NFATc1 Mediates LIPUS-Induced Osteoblast Differentiation through P2X7 in MC3T3-E1 Cells

A spontaneous mutation in Bruton's tyrosine kinase (Btk) induces a defect in B-cell development that results in the immunodeficiency diseases X-linked agammaglobulinemia in humans and X-linked immunodeficiency (Xid) in mice. Here we show an unexpected role of Btk in osteoclast formation. When bone marrow cells derived from Xid mice were stimulated with receptor activator of NF-kappaB ligand, an osteoclast differentiation factor, they did not completely differentiate into mature multinucleated osteoclasts. Moreover, we found that the defects appeared to occur at the stage in which mononuclear preosteoclasts fuse to generate multinucleated cells. Supporting this notion, macrophages from Xid mice also failed to form multinucleated foreign body giant cells. The fusion defect of the Xid mutant osteoclasts was caused by decreased expression of nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclast differentiation, as well as reduced expression of various osteoclast fusion-related molecules, such as the d2 isoform of vacuolar H(+)-ATPase V0 domain and the dendritic cell-specific transmembrane protein. This deficiency was completely rescued by the introduction of a constitutively active form of NFATc1 into bone marrow-derived macrophages. Our data provide strong evidence that Btk plays a critical role in osteoclast multinucleation by modulating the activity of NFATc1.

BackgroundIt is well known that nuclear factor of activated T cells c1 (NFATc1) expression is closely associated with progression of many cancers. And we found that miR‐338 could directly target the NFATc1. However, the precise mechanisms of miR‐338 in non‐small‐cell lung cancer (NSCLC) have not been well clarified. Our study aimed to explore the interaction between NFATc1 and miR‐338 in NSCLC.MethodsQuantitative RT‐PCR was utilized to determine the expressions of NFATc1 and miR‐338 in NSCLC tissues and cell lines. And the cell proliferation and epithelial‐mesenchymal transition (EMT) were assessed to determine the functional roles of miR‐338 and NFATc1 in NSCLC cells. NFATc1 expression was detected using quantitative RT‐PCR and western blotting, respectively. Luciferase reporter assays were performed to validate NFATc1 as a target of miR‐338 in NSCLC cells.ResultsIn this study, our results showed that NFATc1 expression was significantly up‐regulated in NSCLC tissues and cell lines, and the miR‐338 level was dramatically down‐regulated. Moreover high NFATc1 expression was closely associated with low miR‐338 level in NSCLC tissues. Moreover introduction of miR‐338 significantly inhibited proliferation and EMT of NSCLC cells. Bioinformatics analysis predicted that the NFATc1 was a potential target gene of miR‐338. We demonstrated that miR‐338 could directly target NFATc1 by using luciferase reporter assay. Besides, knockdown of NFATc1 had the similar effects with miR‐338 overexpression on NSCLC cells. Up‐regulation of NFATc1 in NSCLC cells partially abolished the inhibitory effects of miR‐338 mimic.ConclusionsOverexpression of miR‐338 inhibited cell proliferation and EMT of NSCLC cells by directly down‐regulating NFATc1 expression.

The expression of nuclear factor of activated T cells c1 (NFATc1) is closely associated with the progression of numerous types of cancer. When NFATc1 expression becomes dysregulated in some types of cancer, this alteration can promote malignant transformation and thereby progression of cancer. NFATc1 expression has been demonstrated to be upregulated in lung cancer cells. This suggests that knockdown of NFATc1 in lung cancer cells may be a therapeutic marker for the treatment of cancer. In the present study, the effects of NFATc1 on the proliferation, apoptosis, invasion and migration of NCI-H1299 and A549 lung cancer cell lines were explored. Lentivirus infection was used to establish a cell model of NFATc1 knockdown in A549 and NCI-H1299 lung cancer cells. Reverse transcription-quantitative PCR was subsequently performed to detect NFATc1 expression in these human lung cancer cells. MTT, wound healing, colony formation and Transwell invasion assays, and flow cytometry were then performed to measure the proliferation, invasion, apoptosis and cell cycle of the cells. Finally, western blot analysis was performed to investigate the mechanism underlying the involvement of NFATc1 in these processes. NFATc1 knockdown was found to significantly inhibit the proliferation, clone formation, migration and invasion of the cells. Furthermore, the cell cycle was arrested at the G1 phase and the expression levels of the target proteins located downstream in the signaling pathway, namely CDK4, c-Myc, ERK, p38 and N-cadherin, were decreased. Following NFATc1 knockdown, the percentages of apoptotic cells were increased, and the expression levels of Bax, cleaved caspase-3 and E-cadherin were also increased. Taken together, the results of the present study suggested that NFATc1 serves an oncogenic role in lung cancer. In terms of the underlying mechanism, NFATc1 promoted the proliferation of lung cancer cells by inhibiting the MAPK and epithelial-to-mesenchymal transition signaling pathways, suggesting that NFATc1 may be a novel target for therapeutic intervention for the treatment of lung cancer.

