Heterogeneous osteoimmune profiles via single-cell transcriptomics in osteoporotic patients who fail bisphosphonate treatment.

Postmenopausal osteoporosis arises from imbalanced osteoclast and osteoblast activity, and mounting evidence suggests a role for the osteoimmune system in bone homeostasis. Bisphosphonate (BP) is an antiresorptive agent, but its treatment failure rate can be as high as 40%. Here, we performed single-cell RNA sequencing on peripheral immune cells from carefully selected postmenopausal women: non-osteoporotic, osteoporosis improved after BP treatment, and BP-failed cases. We found an increase in myeloid cells in patients with osteoporosis (specifically, T cell receptor+ macrophages). Furthermore, lymphoid lineage cells varied significantly, notably elevated natural killer cells (NKs) in the BP-failed group. Moreover, we provide fruitful lists of biomarkers within the immune cells that exhibit condition-dependent differences. The existence of osteoporotic- and BP-failure-specific cellular information flows was revealed by cell-cell interaction analysis. These findings deepen our insight of the osteoporosis pathology enhancing comprehension of the role of immune heterogeneity in postmenopausal osteoporosis and BP treatment failure.

Transmembrane protein 150b attenuates BMP signaling in the Xenopus organizer.

The vertebrate organizer is a specified embryonic tissue that regulates dorsoventral patterning and axis formation. Although numerous cellular signaling pathways have been identified as regulators of the organizer's dynamic functions, the process remains incompletely understood, and as-yet unknown pathways remain to be explored for sophisticated mechanistic understanding of the vertebrate organizer. To identify new potential key factors of the organizer, we performed complementary DNA (cDNA) microarray screening using organizer-mimicking Xenopus laevis tissue. This analysis yielded a list of prospective organizer genes, and we determined the role of six-transmembrane domain containing transmembrane protein 150b (Tmem150b) in organizer function. Tmem150b was expressed in the organizer region and induced by Activin/Nodal signaling. In X. laevis, Tmem150b knockdown resulted in head defects and a shortened body axis. Moreover, Tmem150b negatively regulated bone morphogenetic protein (BMP) signaling, likely via physical interaction with activin receptor-like kinase 2 (ALK2). These findings demonstrated that Tmem150b functions as a novel membrane regulatory factor of BMP signaling with antagonistic effects, contributing to the understanding of regulatory molecular mechanisms of organizer axis function. Investigation of additional candidate genes identified in the cDNA microarray analysis could further delineate the genetic networks of the organizer during vertebrate embryogenesis.

Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1.

Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt signaling essential for embryogenesis and homeostasis. However, whether the antagonism of Wnt signaling that is necessary for vertebrate head formation can be achieved through regulation of Dsh protein stability is unclear. Here, we show that membrane-associated RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes Wnt signaling by regulating the turnover of Dsh protein via ubiquitin-mediated lysosomal degradation in the prospective head region of Xenopus We further found that March2 acquires regional and functional specificities for head formation from the Dsh-interacting protein Dapper1 (Dpr1). Dpr1 stabilizes the interaction between March2 and Dsh in order to mediate ubiquitylation and the subsequent degradation of Dsh protein only in the dorso-animal region of Xenopus embryo. These results suggest that March2 restricts cytosolic pools of Dsh protein and reduces the need for Wnt signaling in precise vertebrate head development.

Tbx2 regulates anterior neural specification by repressing FGF signaling pathway.

