancer development has not been fully understood. This study demonstrates molecular insights into hyperosmolarity effect on OSCC development and shows that NFAT5 transcription factor plays an important functional role in enhancing the oral cancer cell proliferation by inducing the EGFR translocation from the endoplasmic reticulum to the plasma membrane through increase the expression of DPAGT1, an essential enzyme for catalyzing the first committed step of N-linked protein glycosylation. These results suggest that hyperosmolarity-induced intra-nuclear translocation of NFAT5 essential for DPAGT1 activation and EGFR subcellular translocation responsible for OSCC tumor progression.

Bone morphogenetic protein induces bone invasion of melanoma by epithelial-mesenchymal transition via the Smad1/5 signaling pathway.

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial-mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.

Kif1c regulates osteoclastic bone resorption as a downstream molecule of p130Cas.

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c-Src (c-Src-/- ) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk-associated substrate [Cas]) as one of c-Src downstream molecule and osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) mice also exhibited a similar phenotype to c-Src-/- mice, indicating that the c-Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c-Src-/- or p130CasΔOCL-/- mice with wild-type (WT) osteoclasts to identify downstream molecules of c-Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c-Src-/- and p130CasΔOCL-/- osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c-Src-/- and p130CasΔOCL-/- osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL-/- osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). SIGNIFICANCE OF THE STUDY: We previously showed that the c-Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c-Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild-type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas.

Bif-1/Endophilin B1/SH3GLB1 regulates bone homeostasis.

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 -/- ) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 -/- mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 -/- mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 -/- mice. Treatment with ß-glycerophosphate (ß-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 -/- mice. Finally, bone marrow cells from Bif-1 -/- mice showed a significantly higher colony-forming efficacy by the treatment with or without ß-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 -/- mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).

Epb41l5 competes with Delta as a substrate for Mib1 to coordinate specification and differentiation of neurons.

We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

Phospholipase C-related but catalytically inactive proteins regulate ovarian follicle development.

Phospholipase C-related but catalytically inactive proteins PRIP-1 and -2 are inositol-1,4,5-trisphosphate binding proteins that are encoded by independent genes. Ablation of the Prip genes in mice impairs female fertility, which is manifested by fewer pregnancies, a decreased number of pups, and the decreased and increased secretion of gonadal steroids and gonadotropins, respectively. We investigated the involvement of the PRIPs in fertility, focusing on the ovaries of Prip-1 and -2 double-knock-out (DKO) mice. Multiple cystic follicles were observed in DKO ovaries, and a superovulation assay showed a markedly decreased number of ovulated oocytes. Cumulus-oocyte complexes showed normal expansion, and artificial gonadotropin stimulation regulated the ovulation-related genes in a normal fashion, suggesting that the ovulation itself was probably normal. A histological analysis showed atresia in fewer follicles of the DKO ovaries, particularly in the secondary follicle stages. The expression of luteinizing hormone receptor (LHR) was aberrantly higher in developing follicles, and the phosphorylation of extracellular signal-regulated protein kinase, a downstream target of LH-LHR signaling, was higher in DKO granulosa cells. This suggests that the up-regulation of LH-LHR signaling is the cause of impaired follicle development. The serum estradiol level was lower, but estradiol production was unchanged in the DKO ovaries. These results suggest that PRIPs are positively involved in the development of follicles via their regulation of LH-LHR signaling and estradiol secretion. Female DKO mice had higher serum levels of insulin, testosterone, and uncarboxylated osteocalcin, which, together with reduced fertility, are reminiscent of polycystic ovary syndrome in humans.

Phospholipase C-related, but catalytically inactive protein (PRIP) up-regulates osteoclast differentiation via calcium-calcineurin-NFATc1 signaling.

