The long noncoding RNA GAS5 potentiates neuronal injury in Parkinson's disease by binding to microRNA-150 to regulate Fosl1 expression.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been the focus of recent studies of neurodegenerative disorders, including Parkinson's disease (PD). However, the specific mechanism of action of growth arrest-specific 5 (GAS5) in PD has not yet been characterized. First, the GSE8030 and GSE16658 datasets were analyzed to obtain differentially expressed genes (DEGs), followed by the development of a PD mouse model. The effects of shRNA targeting fos-like antigen-1 (shFosl1) and microRNA (miR)-150 agomiR on PD mouse behavior and neuronal injury were evaluated in vitro and in vivo. After the determination of target lncRNAs using bioinformatics tools, cell models were developed in SH-SY5Y and N2a cells using MPP+ to verify the effects of GAS5, miR-150 and Fosl1 on cell viability. Knockdown of Fosl1 and GAS5 or overexpression of miR-150 alleviated neuronal injury in mice after MPTP treatment and significantly increased the activity of SH-SY5Y and N2a cells after MPP treatment. GAS5 bound to miR-150, while miR-150 targeted Fosl1. Fosl1 activated the PTEN/AKT/mTOR pathway, thus promoting apoptosis and inhibiting neuronal activity in the PD model. Overall, our findings illuminated that GAS5 accelerated PD progression by targeting the miR-150/Fosl1 axis.

c-FOS drives reversible basal to squamous cell carcinoma transition.

While squamous transdifferentiation within subpopulations of adenocarcinomas represents an important drug resistance problem, its underlying mechanism remains poorly understood. Here, using surface markers of resistant basal cell carcinomas (BCCs) and patient single-cell and bulk transcriptomic data, we uncover the dynamic roadmap of basal to squamous cell carcinoma transition (BST). Experimentally induced BST identifies activator protein 1 (AP-1) family members in regulating tumor plasticity, and we show that c-FOS plays a central role in BST by regulating the accessibility of distinct AP-1 regulatory elements. Remarkably, despite prominent changes in cell morphology and BST marker expression, we show using inducible model systems that c-FOS-mediated BST demonstrates reversibility. Blocking EGFR pathway activation after c-FOS induction partially reverts BST in vitro and prevents BST features in both mouse models and human tumors. Thus, by identifying the molecular basis of BST, our work reveals a therapeutic opportunity targeting plasticity as a mechanism of tumor resistance.

FosL1 Is a Novel Target of Levetiracetam for Suppressing the Microglial Inflammatory Reaction.

We previously showed that the antiepileptic drug levetiracetam (LEV) inhibits microglial activation, but the mechanism remains unclear. The purpose of this study was to identify the target of LEV in microglial activity suppression. The mouse microglial BV-2 cell line, cultured in a ramified form, was pretreated with LEV and then treated with lipopolysaccharide (LPS). A comprehensive analysis of LEV targets was performed by cap analysis gene expression sequencing using BV-2 cells, indicating the transcription factors BATF, Nrf-2, FosL1 (Fra1), MAFF, and Spic as candidates. LPS increased AP-1 and Spic transcriptional activity, and LEV only suppressed AP-1 activity. FosL1, MAFF, and Spic mRNA levels were increased by LPS, and LEV only attenuated FosL1 mRNA expression, suggesting FosL1 as an LEV target. FosL1 protein levels were increased by LPS treatment and decreased by LEV pretreatment, similar to FosL1 mRNA levels. The FosL1 siRNA clearly suppressed the expression of TNFα and IL-1ß. Pilocarpine-induced status epilepticus increased hippocampus FosL1 expression, along with inflammation. LEV treatment significantly suppressed FosL1 expression. Together, LEV reduces FosL1 expression and AP-1 activity in activated microglia, thereby suppressing neuroinflammation. LEV might be a candidate for the treatment of several neurological diseases involving microglial activation.

Selected Molecular Targets for Antiepileptogenesis.

