ABSTRACT
BACKGROUND: SOX4 is a transcription factor belonging to the SOX (Sry-related High Mobility Group [HMG] box) family and plays a pivotal role in various biological processes at various stages of life. SOX4 is also expressed in the skin in adults and has been reported to be involved in wound healing, tumor formation, and metastasis. METHODS AND RESULTS: In this study, we investigated the role of SOX4 in keratinocyte phenotypic changes. We generated a SOX4-overexpressing keratinocyte cell line that expresses SOX4 in a doxycycline (DOX)-inducible manner. DOX treatment induced a change from a paving stone-like morphology to a spindle-like morphology under microscopic observation. Comprehensive gene analysis by RNA sequencing revealed increased expression of genes related to anatomical morphogenesis and cell differentiation as well as decreased expression of genes related to epithelial formation and keratinization, suggesting that SOX4 induced EMT-like phenotype in keratinocytes. Differentially expressed genes (DEGs) obtained by RNA-seq were confirmed using qRT-PCR. DOX-treated TY-1 SOX4 showed a decrease in the epithelial markers (KRT15, KRT13, KRT5, and CLDN1) and an increase in the mesenchymal marker FN1. Protein expression changes by Western blotting also showed a decrease in the epithelial marker proteins keratin 15, keratin 13, and claudin 1, and an increase in the mesenchymal marker fibronectin. Removal of DOX from DOX-treated cells also restored the epithelial and mesenchymal markers altered by SOX4. CONCLUSION: Our results indicate that SOX4 reversibly induces an EMT-like phenotype in human keratinocytes via suppression of epithelial marker genes.
Subject(s)
Keratinocytes , SOXC Transcription Factors , Skin , Humans , Blotting, Western , Doxycycline , Gene Expression , Phenotype , SOXC Transcription Factors/geneticsABSTRACT
Epithelial-mesenchymal transition (EMT) is a cellular process in which epithelial cells lose their epithelial traits and shift to the mesenchymal phenotype, and is associated with various biological events, such as embryogenesis, wound healing and cancer progression. The transcriptional program that promotes phenotype switching is dynamically controlled by transcription factors during EMT, including Snail (SNAI1), twist family bHLH transcription factor (TWIST) and zinc finger E-box binding homeobox 1 (ZEB1). The present study aimed to investigate the molecular mechanisms underlying EMT in squamous epithelial cells. Western blot analysis and immunocytochemical staining identified Slug (SNAI2) as a transcription factor that is induced during transforming growth factor (TGF)-ß1-mediated EMT in the human keratinocyte cell line HaCaT. The effect of SNAI2 overexpression and knockdown on the phenotypic characteristics of HaCaT cells was evaluated. Filamentous actin staining and western blot analysis revealed that the overexpression of SNAI2 did not induce the observed EMT-related phenotypic changes. In addition, SNAI2 knockdown demonstrated almost no impact on the EMT phenotypes induced by TGF-ß1. Notably, DNA microarray analysis followed by comprehensive bioinformatics analysis revealed that the differentially expressed genes upregulated by TGF-ß1 were significantly enriched in cell adhesion and extracellular matrix binding, whereas the genes downregulated in response to TGF-ß1 were significantly enriched in the cell cycle. No enriched gene ontology term and biological pathways were identified in the differentially expressed gene sets of SNAI2-overexpressing cells. In addition, the candidates for master transcription factors regulating the TGF-ß1-induced EMT were identified using transcription factor enrichment analysis. In conclusion, the results of study demonstrated that SNAI2 does not play an essential role in the EMT of HaCaT cells and identified candidate transcription factors that may be involved in EMT-related gene expression induced by TGF-ß1. These findings may enhance the understanding of molecular events in EMT and contribute to the development of a novel therapeutic approach against EMT in cancers and wound healing.
ABSTRACT
CYLD negatively regulates the NF-κB signaling pathway and osteoclast differentiation largely through antagonizing TNF receptor-associated factor (TRAF)-mediated K63-linkage polyubiquitination in osteoclast precursor cells. CYLD activity is controlled by IκB kinase (IKK), but the molecular mechanism(s) governing CYLD protein stability remains largely undefined. Here, we report that SCFß-TRCP regulates the ubiquitination and degradation of CYLD, a process dependent on prior phosphorylation of CYLD at Ser432/Ser436 by IKK. Furthermore, depletion of ß-TRCP induced CYLD accumulation and TRAF6 deubiquitination in osteoclast precursor cells, leading to suppression of RANKL-induced osteoclast differentiation. Therefore, these data pinpoint the IKK/ß-TRCP/CYLD signaling pathway as an important modulator of osteoclastogenesis.
