Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.

Inhibition of RNase 7 by RNase inhibitor promotes inflammation and Staphylococcus aureus growth: Implications for atopic dermatitis.

BACKGROUND: The antimicrobial ribonuclease RNase 7 is abundantly expressed in the epidermis of lesional skin of atopic dermatitis (AD). Host RNase inhibitor (RI) binds to RNase 7 and blocks its ribonuclease activity. This study aimed to evaluate the impact of RNase 7-RI interactions on AD. METHODS: Cultured human primary keratinocytes, with siRNA-mediated downregulation of RNase 7 and RI, were stimulated with the synthetic RNA polyinosinic-polycytidylic acid (poly I:C). Induction of proinflammatory mediators was analyzed by real-time PCR and ELISA. RI expression in AD non-lesional and lesional skin biopsies and healthy controls was analyzed by real-time PCR and immunostaining. RI protein release in vivo on the AD skin surface was determined by western blot. Antimicrobial and ribonuclease assays were used to investigate the functional role of RI. RESULTS: RNase 7 inhibited the RNA-induced expression of proinflammatory mediators in keratinocytes. Accordingly, downregulation of RNase 7 in keratinocytes enhanced RNA-mediated induction of proinflammatory mediators, whereas downregulation of RI had the opposite effect. RI was released by damaged keratinocytes and epidermis. In vivo expression and release of RI on the skin surface were enhanced in lesional AD skin. Rinsing solution from the surface of lesional AD skin blocked the ribonuclease activity of RNase 7. The anti-Staphylococcus aureus activity of RNase 7 was abrogated by RI. CONCLUSIONS: Our data suggest a novel role of RI as a trigger factor of inflammation in AD by blocking the ribonuclease and antimicrobial activity of RNase 7, thereby enhancing RNA-mediated inflammation and S. aureus growth.

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

Reprogramming of Protein-Targeted Small-Molecule Medicines to RNA by Ribonuclease Recruitment.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

Barnase-Barstar Pair: Contemporary Application in Cancer Research and Nanotechnology.

Barnase is an extracellular ribonuclease secreted by Bacillus amyloliquefaciens that was originally studied as a small stable enzyme with robust folding. The identification of barnase intracellular inhibitor barstar led to the discovery of an incredibly strong protein-protein interaction. Together, barnase and barstar provide a fully genetically encoded toxin-antitoxin pair having an extremely low dissociation constant. Moreover, compared to other dimerization systems, the barnase-barstar module provides the exact one-to-one ratio of the complex components and possesses high stability of each component in a complex and high solubility in aqueous solutions without self-aggregation. The unique properties of barnase and barstar allow the application of this pair for the engineering of different variants of targeted anticancer compounds and cytotoxic supramolecular complexes. Using barnase in suicide gene therapy has also found its niche in anticancer therapy. The application of barnase and barstar in contemporary experimental cancer therapy is reflected in the review.

Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor.

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus.

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.

The role of monocyte chemotactic protein-induced protein 1 (MCPIP1) in angiotensin II-induced macrophage apoptosis and vulnerable plaque formation.

Atherosclerotic plaque rupture is the main cause of acute coronary syndrome (ACS). Angiotensin II (Ang II) and macrophage apoptosis are involved in the pathogenesis of atherosclerosis. However, the underlying mechanisms remain unclear. We aimed to address the role of monocyte chemotactic protein-induced protein 1 (MCPIP1) in Ang II-induced macrophage apoptosis and vulnerable plaque formation. In mouse peritoneal macrophages, Ang II promoted endoplasmic reticulum (ER) stress-dependent macrophage apoptosis. Ang II markedly upregulated the expression of MCPIP1 via activating p38 mitogen-activated protein kinase (p38 MAPK). Treatment with MCPIP1 shRNA downregulated ER stress-related proteins and decreased macrophage apoptosis induced by Ang II. Ang II also activated the AMP-activated protein kinase (AMPK) signaling in macrophages. Inhibition of AMPK reduced macrophage apoptosis by inhibiting the p38 MAPK/MCPIP1/ER stress pathway. Furthermore, blocking the Ang II type 1 receptor (AT1R) with losartan effectively inhibited Ang II-induced macrophage apoptosis and AMPK/p38 MAPK/MCPIP1/ER pathway activation. In the atherosclerotic vulnerable plaque model in mice, losartan inhibited the progression of atherosclerosis and transformed vulnerable plaque into a more stable phenotype. Moreover, losartan markedly decreased the number of CD68+TUNEL+, CD68+MCPIP1+, CD68+p-eIF2α+ and CD68+CHOP+ cells in the lesion area. Taken together, Ang II promotes macrophage apoptosis via the AMPK/p38 MAPK/MCPIP1/ER stress pathway in macrophages via its receptor AT1R, which may contribute to vulnerable plaque formation. Our study clarifies a novel regulatory role of MCPIP1 in Ang II-induced macrophage apoptosis and plaque instability, providing a potential therapeutic target for prevention of ACS.