The term epileptogenesis defines the usually durable process of converting normal brain into an epileptic one. The resistance of a significant proportion of patients with epilepsy to the available pharmacotherapy prompted the concept of a causative treatment option consisting in stopping or modifying the progress of epileptogenesis. Most antiepileptic drugs possess only a weak or no antiepileptogenic potential at all, but a few of them appear promising in this regard; these include, for example, eslicarbazepine (a sodium and T-type channel blocker), lamotrigine (a sodium channel blocker and glutamate antagonist) or levetiracetam (a ligand of synaptic vehicle protein SV2A). Among the approved non-antiepileptic drugs, antiepileptogenic potential seems to reside in losartan (a blocker of angiotensin II type 1 receptors), biperiden (an antiparkinsonian drug), nonsteroidal anti-inflammatory drugs, antioxidative drugs and minocycline (a second-generation tetracycline with anti-inflammatory and antioxidant properties). Among other possible antiepileptogenic compounds, antisense nucleotides have been considered, among these an antagomir targeting microRNA-134. The drugs and agents mentioned above have been evaluated in post-status epilepticus models of epileptogenesis, so their preventive efficacy must be verified. Limited clinical data indicate that biperiden in patients with brain injuries is well-tolerated and seems to reduce the incidence of post-traumatic epilepsy. Exceptionally, in this regard, our own original data presented here point to c-Fos as an early seizure duration, but not seizure intensity-related, marker of early epileptogenesis. Further research of reliable markers of early epileptogenesis is definitely needed to improve the process of designing adequate antiepileptogenic therapies.

Silent FOSL1 Enhances the Radiosensitivity of Glioma Stem Cells by Down-Regulating miR-27a-5p.

Since few reports have mentioned the role of FOSL1 in the radiotherapy sensitivity of glioma, this study would dig deep into this aspect. Cancer stem cells (CSCs) isolated by magnetic bead assay were identified by microscopy, qRT-PCR and western blot. The number of apoptotic cells was counted 72 h after X-ray irradiation to evaluate the sensitivity of cancer cells to radiotherapy. The effects of radiotherapy, FOSL1 and miR-27a-5p on basic cell functions were detected by functional experiments. The expressions of FOSL1, apoptosis-related genes and miR-27a-5p were detected by qRT-PCR and western blot as needed. The differential expression of FOSL1 and the effect of miR-27a-5p on survival rate were analyzed using GEPIA and UALCAN, respectively. FOSL1 was found highly expressed in glioma cells and patients. The appearance of spherical cells and high expressions of CSC-related markers indicated the successful isolation of CSC-like cells. The increment of X-ray dose enhanced the sensitivity of cancer cells to radiotherapy. Radiotherapy down-regulated cell viability and the expressions of FOSL1 and Bcl-2, but up-regulated cell apoptosis and the expressions of cleaved caspase-3 and Bax, which could be partially reversed by overexpressed FOSL1 or further enhanced by shFOSL1. MiR-27a-5p was highly expressed in in patients with glioma, which was associated with poor prognosis, while shFOSL1-inhibited miR-27a-5p expression enhanced the sensitivity of glioma stem cells to radiotherapy. In vivo experiments further verified the results obtained from in vitro experiments. Silent FOSL1 strengthened the radiosensitivity of glioma by down-regulating miR-27a-5p.

A novel visceral pain model of uterine cervix inflammation in rat.

Treatment of visceral pain originating from the uterine cervix is a substantial clinical problem. The underlying mechanisms of such visceral pain remain unclear mainly due to a lack of reliable model. This study aimed to develop and evaluate the performance of a rat model of pain induced by uterine cervix inflammation. Rats were randomized to six groups according to the solution injected into the uterine cervix: normal saline, vehicle, capsaicin (0.3 mg, 0.6 mg, 0.9 mg), capsaicin 0.9 mg + morphine (n = 15 in each group). Spontaneous behaviors after cervical injection were recorded by a computerized video system and analyzed offline. An equation for calculating a novel pain score was derived from particular behaviors, based on Pearson's correlation analysis and regression analysis. c-Fos expression in the spinal cord was detected. The pain score and c-fos expression in the spinal cord were highest in the 0.9 mg capsaicin group and lowest in the normal saline and vehicle groups (P < 0.05). Intrathecal morphine significantly decreased the pain score (P < 0.05) and c-fos expression in the spinal cord (P < 0.05). Injection of capsaicin into the uterine cervix in rats could be a practical model of inflammatory cervical pain, which can be evaluated using our novel pain score. This model will provide further insight into the mechanism underlying visceral pain originating from the uterine cervix.