We previously reported the emerging role of OX40-OX40L interaction in inflammation and atherosclerosis. However, the mechanism by which OX40-OX40L interaction contributes to pathogenesis is poorly understood. This study investigated the effects of OX40-OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE(-/-) mice. Atherosclerotic plaque was induced via rapid perivascular carotid collar placement in ApoE(-/-) mice. The expression levels of OX40, OX40L, and NFATc1 in the lymphocytes were measured via real-time polymerase chain reaction and flow cytometry. The presence of NFATc1 in the atherosclerotic plaque was detected via immunohistochemistry, and the level of IL-4 was measured via enzyme-linked immunosorbent assay. The expression level of NFATc1 significantly increased in atherosclerotic lesion and in the leukocytes from the ApoE(-/-) mice. After stimulating OX40-OX40L interaction, the mRNA and protein expression levels of NFATc1 in the lymphocytes significantly increased. Meanwhile, anti-OX40LmAb significantly suppressed the expression of NFATc1 in the leukocytes and substantially elevated the level of IL-4. NFATc1 inhibitor markedly suppressed IL-4 production. This study suggests that OX40-OX40L interaction regulates the expression of NFATc1, which may play a critical role in atherosclerotic plaque formation, and may therefore have implications with pathophysiology of atherosclerosis.

Interleukin 4 (IL-4) inhibits receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast formation and functional activity in a STAT6-dependent manner. IL-4 down-regulates expression of tartrate-resistant acid phosphatase (TRAP) in mature osteoclasts. To determine whether IL-4 regulates TRAP promoter activity, RAW264.7 cells were transfected with a TRAP promoter-luciferase reporter. Treatment with IL-4 alone modestly enhanced TRAP luciferase activity. However, IL-4 suppressed the ability of RANKL to up-regulate TRAP-luciferase activity, suggesting that IL-4 has multiple effects on TRAP transcription. IL-4 also reduced the RANKL-induced association of RNA polymerase II with the TRAP gene in osteoclasts. The TRAP promoter contains a STAT6-binding motif, and STAT6 bound to the endogenous TRAP promoter after IL-4 treatment. To determine the impact of STAT6 binding, we transfected cells with STAT6VT, a constitutively active STAT6 mutant. STAT6VT alone up-regulated TRAP-luciferase activity; this effect was abrogated by mutating the STAT6 binding site in the minimal TRAP promoter. STAT6VT did not inhibit the potent up-regulation of TRAP promoter activity caused by overexpression of NFATc1, PU.1, and microphthalmia transcription factor, downstream targets of macrophage colony-stimulating factor and RANKL. IL-4 down-regulated the expression of c-Fos and NFATc1 in mature osteoclasts. Knockdown of NFATc1 by short interfering RNA caused TRAP expression to be down-regulated, and ectopic expression of NFATc1 abrogated the IL-4-induced down-regulation of TRAP. These results suggest that STAT6 plays two distinct roles in TRAP expression. The IL-4-induced activation of STAT6 mediates suppression of the RANKL-induced TRAP promoter activity indirectly by inhibiting NFATc1 expression. However, in the absence of RANKL and osteoclast differentiation, STAT6 binds the TRAP promoter after IL-4 treatment and directly enhances TRAP expression.