Phospholipase C-related, but catalytically inactive protein (PRIP) was previously identified as a novel inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ but lacking phospholipase activity. We recently showed that PRIP gene knock-out (KO) in mice increases bone formation and concomitantly decreases bone resorption, resulting in increased bone mineral density and trabecular bone volume. However, the role of PRIP in osteoclastogenesis has not yet been fully elucidated. Here, we investigated the effects of PRIP on bone remodeling by investigating dynamic tooth movement in mice fitted with orthodontic devices. Morphological analysis indicated that the extent of tooth movement was smaller in the PRIP-KO mice than in wild-type mice. Histological analysis revealed fewer osteoclasts on the bone-resorption side in maxillary bones of PRIP-KO mice, and osteoclast formation assays and flow cytometry indicated lower osteoclast differentiation in bone marrow cells isolated from these mice. The expression of genes implicated in bone resorption was lower in differentiated PRIP-KO cells, and genes involved in osteoclast differentiation, such as the transcription factor NFATc1, exhibited lower expression in immature PRIP-KO cells initiated by M-CSF. Moreover, calcineurin expression and activity were also lower in the PRIP-KO cells. The PRIP-KO cells also displayed fewer M-CSF-induced changes in intracellular Ca2+ and exhibited reduced nuclear localization of NFATc1. Up-regulation of intracellular Ca2+ restored osteoclastogenesis of the PRIP-KO cells. These results indicate that PRIP deficiency impairs osteoclast differentiation, particularly at the early stages, and that PRIP stimulates osteoclast differentiation through calcium-calcineurin-NFATc1 signaling via regulating intracellular Ca2.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.

Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium.

Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells.

Lef1 regulates Dusp6 to influence neuromast formation and spacing in the zebrafish posterior lateral line primordium.

The posterior lateral line primordium (PLLp) migrates caudally and periodically deposits neuromasts. Coupled, but mutually inhibitory, Wnt-FGF signaling systems regulate proto-neuromast formation in the PLLp: FGF ligands expressed in response to Wnt signaling activate FGF receptors and initiate proto-neuromast formation. FGF receptor signaling, in turn, inhibits Wnt signaling. However, mechanisms that determine periodic neuromast formation and deposition in the PLLp remain poorly understood. Previous studies showed that neuromasts are deposited closer together and the PLLp terminates prematurely in lef1-deficient zebrafish embryos. It was suggested that this results from reduced proliferation in the leading domain of the PLLp and/or premature incorporation of progenitors into proto-neuromasts. We found that rspo3 knockdown reduces proliferation in a manner similar to that seen in lef1 morphants. However, it does not cause closer neuromast deposition or premature termination of the PLLp, suggesting that such changes in lef1-deficient embryos are not linked to changes in proliferation. Instead, we suggest that they are related to the role of Lef1 in regulating the balance of Wnt and FGF functions in the PLLp. Lef1 determines expression of the FGF signaling inhibitor Dusp6 in leading cells and regulates incorporation of cells into neuromasts; reduction of Dusp6 in leading cells in lef1-deficient embryos allows new proto-neuromasts to form closer to the leading edge. This is associated with progressively slower PLLp migration, reduced spacing between deposited neuromasts and premature termination of the PLLp system.

Involvement of PRIP (Phospholipase C-Related But Catalytically Inactive Protein) in BMP-Induced Smad Signaling in Osteoblast Differentiation.

Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.

Clinicopathological significance of MMP-7, laminin γ2 and EGFR expression at the invasive front of gastric carcinoma.