Therapeutic targeting of FOS in mutant TERT cancers through removing TERT suppression of apoptosis via regulating survivin and TRAIL-R2.

The telomerase reverse transcriptase (TERT) has long been pursued as a direct therapeutic target in human cancer, which is currently hindered by the lack of effective specific inhibitors of TERT. The FOS/GABPB/(mutant) TERT cascade plays a critical role in the regulation of mutant TERT, in which FOS acts as a transcriptional factor for GABPB to up-regulate the expression of GABPB, which in turn activates mutant but not wild-type TERT promoter, driving TERT-promoted oncogenesis. In the present study, we demonstrated that inhibiting this cascade by targeting FOS using FOS inhibitor T-5224 suppressed mutant TERT cancer cells and tumors by inducing robust cell apoptosis; these did not occur in wild-type TERT cells and tumors. Mechanistically, among 35 apoptotic cascade-related proteins tested, the apoptosis induced in this process specifically involved the transcriptional activation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) and inactivation of survivin, two key players in the apoptotic cascade, which normally initiate and suppress the apoptotic cascade, respectively. These findings with suppression of FOS were reproduced by direct knockdown of TERT and prevented by prior knockdown of TRAIL-R2. Further experiments demonstrated that TERT acted as a direct transcriptional factor of survivin, up-regulating its expression. Thus, this study identifies a therapeutic strategy for TERT promoter mutation-driven cancers by targeting FOS in the FOS/GABPB/(mutant) TERT cascade, circumventing the current challenge in pharmacologically directly targeting TERT itself. This study also uncovers a mechanism through which TERT controls cell apoptosis by transcriptionally regulating two key players in the apoptotic cascade.

Hinokitiol inhibits RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced bone loss in vivo.

Osteoporosis is a metabolic bone-loss disease characterized by abnormally excessive osteoclast formation and bone resorption. Identification of natural medicines that can inhibit osteoclastogenesis, bone resorption, and receptor activator of nuclear factor-κB ligand (RANKL)-induced signaling is necessary for improved treatment of osteoporosis. In this study, hinokitiol, a tropolone-related compound extracted from the heart wood of several cupressaceous plants, was found to inhibit RANKL-induced osteoclast formation and bone resorption in vitro. Hinokitiol inhibited early activation of the ERK, p38, and JNK-MAPK pathways, thereby suppressing the activity and expression of downstream factors (c-Jun, c-Fos, and NFATC1). Consistent with the above in vitro findings, hinokitiol treatment protected against ovariectomy-induced bone loss in vivo. Collectively, our results imply that hinokitiol can potentially serve as an effective agent for treating osteoclast-induced osteoporosis.

Analysis of Side Population in Solid Tumor Cell Lines.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

Blocking Fra-1 sensitizes triple-negative breast cancer to PARP inhibitor.

The AP-1 member Fra-1 is overexpressed in TNBC and plays crucial roles in tumor progression and treatment resistance. In a previous large-scale screen, we identified PARP1 to be among 118 proteins that interact with endogenous chromatin-bound Fra-1 in TNBC cells. PARP1 inhibitor (olaparib) is currently in clinical use for treatment of BRCA-mutated TNBC breast cancer. Here, we demonstrate that the Fra-1-PARP1 interaction impacts the efficacy of olaparib treatment. We show that PARP1 interacts with and downregulates Fra-1, thereby reducing AP-1 transcriptional activity. Olaparib treatment, or silencing of PARP1, consequently, increases Fra-1 levels and enhances its transcriptional activity. Increased Fra-1 can have adverse effect, including treatment resistance. We also found that a large fraction of PARP1-regulated genes was dependent on Fra-1. We show that by inhibiting Fra-1/AP-1, non-BRCA-mutated TNBC cells can become sensitized to olaparib treatment. We identify that high PARP1 expression is indicative of a poor clinical outcome in breast cancer patients overall (P = 0.01), but not for HER-2 positive patients. In conclusion, by exploring the functionality of the Fra-1 and PARP1 interaction, we propose that targeting Fra-1 could serve as a combinatory therapeutic approach to improve olaparib treatment outcome for TNBC patients.