Objective: To explore whether CD137-CD137L signaling can promote angiogenesis in atherosclerosis plaque via activating nuclear factor of activated T cells c1 (NFATc1). Methods: Apolipoprotein E knock out mice were divided into the following groups: control group (n=5), CD137 activated group(n=5)and CD137 inhibited group (n=5). Immunohistochemistry was performed to detect the expression of CD31 in aortic plaque. Endothelial cells (bEnd.3) were purchased from ATCC and divided into the following groups: control group, IgG isotype control group, CD137 activated group and CD137 inhibited group. Western blot was used to determine total protein and nucleoprotein expression of NFATc1. The expression level of CD137 protein on the surface of endothelial cells was detected by flow cytometry(FCM) and CD137 protein of lysate of endothelial cells was detected by enzyme-linked immunosorbent assay (ELISA). Transwell assay was used to observe the migration ability of endothelial cells.Matrigel tube formation ability of endothelial cells were tested in the following groups: control group, CD137 activated group, silent NFATc1 + CD137 activated group, CD137 inhibited group, and over expressed NFATc1+ CD137 inhibited group. Results: (1) In vivo, the expression level of CD31 was significantly higher in the aortic plaque of CD137 activated group than in control group(1 191±187 vs. 115±30, P<0.05), while which was significantly downregulated in CD137 inhibited group(450±92, P<0.05). (2) The level of nucleoprotein(3.07±0.03 vs. 1.00±0.00, P<0.05) and total protein(2.18±0.30 vs. 1.00±0.00, P<0.05) of NFATc1 were significantly higher in CD137 activated group than in IgG isotype control group. The level of nucleoprotein(0.82±0.04) and total protein(0.84 ± 0.09) of NFATc1 were significantly lower in CD137 inhibited group than in CD137 activated group(both P<0.05). (3) FCM results showed that the fluorescence intensity of CD137 on the cell membrane was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells(5 163±329 vs. 1 660±162, P<0.05). (4) ELISA examination showed that the level of CD137 protein was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells ((573.4±23.7)pg/mg vs.(69.5±16.7)pg/mg, P<0.05). (5) Migration cell number was remarkably higher in CD137 activated group than in IgG isotype control group(1.19±0.13 vs. 1.00±0.00, P<0.05) and significantly lower in CD137 inhibited group(0.82±0.06)than in control group (P<0.05). (6) Values of the formation of the tube length ((5.76±0.18)mm vs. (4.21±0.11)mm, P<0.05) and branch number (29.38±1.28 vs. 21.13±0.96, P<0.05) were both significantly higher in CD137 activated group than in the control group. The formation of the tube length ((1.90±0.11)mm) and branch number(8.91±0.72)were significantly lower in silent NFATc1 + CD137 activated group than in the CD137 activated group (both P<0.05). The formation of the tube length((1.28±0.34)mm) and branch number(5.07±0.35)were also significantly decreased in the CD137 inhibited group compared with the CD137 activated group (both P<0.05). Compared with the CD137 inhibited group, the formation of the tube length((4.82±0.09)mm) and branch number(24.44±1.05) in the over expressed NFATc1+ CD137 inhibited group was increased (both P<0.05). Conclusion: CD137 can promote the angiogenesis in atherosclerosis plaque by activating NFATc1.

Despite recent insights into prostate cancer (PCa)-associated genetic changes, full understanding of prostate tumorigenesis remains elusive due to complexity of interactions among various cell types and soluble factors present in prostate tissue. We found upregulation of Nuclear Factor of Activated T Cells c1 (NFATc1) in human PCa and cultured PCa cells, but not in normal prostates and non-tumorigenic prostate cells. To understand the role of NFATc1 in prostate tumorigenesis in situ, we temporally and spatially controlled the activation of NFATc1 in mouse prostate and showed that such activation resulted in prostatic adenocarcinoma with features similar to those seen in human PCa. Our results indicate that the activation of a single transcription factor, NFATc1 in prostatic luminal epithelium to PCa can affect expression of diverse factors in both cells harboring the genetic changes and in neighboring cells through microenvironmental alterations. In addition to the activation of oncogenes c-MYC and STAT3 in tumor cells, a number of cytokines and growth factors, such as IL1β, IL6, and SPP1 (Osteopontin, a key biomarker for PCa), were upregulated in NFATc1-induced PCa, establishing a tumorigenic microenvironment involving both NFATc1 positive and negative cells for prostate tumorigenesis. To further characterize interactions between genes involved in prostate tumorigenesis, we generated mice with both NFATc1 activation and Pten inactivation in prostate. We showed that NFATc1 activation led to acceleration of Pten-null–driven prostate tumorigenesis by overcoming the PTEN loss–induced cellular senescence through inhibition of p21 activation. This study provides direct in vivo evidence of an oncogenic role of NFATc1 in prostate tumorigenesis and reveals multiple functions of NFATc1 in activating oncogenes, in inducing proinflammatory cytokines, in oncogene addiction, and in overcoming cellular senescence, which suggests calcineurin-NFAT signaling as a potential target in preventing PCa.

