Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei.

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor that regulates early retinal development.

BACKGROUND: The transcription factor orthodenticle homeobox 2 (OTX2) has critical functions in brain and eye development, and its mutations in humans are related to retinal diseases, such as ocular coloboma and microphthalmia. However, the regulatory mechanisms of OTX2 are poorly identified. OBJECTIVE: The identification of JNK1 as an OTX2 regulatory protein through the protein interaction and phosphorylation. METHODS: To identify the binding partner of OTX2, we performed co-immunoprecipitation and detected with a pooled antibody that targeted effective kinases. The protein interaction between JNK1 and OTX2 was identified with the co-immunoprecipitation and immunocytochemistry. In vivo and in vitro kinase assay of JNK1 was performed to detect the phosphorylation of OTX2 by JNK1. RESULTS: JNK1 directly interacted with OTX2 through the transactivation domain at the c-terminal region. The protein-protein interaction and co-localization between JNK1 and OTX2 were further validated in the developing P0 mouse retina. In addition, we confirmed that the inactivation of JNK1 K55N mutant significantly reduced the JNK1-mediated phosphorylation of OTX2 by performing an immune complex protein kinase assay. CONCLUSION: c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor through the protein-protein interaction.

Mercury Chloride but Not Lead Acetate Causes Apoptotic Cell Death in Human Lung Fibroblast MRC5 Cells via Regulation of Cell Cycle Progression.

Heavy metals are important for various biological systems, but, in excess, they pose a serious risk to human health. Heavy metals are commonly used in consumer and industrial products. Despite the increasing evidence on the adverse effects of heavy metals, the detailed mechanisms underlying their action on lung cancer progression are still poorly understood. In the present study, we investigated whether heavy metals (mercury chloride and lead acetate) affect cell viability, cell cycle, and apoptotic cell death in human lung fibroblast MRC5 cells. The results showed that mercury chloride arrested the sub-G1 and G2/M phases by inducing cyclin B1 expression. In addition, the exposure to mercury chloride increased apoptosis through the activation of caspase-3. However, lead had no cytotoxic effects on human lung fibroblast MRC5 cells at low concentration. These findings demonstrated that mercury chloride affects the cytotoxicity of MRC5 cells by increasing cell cycle progression and apoptotic cell death.

Bisphenol A Exposure Changes the Transcriptomic and Proteomic Dynamics of Human Retinoblastoma Y79 Cells.

Bisphenol A (BPA) is a xenoestrogen chemical commonly used to manufacture polycarbonate plastics and epoxy resin and might affect various human organs. However, the cellular effects of BPA on the eyes have not been widely investigated. This study aimed to investigate the cellular cytotoxicity by BPA exposure on human retinoblastoma cells. BPA did not show cytotoxic effects, such as apoptosis, alterations to cell viability and cell cycle regulation. Comparative analysis of the transcriptome and proteome profiles were investigated after long-term exposure of Y79 cells to low doses of BPA. Transcriptome analysis using RNA-seq revealed that mRNA expression of the post-transcriptional regulation-associated gene sets was significantly upregulated in the BPA-treated group. Cell cycle regulation-associated gene sets were significantly downregulated by exposure to BPA. Interestingly, RNA-seq analysis at the transcript level indicated that alternative splicing events, particularly retained introns, were noticeably altered by low-dose BPA treatment. Additionally, proteome profiling using MALDI-TOF-MS identified a total of nine differentially expressed proteins. These results suggest that alternative splicing events and altered gene/protein expression patterns are critical phenomena affected by long-term low-dose BPA exposure. This represents a novel marker for the detection of various diseases associated with environmental pollutants such as BPA.

Transcriptome Analysis Reveals HgCl2 Induces Apoptotic Cell Death in Human Lung Carcinoma H1299 Cells through Caspase-3-Independent Pathway.

