Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin β1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin β1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.

Treatment of patients with advanced sarcoma remains challenging due to lack of effective medicine, with the development of novel drugs being of keen interest. A pan-PI3K inhibitor, ZSTK474, has been evaluated in clinical trials against a range of advanced solid tumors, with clinical benefit shown in sarcoma patients. In the present study, we developed a panel of 14 human sarcoma cell lines and investigated the antitumor effect of 24 anticancer agents including ZSTK474, other PI3K inhibitors, and those clinically used for sarcoma treatment. ZSTK474 exhibited a similar antiproliferative profile to other PI3K inhibitors but was clearly different from the other drugs examined. Indeed, ZSTK474 inhibited PI3K-downstream pathways, in parallel to growth inhibition, in all cell lines examined, showing proof-of-concept of PI3K inhibition. In addition, ZSTK474 induced apoptosis selectively in Ewing's sarcoma (RD-ES and A673), alveolar rhabdomyosarcoma (SJCRH30) and synovial sarcoma (SYO-1, Aska-SS and Yamato-SS) cell lines, all of which harbor chromosomal translocation and resulting oncogenic fusion genes, EWSR1-FLI1, PAX3-FOXO1 and SS18-SSX, respectively. Finally, animal experiments confirmed the antitumor activity of ZSTK474 in vivo, with superior efficacy observed in translocation-positive cells. These results suggest that ZSTK474 could be a promising drug candidate for treating sarcomas, especially those harboring chromosomal translocation.

Increased global incidence of myopia necessitates establishment of therapeutic approaches against its progression. To explore agents which may control myopia, we screened 207 types of natural compounds and chemical reagents based on an activity of a myopia suppressive factor, early growth response protein 1 (Egr-1) in vitro. Among the candidates, crocetin showed the highest and dose-dependent activation of Egr-1. For in vivo analysis, experimental myopia was induced in 3-week-old C57BL/6 J mice with -30 diopter (D) lenses for 3 weeks. Animals were fed with normal or mixed chow containing 0.003% (n = 19) and 0.03% (n = 7) of crocetin during myopia induction. Refraction and axial length were measured at 3-week-old and the 6-week-old with an infrared photorefractor and a SD-OCT system. Compared to controls (n = 14), crocetin administration showed a significant smaller change of refractive errors (-13.62 ± 8.14 vs +0.82 ± 5.81 D for 0.003%, p < 0.01, -2.00 ± 4.52 D for 0.03%, p < 0.01) and axial elongation (0.27 ± 0.03 vs 0.22 ± 0.04 mm for 0.003%, p < 0.01, 0.23 ± 0.05 mm for 0.03%, p < 0.05). These results suggest that a dietary factor crocetin may have a preventive effect against myopia progression.

Virus-derived double-stranded RNAs (dsRNAs) are sensed in the cytosol by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs). These induce the expression of type I IFN and proinflammatory cytokines through signaling pathways mediated by the mitochondrial antiviral signaling (MAVS) protein. TNF receptor-associated factor (TRAF) family proteins are reported to facilitate the RLR-dependent expression of type I IFN by interacting with MAVS. However, the precise regulatory mechanisms remain unclear. Here, we show the role of FK506-binding protein 51 (FKBP51) in regulating the dsRNA-dependent expression of type I IFN. The binding of FKBP51 to TRAF6 was first identified by "in vitro virus" selection and was subsequently confirmed with a coimmunoprecipitation assay in HEK293T cells. The TRAF-C domain of TRAF6 is required for its interaction, although FKBP51 does not contain the consensus motif for interaction with the TRAF-C domain. Besides TRAF6, we found that FKBP51 also interacts with TRAF3. The depletion of FKBP51 reduced the expression of type I IFN induced by dsRNA transfection or Newcastle disease virus infection in murine fibroblasts. Consistent with this, the FKBP51 depletion attenuated dsRNA-mediated phosphorylations of IRF3 and JNK and nuclear translocation of RelA. Interestingly, dsRNA stimulation promoted the accumulation of FKBP51 in the mitochondria. Moreover, the overexpression of FKBP51 inhibited RLR-dependent transcriptional activation, suggesting a scaffolding function for FKBP51 in the MAVS-mediated signaling pathway. Overall, we have demonstrated that FKBP51 interacts with TRAF proteins and facilitates the expression of type I IFN induced by cytosolic dsRNA. These findings suggest a novel role for FKBP51 in the innate immune response to viral infection.