Importin α3 regulates chronic pain pathways in peripheral sensory neurons.

How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.

Discovery of phenanthridine analogues as novel chemical probes disrupting the binding of DNA to ΔFosB homodimers and ΔFosB/JunD heterodimers.

The transcription factor ΔFosB accumulates in response to chronic insults such as drugs of abuse, L-3,4-dihydroxyphenylalanine (l-DOPA) or stress in specific regions of the brain, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, dyskinesia, and depression. Thus, small molecule chemical probes are urgently needed to investigate biological functions of ΔFosB. Herein we describe the identification of a novel phenanthridine analogue ZL0220 (27) as an active and promising ΔFosB chemical probe with micromolar inhibitory activities against ΔFosB homodimers and ΔFosB/JunD heterodimers.

Endoplasmic reticulum stress promotes inflammation-mediated proteolytic activity at the ocular surface.

A growing body of evidence implicates endoplasmic reticulum (ER) stress in the pathogenesis of chronic inflammatory and autoimmune disorders. Here, we demonstrate that the proinflammatory cytokine TNFα stimulates matrix metalloproteinase 9 (MMP9) at the ocular surface through a c-Fos-dependent mechanism of ER stress. We found positive reactivity of the molecular chaperone BiP/GRP78 in conjunctival epithelium of patients with ocular cicatricial pemphigoid and increased levels of BiP/GRP78, sXBP1 and GRP94 in human corneal epithelial cells treated with TNFα. Pharmacological blockade of ER stress in vitro using dexamethasone or the chemical chaperones TUDCA and 4PBA attenuated MMP9 expression and secretion in the presence of TNFα. Moreover, expression analysis of genes associated with inflammation and autoimmunity identified the c-Fos proto-oncogene as a mediator of ER stress responses in epithelial cells. Substantially less TNFα-induced MMP9 expression occurred when c-Fos signaling was suppressed with a function-blocking antibody. Taken together, these results indicate that activation of ER stress contributes to promote inflammation-mediated proteolytic activity and uncovers a target for restoring tissue homeostasis in ocular autoimmune disease.

Coupling Computational and Intracellular Screening and Selection Toward Co-compatible cJun and cFos Antagonists.

Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico/in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.

The increased marginal zone B cells attenuates early inflammatory responses during sepsis in Gpr174 deficient mice.

GPR174 plays a crucial role in immune responses, but the role of GPR174 in the pathological progress of sepsis remains incompletely understood. In this study, we generated a sepsis model by cecal ligation and puncture (CLP) to investigate the role of GPR174 in regulating functions and underlying mechanism of marginal zone B (MZ B) cells in sepsis. We found that in Gpr174 deficient mice, the number of splenic MZ B cells was increased. Moreover, Gpr174-/- MZ B cells exhibited an enhanced response to LPS stimulation in vitro. By using the CLP-induced sepsis model, we demonstrated that the increased MZ B cells attenuated early inflammatory responses during sepsis. RNA sequencing results revealed that the expression of c-fos in splenic B lymphocytes was upregulated in Gpr174 deficient mice. However, the protective role of increased MZ B cells in Gpr174 deficient mice was weakened by a c-fos-specific inhibitor. Collectively, these findings suggested that GPR174 plays an immunomodulatory role in early immune responses during sepsis through the regulation of MZ B cells.

