Multiple lines of evidence for disruption of nuclear lamina and nucleoporins in FUS amyotrophic lateral sclerosis.

Advanced pathological and genetic approaches have revealed that mutations in fused in sarcoma/translated in liposarcoma (FUS/TLS), which is pivotal for DNA repair, alternative splicing, translation and RNA transport, cause familial amyotrophic lateral sclerosis (ALS). The generation of suitable animal models for ALS is essential for understanding its pathogenesis and developing therapies. Therefore, we used CRISPR-Cas9 to generate FUS-ALS mutation in the non-classical nuclear localization signal (NLS), H517D (mouse position: H509D) and genome-edited mice. Fus WT/H509D mice showed progressive motor impairment (accelerating rotarod and DigiGait system) with age, which was associated with the loss of motor neurons and disruption of the nuclear lamina and nucleoporins and DNA damage in spinal cord motor neurons. We confirmed the validity of our model by showing that nuclear lamina and nucleoporin disruption were observed in lower motor neurons differentiated from patient-derived human induced pluripotent stem cells (hiPSC-LMNs) with FUS-H517D and in the post-mortem spinal cord of patients with ALS. RNA sequence analysis revealed that most nuclear lamina and nucleoporin-linking genes were significantly decreased in FUS-H517D hiPSC-LMNs. This evidence suggests that disruption of the nuclear lamina and nucleoporins is crucial for ALS pathomechanisms. Combined with patient-derived hiPSC-LMNs and autopsy samples, this mouse model might provide a more reliable understanding of ALS pathogenesis and might aid in the development of therapeutic strategies.

Affinity-tagged SMAD1 and SMAD5 mouse lines reveal transcriptional reprogramming mechanisms during early pregnancy.

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci (Smad1HA/HA and Smad5PA/PA). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers (IGFBP1, PRL, FOXO1) and PR-responsive genes (RORB, KLF15). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy.

GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation.

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

ADAD2 functions in spermiogenesis and piRNA biogenesis in mice.

BACKGROUND: Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive. OBJECTIVES: This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2. MATERIALS AND METHODS: Adad2 homozygous mutant mouse lines, Adad2-/- and Adad2Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes. RESULTS: Adad2-/- and Adad2Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs. DISCUSSION AND CONCLUSION: In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Development of transgenic mice overexpressing mouse carbonyl reductase 1.

BACKGROUND: Carbonyl reductase 1 (CBR1) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with broad substrate specificity. CBR1 catalyzes the reduction of numerous carbonyl compounds, including quinones, prostaglandins, menadione, and multiple xenobiotics, while also participating in various cellular processes, such as carcinogenesis, apoptosis, signal transduction, and drug resistance. In this study, we aimed to generate transgenic mice overexpressing mouse Cbr1 (mCbr1), characterize the mCbr1 expression in different organs, and identify changes in protein expression patterns. METHODS AND RESULTS: To facilitate a deeper understanding of the functions of CBR1, we generated transgenic mice overexpressing CBR1 throughout the body. These transgenic mice overexpress 3xFLAG-tagged mCbr1 (3xFLAG-mCbr1) under the CAG promoter. Two lines of transgenic mice were generated, one with 3xFLAG-mCbr1 expression in multiple tissues, and the other, with specific expression of 3xFLAG-mCbr1 in the heart. Pathway and network analysis using transgenic mouse hearts identified 73 proteins with levels of expression correlating with mCbr1 overexpression. The expression of voltage-gated anion channels, which may be directly related to calcium ion-related myocardial contraction, was also upregulated. CONCLUSION: mCbr1 transgenic mice may be useful for further in vivo analyses of the molecular mechanisms regulated by Cbr1; such analyses will provide a better understanding of its effects on carcinogenesis and cardiotoxicity of certain cancer drugs.

Retrovirus-derived RTL5 and RTL6 genes are novel constituents of the innate immune system in the eutherian brain.