Breast cancer is one of the most common cancers in women in the world. Although breast cancer is well treatable at the early stage, patients with distant metastases show a poor prognosis. Data from recent studies using transplantation models indicate that Mint3/APBA3 might promote breast cancer malignancy. However, whether Mint3 indeed contributes to tumor development, progression, or metastasis in vivo remains unclear. To address this, here we examined whether Mint3 depletion affects tumor malignancy in MMTV-PyMT breast cancer model mice. In MMTV-PyMT mice, Mint3 depletion did not affect tumor onset and tumor growth, but attenuated lung metastases. Experimental lung metastasis of breast cancer Met-1 cells derived from MMTV-PyMT mice also decreased in Mint3-depleted mice, indicating that host Mint3 expression affected lung metastasis of MMTV-PyMT-derived breast cancer cells. Further bone marrow transplant experiments revealed that Mint3 in bone marrow-derived cells promoted lung metastasis in MMTV-PyMT mice. Thus, targeting Mint3 in bone marrow-derived cells might be a good strategy for preventing metastasis and improving the prognosis of breast cancer patients.

Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation.

In vitro, topographical and biophysical cues arising from the extracellular matrix (ECM) direct skeletal stem cell (SSC) commitment and differentiation. However, the mechanisms by which the SSC-ECM interface is regulated and the outcome of such interactions on stem cell fate in vivo remain unknown. Here we demonstrate that conditional deletion of the membrane-anchored metalloproteinase MT1-MMP (Mmp14) in mesenchymal progenitors, but not in committed osteoblasts, redirects SSC fate decisions from osteogenesis to adipo- and chondrogenesis. By effecting ECM remodeling, MT1-MMP regulates stem cell shape, thereby activating a β1-integrin/RhoGTPase signaling cascade and triggering the nuclear localization of the transcriptional coactivators YAP and TAZ, which serve to control SSC lineage commitment. These data identify a critical MT1-MMP/integrin/YAP/TAZ axis operative in the stem cell niche that oversees SSC fate determination.

Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-(L)) and an alternatively initiated or spliced (Bcap-(S)) mRNA, and little is known about the differential functions of the BCAP-(L) and BCAP-(S) proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-(L) enhanced IL-6 and IL-10 production but not TNF-α production in TLR ligand-stimulated macrophages. We propose that BCAP-(L) (but not BCAP-(S)) is a negative regulator of the TLR-mediated host defense system in macrophages.

Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies.

HIV-1 latent reservoirs harbouring silenced but replication-competent proviruses are a major obstacle against viral eradication in infected patients. The "shock and kill" strategy aims to reactivate latent provirus with latency reversing agents (LRAs) in the presence of antiretroviral drugs, necessitating the development of effective and efficient LRAs. We screened a chemical library for potential LRAs and identified two dual Polo-like kinase (PLK)/bromodomain inhibitors, BI-2536 and BI-6727 (volasertib), which are currently undergoing clinical trials against various cancers. BI-2536 and BI-6727 significantly reactivated silenced HIV-1 provirus at both the mRNA and protein level in two latently infected model cell lines (ACH2 and U1). BI-2536 dramatically reactivated transcription of latent HIV-1 provirus in peripheral blood mononuclear cells derived from infected patients. Long terminal repeat activation by the inhibitors was associated with bromodomain rather than PLK inhibition. We also found that BI-2536 synergistically activates the latent provirus in combination with SAHA, a histone deacetylase inhibitor, or the non-tumour-promoting phorbol ester prostratin. Our findings strongly suggest that BI-2536 and BI-6727 are potent LRAs for the "shock and kill" HIV-1 eradication strategy.