During early embryogenesis, FGF signals regulate the antero-posterior (AP) patterning of the neural plate by promoting posterior cell fates. In particular, BMP signal-mediated attenuation of FGF pathway plays a critical role in the determination of the anterior neural region. Here we show that Tbx2, a T-box transcriptional repressor regulates anterior neural specification by suppressing FGF8 signaling pathway in Xenopus embryo. Tbx2 is expressed in the anterior edge of the neural plate in early neurulae. Overexpression and knockdown of Tbx2 induce expansion and reduction in the expression of anterior neural markers, respectively. It also suppresses FGF8-induced ERK phosphorylation and neural caudalization. Tbx2, which is a target gene of BMP signal, down-regulates FGF8 signaling by inhibiting the expression of Flrt3, a positive regulator of this pathway. We found that Tbx2 binds directly to the T-box element located in the promoter region of Flrt3 gene, thereby interfering with the activity of the promoter. Consistently, Tbx2 augmentation of anterior neural formation is inhibited by co-expression of Flrt3. Furthermore, disruption of the anterior-most structures such as eyes in Tbx2-depleted embryos can be rescued by inhibition of Flrt3 function or FGF signaling. Taken together, our results suggest that Tbx2 mediates BMP signal to down-regulate FGF signaling pathway by repressing Flrt3 expression for anterior tissue formation.

PLD1 regulates Xenopus convergent extension movements by mediating Frizzled7 endocytosis for Wnt/PCP signal activation.

Phospholipase D (PLD) is involved in the regulation of receptor-associated signaling, cell movement, cell adhesion and endocytosis. However, its physiological role in vertebrate development remains poorly understood. In this study, we show that PLD1 is required for the convergent extension (CE) movements during Xenopus gastrulation by activating Wnt/PCP signaling. Xenopus PLD1 protein is specifically enriched in the dorsal region of Xenopus gastrula embryo and loss or gain-of-function of PLD1 induce defects in gastrulation and CE movements. These defective phenotypes are due to impaired regulation of Wnt/PCP signaling pathway. Biochemical and imaging analysis using Xenopus tissues reveal that PLD1 is required for Fz7 receptor endocytosis upon Wnt11 stimulation. Moreover, we show that Fz7 endocytosis depends on dynamin and regulation of GAP activity of dynamin by PLD1 via its PX domain is crucial for this process. Taken together, our results suggest that PLD1 acts as a new positive mediator of Wnt/PCP signaling by promoting Wnt11-induced Fz7 endocytosis for precise regulation of Xenopus CE movements.

Overexpression of MicA induces production of OmpC-enriched outer membrane vesicles that protect against Salmonella challenge.

Outer membrane vesicles (OMVs) derived from bacteria are promising candidates for subunit vaccines. Stresses that modulate the composition of outer membrane proteins (OMPs) are important for OMV synthesis. Small RNAs (sRNAs) expressed in response to stress regulate OMPs, although the mechanism underlying sRNA-mediated OMV biogenesis and its utility for developing vaccine platforms remains to be elucidated. Here, we characterized the role of a sRNA, MicA, which regulates OmpA, a major OMP involved in both production of OMVs and reactive immunity against Salmonella challenge. A Salmonella strain overexpressing MicA generated more OMVs than a control strain. In addition, OmpC was the major component of MicA-derived OMV proteins. MicA-derived OMVs induced Th1- and Th17-type immune responses in vitro and reduced Salmonella-mediated lethality in a mouse model. Thus, OmpA-regulatory sRNA-derived OMVs may facilitate production of Salmonella-protective vaccines.

Dual functions of DP1 promote biphasic Wnt-on and Wnt-off states during anteroposterior neural patterning.

DP1, a dimerization partner protein of the transcription factor E2F, is known to inhibit Wnt/ß-catenin signalling along with E2F, although the function of DP1 itself was not well characterized. Here, we present a novel dual regulatory mechanism of Wnt/ß-catenin signalling by DP1 independent from E2F. DP1 negatively regulates Wnt/ß-catenin signalling by inhibiting Dvl-Axin interaction and by enhancing poly-ubiquitination of ß-catenin. In contrast, DP1 positively modulates the signalling upon Wnt stimulation, via increasing cytosolic ß-catenin and antagonizing the kinase activity of NLK. In Xenopus embryos, DP1 exerts both positive and negative roles in Wnt/ß-catenin signalling during anteroposterior neural patterning. From subcellular localization analyses, we suggest that the dual roles of DP1 in Wnt/ß-catenin signalling are endowed by differential nucleocytoplasmic localizations. We propose that these dual functions of DP1 can promote and stabilize biphasic Wnt-on and Wnt-off states in response to a gradual gradient of Wnt/ß-catenin signalling to determine differential cell fates.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Role of Tbx2 in defining the territory of the pronephric nephron.