The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant.

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic ß-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes.

PEGylated DC-Chol/DOPE cationic liposomes containing KSP siRNA as a systemic siRNA delivery Carrier for ovarian cancer therapy.

Although siRNA-mediated downregulation technology has been highly successful in suppressing the expression of any disease-related gene, systemic delivery of siRNA for the clinical applications remains challenging, especially in the use of cancer therapy. DC-Chol/DOPE cationic liposomes as one of the most attractive vehicles for gene delivery have been widely exploited for transfection of siRNA into cells, but complexity of systemic delivery has allowed only their direct injection into local targets due to the formation of aggregations with negatively-charged blood components. Herein, we demonstrate the effects of PEGylation on DC-Chol/DOPE cationic liposomes for systemic siRNA delivery in cancer therapy. In contrast to non-PEGylated DC-Chol/DOPE-siRNA lipoplexes, PEGylated DC-Chol/DOPE-siRNA lipoplexes reduce the excretion by kidneys and scavenging in liver, prolonging the circulation time in vivo, and ultimately increase their preferential tumor accumulation. Therefore, systemic injection of PEGylated DC-Chol/DOPE liposomes loaded with siRNA against kinesin spindle protein (KSP) gene exhibited a high level of target gene silencing at tumor sites and substantial suppression of tumor growth. Furthermore, systemically administered PEGylated lipoplexes did not lead to any activation of innate immune responses in the immunocompetent mice. These results suggest the potential of PEGylated DC-Chol/DOPE liposomes as a systemic delivery carrier for siRNA-mediated cancer therapy.

Molecularly imprinted nanoparticles for inhibiting ribonuclease in reverse transcriptase polymerase chain reaction.

Molecularly imprinted nanoparticles (nanoMIPs) are synthesized via a solid-phase approach using RNase as the template. The feasibility of employing the nanoMIPs as RNase inhibitor is successfully demonstrated in reverse transcriptase polymerase chain reaction (RT-PCR) assays, suggesting the tailor-made nanomaterials are very promising for use in routine biological assays.

Collagen Membranes with Ribonuclease Inhibitors for Long-Term Stability of Electrochemical Aptamer-Based Sensors Employing RNA.

Electrochemical aptamer-based (E-AB) sensors offer advantageous analytical detection abilities due to their rapid response time (seconds to minutes), specificity to a target, and selectivity to function in complex media. Ribonucleic acid (RNA) aptamers employed in this class of sensor offer favorable binding characteristics resulting from the ability of RNA to form stable tertiary folds aided by long-range intermolecular interactions. As a result, RNA aptamers can fold into three-dimensional structures more complex than those of their DNA counterparts and consequently exhibit better binding ability to target analytes. Unfortunately, RNA aptamers are susceptible to degradation by nucleases, and for this reason, RNA-based sensors are scarce or require significant sample pretreatment before use in clinically relevant media. Here, we combine the usefulness of a collagen I hydrogel membrane with entrapped ribonuclease inhibitors (RI) to protect small molecule RNA E-AB sensors from endogenous nucleases in complex media. More specifically, the biocompatibility of the naturally polymerized hydrogel with encapsulated RI promotes the protection of an aminoglycoside-binding RNA E-AB sensor up to 6 h, enabling full sensor function in nuclease-rich environments (undiluted serum) without the need for prior sample preparation or oligonucleotide modification. The use of collagen as a biocompatible membrane represents a general approach to compatibly interface E-AB sensors with complex biological samples.