Retrotransposon Gag-like 5 [RTL5, also known as sushi-ichi-related retrotransposon homolog 8 (SIRH8)] and RTL6 (also known as SIRH3) are eutherian-specific genes presumably derived from a retrovirus and phylogenetically related to each other. They, respectively, encode a strongly acidic and extremely basic protein, and are well conserved among the eutherians. Here, we report that RTL5 and RTL6 are microglial genes with roles in the front line of innate brain immune response. Venus and mCherry knock-in mice exhibited expression of RTL5-mCherry and RTL6-Venus fusion proteins in microglia and appeared as extracellular dots and granules in the central nervous system. These proteins display a rapid response to pathogens such as lipopolysaccharide (LPS), double-stranded (ds) RNA analog and non-methylated CpG DNA, acting both cooperatively and/or independently. Experiments using Rtl6 or Rtl5 knockout mice provided additional evidence that RTL6 and RTL5 act as factors against LPS and dsRNA, respectively, in the brain, providing the first demonstration that retrovirus-derived genes play a role in the eutherian innate immune system. Finally, we propose a model emphasizing the importance of extra-embryonic tissues as the origin site of retrovirus-derived genes. This article has an associated 'The people behind the papers' interview.

T Cell-Intrinsic Vitamin A Metabolism and Its Signaling Are Targets for Memory T Cell-Based Cancer Immunotherapy.

Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T-cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

Establishment of mouse model of inherited PIGO deficiency and therapeutic potential of AAV-based gene therapy.

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is caused by mutations in GPI biosynthesis genes. The mechanisms of its systemic, especially neurological, symptoms are not clarified and fundamental therapy has not been established. Here, we report establishment of mouse models of IGD caused by PIGO mutations as well as development of effective gene therapy. As the clinical manifestations of IGD are systemic and lifelong lasting, we treated the mice with adeno-associated virus for homology-independent knock-in as well as extra-chromosomal expression of Pigo cDNA. Significant amelioration of neuronal phenotypes and growth defect was achieved, opening a new avenue for curing IGDs.

Kastor and Polluks polypeptides encoded by a single gene locus cooperatively regulate VDAC and spermatogenesis.

Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility.

MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells.

The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice.

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules and some attached projections. Radial spokes (RSs) facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 radial spoke proteins (RSP1-RSP23), although the roles of some radial spoke proteins remain unknown. Recently, RSP15 has been reported to be bound to the stalk of RS2, but its homolog in mammals has not been identified. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A and RSPH3B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was not significantly affected. These data indicate that LRRC23 is a key regulator that underpins the integrity of the RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. This article has an associated First Person interview with the first author of the paper.

Rubicon prevents autophagic degradation of GATA4 to promote Sertoli cell function.

Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon, a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon-null mice also had lower levels of mRNAs of Sertoli cell-related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.

Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment.

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.

Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis.

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.

A novel tissue specific alternative splicing variant mitigates phenotypes in Ets2 frame-shift mutant models.

E26 avian leukemia oncogene 2, 3' domain (Ets2) has been implicated in various biological processes. An Ets2 mutant model (Ets2db1/db1), which lacks the DNA-binding domain, was previously reported to exhibit embryonic lethality caused by a trophoblast abnormality. This phenotype could be rescued by tetraploid complementation, resulting in pups with wavy hair and curly whiskers. Here, we generated new Ets2 mutant models with a frame-shift mutation in exon 8 using the CRISPR/Cas9 method. Homozygous mutants could not be obtained by natural mating as embryonic development stopped before E8.5, as previously reported. When we rescued them by tetraploid complementation, these mice did not exhibit wavy hair or curly whisker phenotypes. Our newly generated mice exhibited exon 8 skipping, which led to in-frame mutant mRNA expression in the skin and thymus but not in E7.5 Ets2em1/em1 embryos. This exon 8-skipped Ets2 mRNA was translated into protein, suggesting that this Ets2 mutant protein complemented the Ets2 function in the skin. Our data implies that novel splicing variants incidentally generated after genome editing may complicate the phenotypic analysis but may also give insight into the new mechanisms related to biological gene functions.

Astrocytic cAMP modulates memory via synaptic plasticity.

Astrocytes play a key role in brain homeostasis and functions such as memory. Specifically, astrocytes express multiple receptors that transduce signals via the second messenger cAMP. However, the involvement of astrocytic cAMP in animal behavior and the underlying glial-neuronal interactions remains largely unknown. Here, we show that an increase in astrocytic cAMP is sufficient to induce synaptic plasticity and modulate memory. We developed a method to increase astrocytic cAMP levels in vivo using photoactivated adenylyl cyclase and found that increased cAMP in hippocampal astrocytes at different time points facilitated memory formation but interrupted memory retention via NMDA receptor-dependent plasticity. Furthermore, we found that the cAMP-induced modulation of memory was mediated by the astrocyte-neuron lactate shuttle. Thus, our study unveils a role of astrocytic cAMP in brain function by providing a tool to modulate astrocytic cAMP in vivo.