NFATc1 (nuclear factor of activated T-cells c1), a key transcription factor, plays a role in regulating expression of osteoclast-specific downstream target genes such as TRAP (tartrate-resistant acid phosphatase) and OSCAR (osteoclast-associated receptor). It has been shown that RANKL [receptor activator of NF-κB (nuclear factor κB) ligand] induces NFATc1 expression during osteoclastogenesis at a transcriptional level. In the present study, we demonstrate that RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via HATs (histone acetyltransferases), such as p300 and PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor], thereby stabilizing NFATc1 proteins. PCAF physically interacts with NFATc1 and directly induces NFATc1 acetylation and stability, subsequently increasing the transcriptional activity of NFATc1. In addition, RANKL-mediated NFATc1 acetylation is increased by the HDAC (histone deacetylase) inhibitors sodium butyrate and scriptaid. Overexpression of HDAC5 reduces RANKL- or PCAF-mediated NFATc1 acetylation, stability and transactivation activity, suggesting that the balance between HAT and HDAC activities might play a role in the regulation of NFATc1 levels. Furthermore, RANKL and p300 induce PCAF acetylation and stability, thereby enhancing the transcriptional activity of NFATc1. Down-regulation of PCAF by siRNA (small interfering RNA) decreases NFATc1 acetylation and stability, as well as RANKL-induced osteoclastogenesis. Taken together, the results of the present study demonstrate that RANKL induces HAT-mediated NFATc1 acetylation and stability, and subsequently increases the transcriptional activity of NFATc1 during osteoclast differentiation.

BackgroundOur previous studies have shown that OX40-OX40L interaction regulates the expression of nuclear factor of activated T cells c1(NFATc1) in ApoE−/− mice during atherogenesis. The aim of this study was to investigate whether OX40-OX40L interaction promotes Th cell activation via NFATc1 in ApoE−/− mice.Methods and ResultsThe lymphocytes isolated from spleen of ApoE−/− mice were cultured with anti-CD3 mAb in the presence or absence of anti-OX40 or anti-OX40L antibodies. The expression of NFATc1 mRNA and protein in isolated lymphocytes were measured by real time PCR (RT-PCR) and flow cytometry (FCM), respectively. The proliferation of lymphocytes was analyzed by MTT method,and the expression of IL-2, IL-4 and IFN-γ in the cultured cells and supernatant were measured by RT-PCR and enzyme-linked immunosorbent assary (ELISA), respectively. After stimulating OX40-OX40L signal pathway, the expression of NFATc1 and the proliferation of leukocytes were significantly increased. Anti-OX40L suppressed the expression of NFATc1 in lymphocytes of ApoE−/− mice. Anti-OX40L or the NFATc1 inhibitor (CsA) markedly suppressed the cell proliferation induced by anti-OX40. Moreover, the expression of IL-2 and IFN-γ was increased in lymphocytes induced by OX40-OX40L interaction. Blocking OX40-OX40L interaction or NFATc1 down-regulated the expression of IL-2 and IFN-γ, but didn’t alter the expression of IL-4 in supernatants.ConclusionThese results suggest that OX40-OX40L interaction promotes the proliferation and activation of lymphocytes through NFATc1.