Mercury is one of the detrimental toxicants that can be found in the environment and exists naturally in different forms; inorganic and organic. Human exposure to inorganic mercury, such as mercury chloride, occurs through air pollution, absorption of food or water, and personal care products. This study aimed to investigate the effect of HgCl2 on cell viability, cell cycle, apoptotic pathway, and alters of the transcriptome profiles in human non-small cell lung cancer cells, H1299. Our data show that HgCl2 treatment causes inhibition of cell growth via cell cycle arrest at G0/G1- and S-phase. In addition, HgCl2 induces apoptotic cell death through the caspase-3-independent pathway. Comprehensive transcriptome analysis using RNA-seq indicated that cellular nitrogen compound metabolic process, cellular metabolism, and translation for biological processes-related gene sets were significantly up- and downregulated by HgCl2 treatment. Interestingly, comparative gene expression patterns by RNA-seq indicated that mitochondrial ribosomal proteins were markedly altered by low-dose of HgCl2 treatment. Altogether, these data show that HgCl2 induces apoptotic cell death through the dysfunction of mitochondria.

Ablation of SMUG1 Reduces Cell Viability and Increases UVC-Mediated Apoptosis in Hepatocarcinoma HepG2 Cells.

Uracil is an unavoidable aberrant base in DNA sequences, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires multiple biochemical steps with conformational changes of DNA that inhibit DNA replication and interfere with transcription. However, the relevance of uracil in DNA for cellular physiology and transcriptional regulation is not fully understood. We investigated the functional roles of SMUG1 using knock-down (KD) and knock-out (KO) models. The proliferation ratio of SMUG1 KD and KO cells was decreased compared to WT control cells, and the cell cycle was arrested in the G2/M phases before the transition to mitosis. The apoptotic cell death was increased in KD and KO cell lines through the increase of BAX and active caspase 3 expression. Phospho-gamma-H2AX expression, which reflected accumulated DNA damage, was also increased in KO cells. Moreover, the apoptotic cells by DNA damage accumulation were markedly increased in SMUG1 KD and KO cells after ultraviolet C irradiation. Transcriptomic analysis using RNA-seq revealed that SMUG1 was involved in gene sets expression including cell cycle transition and chromatin silencing. Together, the results implicate SMUG1 as a critical factor in cell cycle and transcriptional regulation.

Ethylparaben induces apoptotic cell death in human placenta BeWo cells via the Caspase-3 pathway.

Parabens are generally used as preservatives in foods, pharmaceuticals, and various other commercial products. Among them, ethylparaben has weaker estrogenic characteristics than endogenous estrogen. However, growing evidence indicates that ethylparaben has an adverse effect on various human tissues. Here, we investigated whether ethylparaben induces cell death by affecting cell viability, cell proliferation, cell cycle, and apoptosis using the human placenta cell line BeWo. Ethylparaben significantly decreased cell viability in a dose-dependent manner. It caused cell cycle arrest at sub-G1 by reducing the expression of cyclin D1, whereas it decreased the cell proportion at the G0/G1 and S phases. Furthermore, we verified that ethylparaben induces apoptotic cell death by enhancing the activity of Caspase-3. Taken together, our results suggest that ethylparaben exerts cytotoxic effects in human placental BeWo cells via cell cycle arrest and apoptotic pathways.

Transcriptome dynamics of alternative splicing events revealed early phase of apoptosis induced by methylparaben in H1299 human lung carcinoma cells.

Methylparaben is most frequently used as an antimicrobial preservative in pharmaceuticals and foods. Methylparaben has been subjected to toxicological studies owing to the increasing concern regarding its possible impact on the environment and human health. However, the cytotoxicity and underlying mechanisms of methylparaben exposure in human lung cells have not been explored. Here, we investigated the effect of methylparaben on cell cycle, apoptotic pathways, and changes in the transcriptome profiles in human lung cells. Our results demonstrate that treatment with methylparaben causes inhibition of cell growth. In addition, methylparaben induced S- and G2/M-phase arrest as a result of enhanced apoptosis. Transcriptome analysis using RNA-seq revealed that mRNA expression of ER stress- and protein misfolding-related gene sets was upregulated in methylparaben-treated group. RNA splicing- and maturation-related gene sets were significantly down-regulated by methylparaben treatment. Interestingly, RNA-seq analysis at the transcript level revealed that alternative splicing events, especially retained intron, were markedly changed by a low dose of methylparaben treatment. Altogether, these data show that methylparaben induces an early phase of apoptosis through cell cycle arrest and downregulation of mRNA maturation.