Metformin reduces c-Fos and ATF3 expression in the dorsal root ganglia and protects against oxaliplatin-induced peripheral sensory neuropathy in mice.

Oxaliplatin is a third-generation platinum drug commonly used as the first line treatment of metastatic colorectal cancer. Oxaliplatin-based anticancer regimens course with dose-limiting neurotoxicity. The pharmacological strategies used to manage such side effect are not totally effective. Metformin is an anti-diabetic drug that is described to negatively modulate painful diabetic neuropathy. Then, this study aimed to assess the effect of metformin in the oxaliplatin-induced peripheral sensory neuropathy in mice. For that purpose, Swiss male mice were injected with oxaliplatin (1, 2 or 4 mg/kg, i.v., twice a week with a total of nine injections) alone or in combination with daily administration of metformin (250 mg/kg, p.o.). Thermal and mechanical nociceptive tests were performed once a week for five weeks. Then, the animals were euthanized on day 35 post-first injection of oxaliplatin and the dorsal root ganglia were harvested for the assessment of c-Fos and ATF3 expressions. Oxaliplatin caused a nociceptive response accompanied by the increased expression of c-Fos and ATF3 in the dorsal root ganglia and spinal cord. In addition, the oxaliplatin-associated nociception was significantly attenuated by metformin (P < 0.05), which also reduced the expression of c-Fos and ATF3 (P < 0.05). Therefore, metformin protected from the peripheral sensory neuropathy induced by oxaliplatin, which was confirmed by the reduction of c-Fos and ATF3 expression, two known neuronal activation and damage markers, respectively.

MiR-501-3p Forms a Feedback Loop with FOS, MDFI, and MyoD to Regulate C2C12 Myogenesis.

Skeletal muscle plays an essential role in maintaining body energy homeostasis and body flexibility. Loss of muscle mass leads to slower wound healing and recovery from illness, physical disability, poor quality of life, and higher health care costs. So, it is critical for us to understand the mechanism of skeletal muscle myogenic differentiation for maintaining optimal health throughout life. miR-501-3p is a novel muscle-specific miRNA, and its regulation mechanism on myoblast myogenic differentiation is still not clear. We demonstrated that FOS was a direct target gene of miR-501-3p, and MyoD regulated miR-501-3p host gene Clcn5 through bioinformatics prediction. Our previous laboratory experiment found that MDFI overexpression promoted C2C12 myogenic differentiation and MyoD expression. The database also showed there is an FOS binding site in the MDFI promoter region. Therefore, we hypothesize that miR-501-3p formed a feedback loop with FOS, MDFI, and MyoD to regulate myoblast differentiation. To validate our hypothesis, we demonstrated miR-501-3p function in the proliferation and differentiation period of C2C12 cells by transfecting cells with miR-501-3p mimic and inhibitor. Then, we confirmed there is a direct regulatory relationship between miR-501-3p and FOS, MyoD and miR-501-3p, FOS and MDFI through QPCR, dual-luciferase reporter system, and ChIP experiments. Our results not only expand our understanding of the muscle myogenic development mechanism in which miRNA and genes participate in controlling skeletal muscle development, but also provide treatment strategies for skeletal muscle or metabolic-related diseases in the future.

Idelalisib inhibits osteoclast differentiation and pre-osteoclast migration by blocking the PI3Kδ-Akt-c-Fos/NFATc1 signaling cascade.