To investigate if miR-145a-5p participates the modulation process of CD137 signaling on the expression of nuclear factor of activated T cells c1 (NFATc1) in ApoE(-)/(-) mice. Atherosclerotic plaque model was produced by perivascular carotid collar placement in ApoE(-)/(-) mice. After surgery, the mice were randomly divided into the following groups: CD137 activated group (CD137 group, n = 6), CD137 inhibited group (anti-CD137 group, n = 6) and control group (n = 6). The mRNA expression of miR-145a-5p in plaque and cells was measured by real-time quantitative PCR (RT-PCR). Immunofluorescence was used to observe the distribution of NFATc1 in plaque and the expression of NFATc1 at mRNA and protein levels were detected by qRT-PCR, Western blot, respectively. The mouse vascular smooth muscle cells (VSMCs) were isolated and transfected with miR-145a-5p mimics or inhibitors by Lipofectamine. The eukaryotic expression vector and luciferase vector including p3xFLAG-NFATc1, p3xFLAG-NFATc1-3'UTR, psicheck2-NFATc1, psicheck2-NFATc1-Mut were constructed through molecular cloning and homologous recombination techniques, 293T cells were transfected with the miR-145a-5p mimics or inhibitors and the protein level and fluorescence intensity were then measured, respectively. In vivo or in vitro, the level of miR-145a-5p was significantly decreased (0.21 ± 0.06 vs. 1.00 ± 0.00, P < 0.05, 0.22 ± 0.07 vs. 0.50 ± 0.12, P < 0.05) while the opposite effects were observed in anti-CD137 group. NFATc1 expression was decreased or increased in VSMCs transfected with miR-145a-5p mimics or inhibitors, respectively (all P < 0.05). miR-145a-5p mimics decreased the expression of p3xFLAG-NFATc1-3'UTR and the fluorescence intensity (0.56 ± 0.08 vs. 1.00 ± 0.00, P < 0.05). CD137 signaling participates the regulation process on the expression of NFATc1 through miR-145a-5p in ApoE(-)/(-) mice.

NFATc1 (nuclear factor of activated T‑cells c1) is associated with malignancy in several cancer models. However, the expression and function of NFATc1 in ovarian cancer remain elusive. In the present study, we investigated the role of NFATc1 in human epithelial ovarian cancer (EOC) using human ovarian adenocarcinoma SKOV3 cells and patient characteristics. NFATc1 expression was silenced by siRNA in the SKOV3 ovarian cancer cell line and in human ovarian cancer nude mouse xenografts. Real‑time PCR, western blotting, immunohistochemical staining, MTT, flow cytometry, transwell, erasion trace and mouse assays were used to detect NFATc1 expression, cell proliferation, apoptosis, cell invasion and migration, tumor growth and angiogenesis. Survival analysis was performed to assess the correlation between NFATc1 expression and survival. NFATc1 was overexpressed in the SKOV3 ovarian cancer cell line and in human serous/mucinous ovarian cancer tissues. The silencing of NFATc1 expression by siRNA reduced cell proliferation and migration and promoted apoptosis invitro and decreased the ovarian cancer cell tumorigenesis invivo in nude mice. NFATc1 overexpression in high‑grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival and of early relapse (P<0.01) in a univariate analysis. Our present data provide evidence that NFATc1 is overexpressed in human serous/mucinous ovarian cancer and is associated with a poor prognosis. NFATc1 silencing regulates the cell cycle, apoptosis, invasion and migration. NFATc1 thus has the potential to be a therapeutic target and to be used in EOC diagnosis and prognosis.

NFATc1 Links EGFR Signaling to Induction of Sox9 Transcription and Acinar–Ductal Transdifferentiation in the Pancreas

We investigated the effect of nuclear factor of activated T cells c1 (NFATc1) on the growth and vascular generation of human ovarian carcinoma SKOV3 cell-transplanted tumors in nude mice and explored the possible underlying mechanism. NFATc1 siRNA was transfected into the SKOV3 cells, which were then subjected to immunofluorescence tests and real-time reverse transcription polymerase chain reaction (RT-PCR) to determine the transfection-induced inhibition rate. The tumor volumes in the nude mice in all groups were measured to determine the in vivo antitumor effect of NFATc1 siRNA. Immunohistochemical (IHC) methods were employed to detect NFATc1 expression in tumor tissue, combined with cytokeratin (CK) staining to label the epithelial origin of the tumor tissue. CD34 and podoplanin were used as markers for labeling microvessels and microlymphatic vessels, respectively. The densities of microvessels and microlymphatic vessels in each group were calculated and statistically analyzed. RT-PCR and western blotting were performed to detect the protein and mRNA expression levels of NFATc1, the ELR+ CXC chemokine interleukin (IL)-8, fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor BB (PDGF BB) in xenografted tumor tissue in all groups. NFATc1 was highly expressed in tumor tissue in the control groups. The intervention group exhibited a tumor growth inhibition rate of 57.08% and presented a lower tumor weight and volume compared with the two control groups. In the control groups, the microvessel densities were 12.00 ± 1.65 and 11.47 ± 0.32, respectively, and the microlymphatic vessel densities were 10.03 ± 0.96 and 9.95 ± 1.12; these values were significantly higher than in the intervention group. RT-PCR and western blot shows that NFATc1 siRNA could markedly suppress the expression of IL-8, FGF-2 and PDGF BB at the mRNA and the protein level. In conclusion, it was shown that NFATc1 siRNA significantly suppresses the growth and vascular generation of SKOV3 human ovarian carcinoma cell-transplanted tumors subcutaneously xenografted into nude mice. The downregulation of the expression of IL-8, FGF-2 and PDGF BB may be one of the mechanisms underlying the above inhibitory effects.