BACKGROUND: For several types of cancer, including gastric cancer (GC), tumor cells at the invasive front are considered to have a more aggressive behavior compared with those in the more central region. The aim of the present study was to analyze the expression of MMP-7, laminin γ2 and EGFR in a large number of GCs and to investigate how these expression patterns correlate with clinicopathologic parameters, infiltrative patterns, histology or mucin phenotype. METHODS: We immunohistochemically examined the expression of MMP-7, laminin γ2 and EGFR using a tissue microarray analysis of 790 GCs, and evaluated their clinicopathological significance. RESULTS: MMP-7, cytoplasmic laminin γ2, extracellular laminin γ2 and EGFR expression were observed in 25, 25, 8 and 21 % of the 790 GC cases, respectively. Expression of MMP-7, cytoplasmic laminin γ2 and EGFR was associated with advanced T grade, N grade and tumor stage. Extracellular laminin γ2 expression was not associated with any clinicopathologic parameters, infiltrative patterns, histology or mucin phenotype. Furthermore, we investigated the correlations of MMP-7, laminin γ2 and EGFR expression. MMP-7 expression was significantly more frequent in positive expression of cytoplasmic laminin γ2 than negative cases, and EGFR expression was significantly more frequent in positive expression of cytoplasmic laminin γ2 and MMP-7. CONCLUSIONS: Molecular expression of MMP-7, laminin γ2 or EGFR, and their combinations, may be associated with GC tumor aggressiveness. Assessment of expression of these molecules at the invasive front of primary tumors is clinically significant in predicting the malignant behavior of GC.

Use of fibrin monomer complex for screening for venous thromboembolism in the late pregnancy and post-partum period.

AIM: We measured fibrin monomer complex (FMC) levels in all subjects who gave birth at our hospital and evaluated the feasibility of using FMC for screening for venous thromboembolism (VTE) in patients during late pregnancy and the post-partum period. METHODS: From August 2010 to January 2012, all women who gave birth at our hospital were included. FMC and D-dimer levels were determined during the late pregnancy and post-partum periods. Compression ultrasonography of the lower extremities was performed in women with high FMC values. RESULTS: Of the 673 women enrolled, measurements were performed in 595 women (88.4%) during late pregnancy and in 610 women (90.6%) during the post-partum period. The FMC levels were normal during late pregnancy in 400 women (67.2%) and during the post-partum period in 399 women (78.5%) having vaginal delivery and 83 women (81.4%) who underwent a cesarean section. The FMC levels were abnormal during late pregnancy in 50 women (8.4%) and during the post-partum period in nine women (1.8%) having vaginal delivery and in none (0%) who underwent a cesarean section. Ultrasonography detected thrombi in three (6.0%) women during late pregnancy. The FMC levels were strongly correlated with D-dimer levels (R = 0.726, P < 0.0001, in late pregnancy; and R = 0.888, P < 0.0001, in the post-partum period following vaginal delivery). CONCLUSION: FMC levels could identify pregnancy-related abnormalities requiring compression ultrasonography examination, without changing the cut-off values for non-pregnant individuals. Thus, this marker may be used to screen for VTE.

Prickle2 regulates apical junction remodeling and tissue fluidity during vertebrate neurulation.

The process of folding the flat neuroectoderm into an elongated neural tube depends on tissue fluidity, a property that allows epithelial deformation while preserving tissue integrity. Neural tube folding also requires the planar cell polarity (PCP) pathway. Here, we report that Prickle2 (Pk2), a core PCP component, increases tissue fluidity by promoting the remodeling of apical junctions (AJs) in Xenopus embryos. This Pk2 activity is mediated by the unique evolutionarily conserved Ser-Thr-rich region (STR) in the carboxyterminal half of the protein. Mechanistically, the effects of Pk2 require Rac1 and are accompanied by increased cadherin dynamics and destabilization of tricellular junctions, the hotspots of AJ remodeling. Notably, Pk2 depletion leads to the accumulation of mediolaterally oriented cells in the neuroectoderm, whereas the overexpression of Pk2 or Pk1 containing the Pk2-derived STR promotes cell elongation along the anteroposterior axis. We propose that Pk2-dependent regulation of tissue fluidity contributes to anteroposterior tissue elongation in response to extrinsic cues.

Phosphorylation of Serine 536 of p65(RelA) Downregulates Inflammatory Responses.

Nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of various genes involved in inflammatory diseases and immune responses. Recently, a novel transcriptional regulatory mechanism of NF-κB involving the phosphorylation of serine 536 (534 in mice; S534) of its p65 subunit was reported; however, further research is required to elucidate the physiological role of S534 phosphorylation. Therefore, we generated S534A knock-in (KI) mice, in which the S534 of p65 was substituted with alanine. Similar to the wild-type (WT) mice, S534A KI mice developed normally. After stimulation with tumor necrosis factor α (TNFα), mouse embryonic fibroblasts (MEFs) derived from S534A KI mice exhibited increased target gene expression compared with that in the WT MEFs, which was induced by long-term binding of p65 to DNA. According to comprehensive gene expression analysis after stimulation with TNFα, the expression of genes p65ted to inflammatory and immune responses was increased, and the expression of genes p65ted to lipolysis was decreased in S534A KI MEFs. Analyses of a periodontal disease model established using WT and S534A KI mice revealed that alveolar bone resorption was enhanced in S534A KI mice owing to an increase in the number of osteoclasts, which was not attributed to the differentiation of osteoclast precursor cells but to an increased expression of interleukin-1ß and receptor activator of NF-κB ligand in the periodontal tissue. Hence, phosphorylation of S536 negatively regulates inflammatory responses in vitro and in vivo.

Activation of NF-κB signaling regulates ovariectomy-induced bone loss and weight gain.

Postmenopausal women experience bone loss and weight gain. To date, crosstalk between estrogen receptor signals and nuclear factor-κB (NF-κB) has been reported, and estrogen depletion enhances bone resorption by osteoclasts via NF-κB activation. However, it is unclear when and in which tissues NF-κB is activated after menopause, and how NF-κB acts as a common signaling molecule for postmenopausal weight gain and bone loss. Therefore, we examined the role of NF-κB in bone and energy metabolism following menopause. NF-κB reporter mice, which can be used to measure NF-κB activation in vivo, were ovariectomized (OVX) and the luminescence intensity after OVX increased in the metaphyses of the long bones and perigonadal white adipose tissue, but not in the other tissues. OVX was performed on wild-type (WT) and p65 mutant knock-in (S534A) mice, whose mutation enhances the transcriptional activity of NF-κB. Weight gain with worsening glucose tolerance was significant in S534A mice after OVX compared with those of WT mice. The bone density of the sham group in WT or S534A mice did not change, whereas in the S534A-OVX group it significantly decreased due to the suppression of bone formation and increase in bone marrow adipocytes. Disulfiram, an anti-alcoholic drug, suppressed OVX-induced activation of NF-κB in the metaphyses of long bones and white adipose tissue (WAT), as well as weight gain and bone loss. Overall, the activation of NF-κB in the metaphyses of long bones and WAT after OVX regulates post-OVX weight gain and bone loss.

Phospholipase C-related catalytically inactive protein, a novel microtubule-associated protein 1 light chain 3-binding protein, negatively regulates autophagosome formation.

Upon starvation, cells undergo autophagy, an intracellular bulk-degradation process, to provide the required nutrients. Here, we observed that phospholipase C-related catalytically inactive protein (PRIP) binds to microtubule-associated protein 1 light chain 3 (LC3), a mammalian autophagy-related initiator that regulates the autophagy pathway. Then, we examined the involvement of PRIP in the nutrient depletion-induced autophagy pathway. Enhanced colocalization of PRIP with LC3 was clearly seen in nutrient-starved mouse embryonic fibroblasts under a fluorescent microscope, and interaction of the proteins was revealed by immunoprecipitation experiments with an anti-LC3 antibody. Under starvation conditions, there were more green fluorescent protein fused-LC3 dots in mouse embryonic fibroblasts from PRIP-deficient mice than in fibroblasts from wild type cells. The formation of new dots in a single cell increased, as assessed by time-lapse microscopy. Furthermore, the increase in autophagosome formation in PRIP-deficient cells was notably inhibited by exogenously overexpressed PRIP. Taken together, PRIP is a novel LC3-binding protein that acts as a negative modulator of autophagosome formation.