Nuclear factor (NF)-κB-inducing kinase (NIK) is a serine/threonine kinase that activates NF-κB pathways, thereby regulating a wide variety of immune systems. Aberrant NIK activation causes tumor malignancy, suggesting a requirement for precise regulation of NIK activity. To explore novel interacting proteins of NIK, we performed in vitro virus screening and identified the catalytic subunit Aα isoform of serine/threonine phosphatase calcineurin (CnAα) as a novel NIK-interacting protein. The interaction of NIK with CnAα in living cells was confirmed by co-immunoprecipitation. Calcineurin catalytic subunit Aβ isoform (CnAβ) also bound to NIK. Experiments using domain deletion mutants suggested that CnAα and CnAβ interact with both the kinase domain and C-terminal region of NIK. Moreover, the phosphatase domain of CnAα is responsible for the interaction with NIK. Intriguingly, we found that TRAF3, a critical regulator of NIK activity, also binds to CnAα and CnAβ. Depletion of CnAα and CnAβ significantly enhanced lymphotoxin-β receptor (LtβR)-mediated expression of the NIK-dependent gene Spi-B and activation of RelA and RelB, suggesting that CnAα and CnAβ attenuate NF-κB activation mediated by LtβR-NIK signaling. Overall, these findings suggest a possible role of CnAα and CnAβ in modifying NIK functions.

Cellular migration, coupled with the degradation of the extracellular matrix (ECM), is a key step in tumor invasion and represents a promising therapeutic target in malignant tumors. Focal adhesions (FAs) and invadopodia, which are distinct actin-based cellular structures, play key roles in cellular migration and ECM degradation, respectively. The molecular machinery coordinating the dynamics between FAs and invadopodia is not fully understood, although several lines of evidence suggest that the disassembly of FAs is an important step in triggering the formation of invadopodia. In a previous study, we identified the ZF21 protein as a regulator of both FA turnover and invadopodia-dependent ECM degradation. ZF21 interacts with multiple factors for FA turnover, including focal adhesion kinase (FAK), microtubules, m-Calpain, and Src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2). In particular, the dephosphorylation of FAK by ZF21 is a key event in tumor invasion. However, the precise role of ZF21 binding to FAK remains unclear. We established a method to disrupt the interaction between ZF21 and FAK using the FAK-binding NH2 -terminal region of ZF21. Tumor cells expressing the ZF21-derived polypeptide had significantly decreased FA turnover, migration, invadopodia-dependent ECM degradation, and Matrigel invasion. Furthermore, the expression of the polypeptide inhibited an early step of experimental lung metastasis in mice. These findings indicate that the interaction of ZF21 with FAK is necessary for FA turnover as well as ECM degradation at the invadopodia. Thus, ZF21 is a potential regulator that coordinates the equilibrium between FA turnover and invadopodia activity by interacting with FAK.

Cancer cells adapt to various stress conditions by optimizing gene expression profiles via transcriptional and translational regulation. However, whether and how EXOSC9, a component of the RNA exosome complex, regulates adaptation to stress conditions and tumorigenicity in cancer cells remain unclear. Here, we examined the effects of EXOSC9 depletion on cancer cell growth under various stress conditions. EXOSC9 depletion attenuated growth and survival under various stress conditions in cancer cells. Interestingly, this also decreased the number of P-bodies, which are messenger ribonucleoprotein particles (mRNPs) required for stress adaptation. Meanwhile, EXOSC2/EXOSC4 depletion also attenuated P-body formation and stress resistance with decreased EXOSC9 protein. EXOSC9-mediated stress resistance and P-body formation were found to depend on the intact RNA-binding motif of this protein. Further, RNA-seq analyses identified 343 EXOSC9-target genes, among which, APOBEC3G contributed to defects in stress resistance and P-body formation in MDA-MB-231 cells. Finally, EXOSC9 also promoted xenografted tumor growth of MDA-MB-231 cells in an intact RNA-binding motif-dependent manner. Database analyses further showed that higher EXOSC9 activity, estimated based on the expression of 343 target genes, was correlated with poorer prognosis in some cancer patients. Thus, drugs targeting activity of the RNA exosome complex or EXOSC9 might be useful for cancer treatment.

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

Pathological angiogenesis contributes to cancer progression and chronic inflammatory diseases. In inflammatory bowel disease, the microvasculature expands by intussusceptive angiogenesis (IA), a poorly characterized mechanism involving increased blood flow and splitting of pre-existing capillaries. In this report, mice lacking the protease MT1-MMP in endothelial cells (MT1iΔEC ) presented limited IA in the capillary plexus of the colon mucosa assessed by 3D imaging during 1% DSS-induced colitis. This resulted in better tissue perfusion, preserved intestinal morphology, and milder disease activity index. Combined in vivo intravital microscopy and lentiviral rescue experiments with in vitro cell culture demonstrated that MT1-MMP activity in endothelial cells is required for vasodilation and IA, as well as for nitric oxide production via binding of the C-terminal fragment of MT1-MMP substrate thrombospondin-1 (TSP1) to CD47/αvβ3 integrin. Moreover, TSP1 levels were significantly higher in serum from IBD patients and in vivo administration of an anti-MT1-MMP inhibitory antibody or a nonamer peptide spanning the αvβ3 integrin binding site in TSP1 reduced IA during mouse colitis. Our results identify MT1-MMP as a new actor in inflammatory IA and a promising therapeutic target for inflammatory bowel disease.