To observe whether CD137 signaling could affect the nuclear factor of activated T cells c1 (NFATc1) expression through nuclear factor-κB (NF-κB) pathway in mice aortic vascular smooth muscle cells (VSMCs). Adherence methods for tissues explants were used for primary culture of mouse aortic VSMCs. The mRNA expression of CD137 and NFATc1 was detected by real-time quantitative PCR (RT-qPCR). The VSMCs protein expression of IκB-α, NF-κB p65, phospo-p65 and NFATc1 was determined by Western blot. The level of CD137 was measured by Flow Cytometry (FCM). (1) The mRNA and protein expression of CD137 in VSMCs was significantly upregulated at 24 h after co-culture with TNF-α (10 ng/ml, all P < 0.05). (2) Compared with the control group, the level of p-NF-κB p65 in cytoplasm and nucleus was significantly increased (8.34 ± 0.28 vs. 1, P < 0.05, and 2.64 ± 0.42 vs. 1, P < 0.05) while the level of IκB-α was reduced (1 vs. 2.70 ± 0.28, P < 0.05) after co-treatment with agonist-CD137 mAb, above effects were partly blocked by adding specific NF-κB inhibitor PDTC (30 µmol/L: 1.15 ± 0.14 vs. 8.34 ± 0.28, P < 0.05, and 2.09 ± 0.12 vs. 2.64 ± 0.42, P < 0.05, and 1.78 ± 0.74 vs. 1, P < 0.05). (3) The mRNA (2.07 ± 0.09 vs. 1, P < 0.05) and protein (1.75 ± 0.07 vs. 1, P < 0.05) expression of NFATc1 was significantly upregulated by agonist CD137mAb compared with the control group, and these effects could be reversed by PDTC (1.15 ± 0.07 vs. 2.07 ± 0.09, P < 0.05, and 0.90 ± 0.11 vs. 1.75 ± 0.07, P < 0.05). CD137 signaling could affect the NFATc1expression in VSMCs through NF-kappaB pathway.

Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing. However, it remains unclear which skeletal cells, in particular osteocytes or osteoblasts, primarily respond to LIPUS stimulation and how they contribute to fracture healing. To examine this, we utilized medaka, whose bone lacks osteocytes, and zebrafish, whose bone has osteocytes, as in vivo models. Fracture healing was accelerated by ultrasound stimulation in zebrafish, but not in medaka. To examine the molecular events induced by LIPUS stimulation in osteocytes, we performed RNA sequencing of a murine osteocytic cell line exposed to LIPUS. 179 genes reacted to LIPUS stimulation, and functional cluster analysis identified among them several molecular signatures related to immunity, secretion, and transcription. Notably, most of the isolated transcription-related genes were also modulated by LIPUS in vivo in zebrafish. However, expression levels of early growth response protein 1 and 2 (Egr1, 2), JunB, forkhead box Q1 (FoxQ1), and nuclear factor of activated T cells c1 (NFATc1) were not altered by LIPUS in medaka, suggesting that these genes are key transcriptional regulators of LIPUS-dependent fracture healing via osteocytes. We therefore show that bone-embedded osteocytes are necessary for LIPUS-induced promotion of fracture healing via transcriptional control of target genes, which presumably activates neighboring cells involved in fracture healing processes.