Despite extensive study of the development of the nephron, which is the functional unit of the kidney, the molecular mechanisms underlying the determination of nephron size remain largely unknown. Using the Xenopus pronephros, we demonstrate here that Tbx2, a T-box transcriptional repressor, functions to demarcate the territory of the pronephric nephron. Tbx2 is specifically expressed around three distinct components of the pronephric nephron: the tubule, duct and glomus. Gain of function of Tbx2 inhibits nephric mesoderm formation. Conversely, Tbx2 loss of function expands the boundary of each component of the pronephric nephron, resulting in an enlarged pronephros. BMP signals induce Tbx2 in the non-nephric mesoderm, which inhibits the expression of the nephric markers Hey1 and Gremlin. Importantly, these pronephric molecules repress Tbx2 expression by antagonizing BMP signals in the nephric mesoderm. These results suggest that the negative regulatory loops between BMP/Tbx2 and Gremlin or Hey1 are responsible for defining the territory of the pronephric nephron.

Role of Sp5 as an essential early regulator of neural crest specification in xenopus.

BACKGROUND: The neural crest (NC) is a multipotent embryonic cell population, which is induced by an integration of secreted signals including BMP, Wnt, and FGF and, subsequently, NC cell fates are specified by a regulatory network of specific transcription factors. This study was undertaken to identify a role of Sp5 transcription factor in vertebrates. RESULTS: Xenopus Sp5 is expressed in the prospective neural crest regions from gastrulation through the tadpole stages in early development. Knockdown of Sp5 caused severe defects in craniofacial cartilage, pigmentation, and dorsal fin. Gain- and loss-of-function of Sp5 led to up- and down-regulation of the expression of NC markers in the neural fold, respectively. In contrast, Sp5 had no effect on neural induction and patterning. Sp5 regulated the expression of neural plate border (NPB) specifiers, Msx1 and Pax3, and these regulatory factors recovered the expression of NC marker in the Sp5-deficient embryos. Depletion of Sp5 impaired NC induction by Wnt/ß-catenin or FGF signal, whereas its co-expression rescued NC markers in embryos in which either signal was blocked. CONCLUSIONS: These results suggest that Sp5 functions as a critical early factor in the genetic cascade to regulate NC induction downstream of Wnt and FGF pathways.

Selective fluorescent detection of RNA in living cells by using imidazolium-based cyclophane.

A water-soluble imidazolium-based fluorescent chemosensor senses RNA selectively through fluorescence enhancement over other biologically relevant biomolecules in aqueous solution at physiological pH 7.4. Fluorescence image detection of RNA in living cells such as onion cells, HeLa cells, and animal model cells was successfully demonstrated which displays a chelation-enhanced fluorescence effect. These affinities can be attributed to the strong electrostatic (C-H)(+)···A(-) ionic H-bonding and the aromatic moiety driven π-stacking of imidazolium-based cyclophane with the size-complementary major groove of RNA.

BMP signal attenuates FGF pathway in anteroposterior neural patterning.