Subject(s)
SKP Cullin F-Box Protein Ligases/genetics , Tumor Suppressor Proteins/genetics , Animals , Deubiquitinating Enzyme CYLD , HeLa Cells , Humans , Male , Mice , Osteoclasts , SKP Cullin F-Box Protein Ligases/metabolism , Signal Transduction , Transfection , Tumor Suppressor Proteins/metabolism , UbiquitinationABSTRACT
Nitrogen-containing bisphosphonates have been well known to be inhibited farnesyl diphosphate synthase (FDPS), an enzyme in mevalonic acid metabolism, resulting in disturbance in polymerization of cytoskeleton structure in bone resorption and promotion of apoptosis in mature osteoclasts. Although bisphosphonates have been reported to activate ion transporters in native epithelium and Xenopus oocytes, little is known whether bisphosphonates affect acid hydrochronic acid extrusion in osteoclasts during bone resorption. The aim of this study was to determine the role of bisphosphonates on inhibition of hydrochronic acid extrusion in osteoclasts. Effects of zoledronic acid, a nitrogen-containing bisphosphonate, on the Cl(-) current activated by extracellular acidification were examined in two types of osteoclasts derived from RAW264.7 cells and mouse bone marrow macrophages (BMMs). Extracellular acidification induced outwardly rectifying Cl(-) currents in mouse osteoclasts. Zoledronic acid dose-dependently inhibited the acid-activated Cl(-) current. The non-nitrogen bisphosphonate etidronic acid had no effect on the acid-activated Cl(-) current. Tetracycline-induced FDPS silencing caused a significant decrease in the Cl(-) current. The inhibitor of geranylgeranyl transferase suppressed the Cl(-) current. By contrast, the inhibitory action of zoledronic acid was rescued by addition of geranylgeranyl acid, a derivative of mevalonic acid. The activity of acid-activated Cl(-) currents was dependent on expression of ClC-7 during osteoclastogenesis. These results suggest that nitrogen-containing bisphosphonates suppress the activity of osteoclastic acid-activated Cl(-) currents through FDPS inhibition, suggesting the inhibition of Cl(-) extrusion activity.
Subject(s)
Chlorides/physiology , Diphosphonates/pharmacology , Geranyltranstransferase/antagonists & inhibitors , Osteoclasts/drug effects , Alkyl and Aryl Transferases/antagonists & inhibitors , Animals , Bone Marrow Cells/cytology , Cell Line , Cells, Cultured , Diphosphonates/chemistry , Diterpenes/pharmacology , Gene Silencing , Geranyltranstransferase/genetics , Geranyltranstransferase/metabolism , Male , Mice , Nitrogen/chemistry , Osteoclasts/physiology , RNA, Small Interfering/geneticsABSTRACT
To investigate the recognition sites of tRNA(Thr) for threonyl-tRNA synthetase (ThrRS) from an extreme thermophilic and aerobic archaeon, Aeropyrum pernix K1, threonylation experiments using various in vitro mutant transcripts of tRNA(Thr) were examined. The results indicated that A. pernix ThrRS did recognize the first three base pairs of acceptor stem in addition to the second and the third letters of anticodon of tRNA(Thr), in spite of its N-terminal truncated unique structure. Discriminator base was not involved in recognition by A. pernix ThRS. These determinants were confirmed by the identity switching experiments from the in vitro mutants of A. pernix tRNA(Pro) and tRNA(Asn).
Subject(s)
Archaea/enzymology , RNA, Transfer, Thr/metabolism , Threonine-tRNA Ligase/metabolism , RNA, Transfer, Thr/geneticsABSTRACT
Recognition sites of tyrosine tRNA for tyrosyl-tRNA synthetase from Escherichia coli and extreme thermophilic archaeon, Aeropyrum pernix K1 were examined using various in vitro transcripts. With respect to the long variable arm in E. coli tyrosine tRNA, some base pairs in length was required for tyrosylation. None of the recognition sites were found in the acceptor stem, except the discriminator base A73 in E. coli tyrosine tRNA. In case of A. pernix tyrosine tRNA, C1-G72 base pair and discriminator base A73 in the acceptor region as well as anticodon were base specifically involved in tyrosylation by A. pernix tyrosyl-tRNA synthetase.