NAIL: an evolutionarily conserved lncRNA essential for licensing coordinated activation of p38 and NFκB in colitis.

OBJECTIVE: NFκB is the key modulator in inflammatory disorders. However, the key regulators that activate, fine-tune or shut off NFκB activity in inflammatory conditions are poorly understood. In this study, we aim to investigate the roles that NFκB-specific long non-coding RNAs (lncRNAs) play in regulating inflammatory networks. DESIGN: Using the first genetic-screen to identify NFκB-specific lncRNAs, we performed RNA-seq from the p65-/- and Ikkß-/- mouse embryonic fibroblasts and report the identification of an evolutionary conserved lncRNA designated mNAIL (mice) or hNAIL (human). hNAIL is upregulated in human inflammatory disorders, including UC. We generated mNAILΔNFκB mice, wherein deletion of two NFκB sites in the proximal promoter of mNAIL abolishes its induction, to study its function in colitis. RESULTS: NAIL regulates inflammation via sequestering and inactivating Wip1, a known negative regulator of proinflammatory p38 kinase and NFκB subunit p65. Wip1 inactivation leads to coordinated activation of p38 and covalent modifications of NFκB, essential for its genome-wide occupancy on specific targets. NAIL enables an orchestrated response for p38 and NFκB coactivation that leads to differentiation of precursor cells into immature myeloid cells in bone marrow, recruitment of macrophages to inflamed area and expression of inflammatory genes in colitis. CONCLUSION: NAIL directly regulates initiation and progression of colitis and its expression is highly correlated with NFκB activity which makes it a perfect candidate to serve as a biomarker and a therapeutic target for IBD and other inflammation-associated diseases.

PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins.

PGAP6, also known as TMEM8A, is a phospholipase A2 with specificity to glycosylphosphatidylinositol (GPI) and expressed on the surface of various cells. CRIPTO, a GPI-anchored co-receptor for a morphogenic factor Nodal, is a sensitive substrate of PGAP6. PGAP6-mediated shedding of CRIPTO plays a critical role in an early stage of embryogenesis. In contrast, CRYPTIC, a close family member of CRIPTO, is resistant to PGAP6. In this report, chimeras between CRIPTO and CRYPTIC and truncate mutants of PGAP6 were used to demonstrate that the Cripto-1/FRL1/Cryptic domain of CRIPTO is recognized by an N-terminal domain of PGAP6 for processing. We also report that among 56 human GPI-anchored proteins tested, only glypican 3, prostasin, SPACA4, and contactin-1, in addition to CRIPTO, are sensitive to PGAP6, indicating that PGAP6 has a narrow specificity toward various GPI-anchored proteins.

Protocadherin-7 contributes to maintenance of bone homeostasis through regulation of osteoclast multinucleation.

Osteoclasts are hematopoietic-derived cells that resorb bone. They are required to maintain proper bone homeostasis and skeletal strength. Although osteoclast differentiation depends on receptor activator of NF-κB ligand (RANKL) stimulation, additional molecules further contribute to osteoclast maturation. Here, we demonstrate that protocadherin-7 (Pcdh7) regulates formation of multinucleated osteoclasts and contributes to maintenance of bone homeostasis. We found that Pcdh7 expression is induced by RANKL stimulation, and that RNAi-mediated knockdown of Pcdh7 resulted in impaired formation of osteoclasts. We generated Pcdh7-deficient mice and found increased bone mass due to decreased bone resorption but without any defect in bone formation. Using an in vitro culture system, it was revealed that formation of multinucleated osteoclasts is impaired in Pcdh7-deficient cultures, while no apparent defects were observed in differentiation and function of Pcdh7-deficient osteoblasts. Taken together, these results reveal an osteoclast cell-intrinsic role for Pcdh7 in maintaining bone homeostasis. [BMB Reports 2020; 53(9): 472-477].