In vertebrate early development, the neural tissue is specified along the antero-posterior (A-P) axis by the activity of graded patterning signals such as Wnt, Nodal and FGF. Attenuation of these signals has been shown to play critical roles in the determination of anterior neural region, but it remains poorly understood how FGF action is counteracted in the neural plate. Here, we show that BMP signal acts as an antagonist of FGF signaling for AP neural patterning in Xenopus embryo. During the neurula stages, BMP signal was up-regulated in the anterior neural plate, displaying a graded pattern along the AP axis. Inhibition of the late BMP signaling after mid-gastrulation abrogated the expression of anterior neural markers. We found that BMP signaling interfered with FGFs-induced ERK phosphorylation and neural caudalization. This inhibitory action of BMP signal involved repression of the expression of Flrt3, a positive regulator of FGF signaling. Furthermore, the gain- and loss-of-function of Flrt3 inhibited and expanded the expression of forebrain marker genes, respectively. Together, these results demonstrate that BMP signal can down-regulate FGF pathway via inhibition of Flrt3 expression for anterior neural formation, revealing stage-specific roles of BMP signaling and its novel crosstalk with FGF pathway in neural development.

ß-Arrestin 1 mediates non-canonical Wnt pathway to regulate convergent extension movements.

ß-Arrestins are multifaceted proteins that play critical roles in termination of G protein-coupled receptor (GPCR) signaling by inducing its desensitization and internalization as well as in facilitation of many intracellular signaling pathways. Here, we examine using Xenopus embryos whether ß-arrestin 1 might act as a mediator of ß-catenin-independent Wnt (non-canonical) signaling. Xenopus ß-arrestin 1 (xßarr1) is expressed in the tissues undergoing extensive cell rearrangements in early development. Gain- and loss-of-function analyses of xßarr1 revealed that it regulates convergent extension (CE) movements of mesodermal tissue with no effect on cell fate specification. In addition, rescue experiments showed that xßarr1 controls CE movements downstream of Wnt11/Fz7 signal and via activation of RhoA and JNK. In line with this, xßarr1 associated with key Wnt components including Ryk, Fz, and Dishevelled. Furthermore, we found that xßarr1 could recover CE movements inhibited by xßarr2 knockdown or its endocytosis defective mutant. Overall, these results suggest that ß-arrestin 1 and 2 share interchangeable endocytic activity to regulate CE movements downstream of the non-canonical Wnt pathway.

Dab2 is pivotal for endothelial cell migration by mediating VEGF expression in cancer cells.

Although angiogenesis is crucial for tumor growth and metastasis, the molecular mechanisms controlling this process are not clearly understood. Here, we explore the role of Dab2 in tumor angiogenesis. We found that Dab2 is expressed in several cancer cells, including A549 lung cancer cells, but it is hardly detectable in SW480 colon cancer cells. Migration and Erk phosphorylation were enhanced in human umbilical vein endothelial cells (HUVECs) treated with the conditioned medium obtained from Dab2-overexpressing SW480 stable cells. In addition, vascular endothelial growth factor (VEGF) protein was strongly detected in conditioned medium derived from Dab2-overexpressing SW480 cells, and Erk phosphorylation enhanced by Dab2(+) CM was restored by VEGF inhibition. Moreover, Dab2 depletion in A549 cells led to a decrease in HUVEC migration and Erk phosphorylation. Furthermore, we show that Dab2 is required for the TGFß-induced gene expression of angiogenic factors such as VEGF and FGF2. Taken together, these results suggest that Dab2, which is expressed in cancer cells, is pivotal for endothelial cell migration by affecting VEGF expression.

RNF2 regulates Wnt/ß-catenin signaling via TCF7L1 destabilization.

The Wnt signaling pathway is a crucial regulator of various biological processes, such as development and cancer. The downstream transcription factors in this pathway play a vital role in determining the threshold for signaling induction and the length of the response, which vary depending on the biological context. Among the four transcription factors involved in canonical Wnt/ß-catenin signaling, TCF7L1 is known to possess an inhibitory function; however, the underlying regulatory mechanism remains unclear. In this study, we identified the E3 ligase, RNF2, as a novel positive regulator of the Wnt pathway. Here, we demonstrate that RNF2 promotes the degradation of TCF7L1 through its ubiquitination upon activation of Wnt signaling. Loss-of-function studies have shown that RNF2 consistently destabilizes nuclear TCF7L1 and is required for proper Wnt target gene transcription in response to Wnt activation. Furthermore, our results revealed that RNF2 controls the threshold, persistence, and termination of Wnt signaling by regulating TCF7L1. Overall, our study sheds light on the previously unknown degradation mechanism of TCF7L1 by a specific E3 ligase, RNF2, and provides new insights into the variability in cellular responses to Wnt activation.