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid-tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid-tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T-cell infiltration at tumor sites and programmed death receptor-1 (PD-1) upregulation in CD8+ T cells in MMTV-PyMT/Asxl1-MT mice. In addition, we found that Asxl1-MT induced T-cell dysregulation, including aberrant intrathymic T-cell development, decreased CD4/CD8 ratio, and naïve-memory imbalance in peripheral T cells. These results indicate that Asxl1-MT perturbs T-cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1-mutated blood cells exacerbate solid-tumor progression in ASXL1-CHIP carriers.

Secondary lymphoid organs are critical for regulating acquired immune responses. The aim of this study was to characterize the impact of spaceflight on secondary lymphoid organs at the molecular level. We analysed the spleens and lymph nodes from mice flown aboard the International Space Station (ISS) in orbit for 35 days, as part of a Japan Aerospace Exploration Agency mission. During flight, half of the mice were exposed to 1 g by centrifuging in the ISS, to provide information regarding the effect of microgravity and 1 g exposure during spaceflight. Whole-transcript cDNA sequencing (RNA-Seq) analysis of the spleen suggested that erythrocyte-related genes regulated by the transcription factor GATA1 were significantly down-regulated in ISS-flown vs. ground control mice. GATA1 and Tal1 (regulators of erythropoiesis) mRNA expression was consistently reduced by approximately half. These reductions were not completely alleviated by 1 g exposure in the ISS, suggesting that the combined effect of space environments aside from microgravity could down-regulate gene expression in the spleen. Additionally, plasma immunoglobulin concentrations were slightly altered in ISS-flown mice. Overall, our data suggest that spaceflight might disturb the homeostatic gene expression of the spleen through a combination of microgravity and other environmental changes.

Myocardial fibrosis is often associated with cardiac hypertrophy; indeed, fibrosis is one of the most critical factors affecting prognosis. We aimed to identify the molecules involved in promoting fibrosis under hypertrophic stimuli. We previously established a rat model of cardiac hypertrophy by pulmonary artery banding, in which approximately half of the animals developed fibrosis in the right ventricle. Here, we first comprehensively analyzed mRNA expression in the right ventricle with or without fibrosis in pulmonary artery banding model rats by DNA microarray analysis (GSE141650 at NCBI GEO). The expression levels of 19 genes were up-regulated more than 1.5-fold in fibrotic hearts compared with non-fibrotic hearts. Among them, fibrosis growth factor (FGF) 23 showed one of the biggest increases in expression. Real-time PCR analysis also revealed that, among the FGF receptor (FGFR) family, FGFR1 was highly expressed in fibrotic hearts. We then found that FGF23 was expressed predominantly in cardiomyocytes, while FGFR1 was predominantly expressed in fibroblasts in the rat ventricle. Next, we added FGF23 and transforming growth factor (TGF)-β1 (10-50 ng/mL of each) to isolated fibroblasts from normal adult rat ventricles and cultured them for three days. While FGF23 itself did not directly affect the expression levels of any fibrosis-related mRNAs, FGF23 enhanced the effect of TGF-β1 on increasing the expression levels of α-smooth muscle actin (α-SMA) mRNA. This increase in xx-SMA mRNA levels due to the combination of TGF-β1 and FGF23 was attenuated by the inhibition of FGFR1 or the knockdown of FGFR1 in fibroblasts. Thus, FGF23 synergistically promoted the activation of fibroblasts with TGF-β1, transforming fibroblasts into myofibroblasts via FGFR1. Thus, we identified FGF23 as a paracrine factor secreted from cardiomyocytes to promote cardiac fibrosis under conditions in which TGF-β1 is activated. FGF23 could be a possible target to prevent fibrosis following myocardial hypertrophy.