Since increased number of osteoclasts could lead to impaired bone structure and low bone mass, which are common characteristics of bone disorders including osteoporosis, the pharmacological inhibition of osteoclast differentiation is one of therapeutic strategies for preventing and/or treating bone disorders and related facture. However, little data are available regarding the functional relevance of phosphoinositide 3-kinase (PI3K) isoforms in the osteoclast differentiation process. To elucidate the functional involvement of PI3Kδ in osteoclastogenesis, here we investigated how osteoclast differentiation was influenced by idelalisib (also called CAL-101), which is p110δ-selective inhibitor approved for the treatment of specific human B cell malignancies. Here, we found that receptor activator of nuclear factor kappa B ligand (RANKL) induced PI3Kδ protein expression, and idelalisib inhibited RANKL-induced osteoclast differentiation. Next, the inhibitory effect of idelalisib on RANKL-induced activation of the Akt-c-Fos/NFATc1 signaling cascade was confirmed by western blot analysis and real-time PCR. Finally, idelalisib inhibited pre-osteoclast migration in the last stage of osteoclast differentiation through down-regulation of the Akt-c-Fos/NFATc1 signaling cascade. It may be possible to expand the clinical use of idelalisib for controlling osteoclast differentiation. Together, the present results contribute to our understanding of the clinical value of PI3Kδ as a druggable target and the efficacy of related therapeutics including osteoclastogenesis.

Activation of protein kinase C accelerates murine osteoclastogenesis partly via transactivation of RANK gene through functional AP-1 responsive element in RANK gene promoter.

Receptor activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts. We have cloned a basic promoter region of the mouse RANK gene and have analyzed the transcription machinery by transcription factors such as PU.1 (-480), and MITF (-100). Here, we examined the regulatory mechanisms of RANK gene transcription through AP-1 binding site, agagctca (-240). RANK mRNA expression in pre-osteoclastic RAW264.7 cells was induced by Phorbol12-myristate13-acetate (PMA) and suppressed by protein kinase C (PKC) inhibitor calphostin C. In RAW264.7 cells, Fos knockdown by siRNA blocked the inducible effect of PMA on RANK expression. By EMSA, an oligonucleotide (-246/-238) showed DNA protein binding, the specificity of which was confirmed by block-shift assay with an anti-Fos antibody and by the addition of the excess of a cold consensus probe. Co-transfection with a Fos expression vector showed that Fos increased RANK promoter activity 6-fold in RAW264.7 cells, and the addition of PU.1 and MITF superinduced the activity more than twenty-fold by the addition of PU.1 and MITF. Mutagenesis of the putative AP-1 site (-240) blocked the inducible effect of Fos on promoter activity. Taken together, these results indicate that during the differentiation of bone marrow mono-nucleated cells into osteoclast precursors, RANK transcription is positively regulated by Fos/AP-1 through the binding element of its gene promoter, supporting the concept that Fos activation by continuous CSF-1 stimulation on macrophages triggers initial expression of RANK and, later, a positive feedback loop by RANKL-RANK interaction.

Histone deacetylase inhibitor CI-994 inhibits osteoclastogenesis via suppressing NF-κB and the downstream c-Fos/NFATc1 signaling pathways.

[4-(acetylamino)-N-(2-amino-phenyl) benzamide] (CI-994) is a histone deacetylase 1-3 specific inhibitor that has been shown to indirectly increase the production of Dickkopf-1, which is an inhibitor of osteoclastic development. However, whether CI-994 has an influence on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis is still unclear; in our study, this mechanism was investigated. In an in vitro study, using a tartrate-resistant acid phosphatase (TRAP) stain, an F-actin ring, bone absorption test, quantitative PCR and Western blotting, the role of CI-994 in osteoclastogenesis and the expression of related genes and proteins were investigated. In an in vivo study, the effect of CI-994 on osteolysis was evaluated using a titanium particle-induced murine calvarial osteolysis model. Our results indicated that CI-994 inhibited osteoclast differentiation and the function of bone resorption without cytotoxic effects. Moreover, CI-994 inhibited the expression of osteoclast-related genes, including ACP5, CTSK, NFATc1, c-Fos, DC-STAMP and V-ATPase-d2. Furthermore, CI-994 suppressed the phosphorylation of IκBα and p65 and the expression of downstream c-Fos and NFATc1. Consistent with the in vitro results described above, our in vivo experiment indicated that CI-994 inhibited Ti-induced osteolysis. In conclusion, CI-994 inhibited osteoclastogenesis by suppressing NF-κB and the downstream c-Fos/NFATc1 signaling pathway. Thus, this study showed the possibility of using CI-994 for the treatment of exorbitant osteoclastic bone resorption.