The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements.

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph-ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.

Rab3d is required for Xenopus anterior neurulation by regulating Noggin secretion.

Rab3d is a member of the Ras-related small GTPase family of secretory Rab, Rab3. In this study, we showed that Xenopus Rab3d is expressed specifically in the anterior border of the neural plate when the neural plate converges and folds to initiate neural tube formation. Morpholino-mediated knockdown of Rab3d resulted in neurulation defects both in neural plate convergence and folding. Interestingly, perturbation of BMP signaling rescued neurulation defects of Rab3d morphants, suggesting that Rab3d inhibits BMP signaling during neurulation. By secretion assay in the Xenopus animal cap, we found that Rab3d specifically regulates secretion of a BMP antagonist, Noggin, but not Chordin and Wnts. We also found that Rab3d is co-localized with Noggin and that this interaction is dependent on the GTP/GDP cycle of Rab3d. Collectively, these findings suggest that Rab3d-mediated secretion regulation of a BMP antagonist, Noggin, is one of the mechanisms of BMP antagonism during Xenopus anterior neurulation.

UCHL5 controls ß-catenin destruction complex function through Axin1 regulation.

Wnt/ß-catenin signaling is crucially involved in many biological processes, from embryogenesis to cancer development. Hence, the complete understanding of its molecular mechanism has been the biggest challenge in the Wnt research field. Here, we identified ubiquitin C-terminal hydrolase like 5 (UCHL5), a deubiquitinating enzyme, as a novel negative regulator of Wnt signaling, upstream of ß-catenin. The study further revealed that UCHL5 plays an important role in the ß-catenin destruction complex, as it physically interacts with multiple domains of Axin1 protein. Our functional analyses also elucidated that UCHL5 is required for both the stabilization and the polymerization of Axin1 proteins. Interestingly, although these events are governed by deubiquitination in the DIX domain of Axin1 protein, they do not require the deubiquitinating activity of UCHL5. The study proposes a novel molecular mechanism of UCHL5 potentiating the functional activity of Axin1, a scaffolder of the ß-catenin destruction complex.

Identification of a novel negative regulator of activin/nodal signaling in mesendodermal formation of Xenopus embryos.

Phosphotyrosine binding (PTB) domains, which are found in a large number of proteins, have been implicated in signal transduction mediated by growth factor receptors. However, the in vivo roles of these PTB-containing proteins remain to be investigated. Here, we show that Xdpcp (Xenopus dok-PTB containing protein) has a pivotal role in regulating mesendoderm formation in Xenopus, and negatively regulates the activin/nodal signaling pathway. We isolated cDNA for xdpcp and examined its potential role in Xenopus embryogenesis. We found that Xdpcp is strongly expressed in the animal hemisphere at the cleavage and blastula stages. The overexpression of xdpcp RNA affects activin/nodal signaling, which causes defects in mesendoderm formation. In addition, loss of Xdpcp function by injection of morpholino oligonucleotides leads to the expansion of the mesodermal territory. Moreover, we found that axis duplication by ventrally forced expression of activin is recovered by coexpression with Xdpcp. In addition, Xdpcp inhibits the phosphorylation and nuclear translocation of Smad2. Furthermore, we also found that Xdpcp interacts with Alk4, a type I activin receptor, and inhibits activin/nodal signaling by disturbing the interaction between Smad2 and Alk4. Taken together, these results indicate that Xdpcp regulates activin/nodal signaling that is essential for mesendoderm specification.

Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the ß-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2-SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.