Exploring the parameters of post-segregational killing using heterologous expression of secreted toxin barnase and antitoxin barstar in an Escherichia coli case study.

Post-segregational killing (PSK) is a phenotype determined by plasmids using a toxin and an antitoxin gene pair. Loss of the genes depletes the cell's reserve of antitoxin and allows the toxin to act upon the cell. PSK benefits mobile elements when it increases reproductive success relative to other mobile competitors. A side effect of PSK is that plasmids become refractory to displacement from the cell during growth as a monoculture. Most PSK systems use a cytoplasmic toxin, but the external toxins of bacteriocins also have a PSK-like effect. It may be that any toxin and antitoxin gene pair can demonstrate PSK when it is on a plasmid. The secreted ribonuclease barnase and its protein inhibitor barstar have features in common with PSK modules, though their native context is chromosomal. We hypothesized that their recruitment to a plasmid could produce an emergent PSK phenotype. Others had shown that secreted barnase could exert a lethal effect on susceptible bacteria similarly to bacteriocins. However, barnase toxicity did not occur under the conditions tested, suggesting that barnase is toxic to neighbouring cells only under very specific conditions. Bacteriocins are only produced under some conditions, and some conditionality on toxin function or release may be advantageous in general to PSKs with external toxins because it would prevent killing of potential plasmid-naive hosts. Too much conditionality, however, would limit how advantageous the gene pair was to mobile elements, making the genes unlikely to be recruited as a PSK system.

Modulation of influenza A virus replication by microRNA-9 through targeting MCPIP1.

MicroRNAs (miRNAs), a family of small non-coding RNAs controlling translation and transcription of its target genes, play important roles in the regulation of various biological processes, including viral infection. Influenza A viruses (IAV) infection alters expression of cellular miRNAs, which in turn can modify the cellular environment to facilitate efficient virus replication. In this study, we showed that IAV infection significantly induced miR-9 expression in A549 cells, which occurred earlier than drastic expression of viral matrix (M) and nucleoprotein (NP) genes. Overexpression of miR-9 enhanced viral gene expression and production of infectious progeny, while knockdown of miR-9 significantly inhibited IAV replication in A549 cells. Recent studies have revealed antiviral potential of monocyte chemoattractant protein 1-induced protein 1 (MCPIP1), a PIN-like RNase capable of targeting and degrading viral RNA. Subsequently, we comprehensively confirmed that MCPIP1 functionally inhibited viral M and NP genes expression and progeny production, and also was regulated by miR-9 in A549 cells. Furthermore, MCPIP1 overexpression abrogated miR-9-induced IAV replication. Taken together, our findings indicate a new role of miR-9 induction in IAV infection and suggest IAV may hijack cellular miR-9 to benefit the viral life cycle. J. Med. Virol. 89:41-48, 2017. © 2016 Wiley Periodicals, Inc.

Neogambogic acid prevents silica-induced fibrosis via inhibition of high-mobility group box 1 and MCP-1-induced protein 1.

BACKGROUND: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2); early stages are characterized by alveolar inflammation, and later stages are characterized by progressive lung fibrosis. Mounting evidence indicates that high-mobility group box 1 (HMGB1) is involved in pulmonary fibrosis. Whether neogambogic acid (NGA) inhibits macrophage and fibroblast activation induced by SiO2 by targeting HMGB1 remains unclear. METHODS AND RESULTS: Experiments using cultured mouse macrophages (RAW264.7 cells) demonstrated that SiO2 treatment induces the expression of HMGB1 in a time- and dose-dependent manner via mitogen-activated protein kinases (MAPKs) and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway; in turn, this expression causes macrophage apoptosis and fibroblast activation. Pretreating macrophages with NGA inhibited the HMGB1 expression induced by SiO2 and attenuated both macrophage apoptosis and fibroblast activation. Moreover, NGA directly inhibited MCP-1-induced protein 1 (MCPIP1) expression, as well as markers of fibroblast activation and migration induced by SiO2. Furthermore, the effects of NGA on macrophages and fibroblasts were confirmed in vivo by exposing mice to SiO2. CONCLUSION: NGA can prevent SiO2-induced macrophage activation and apoptosis via HMGB1 inhibition and SiO2-induced fibrosis via the MCPIP1 pathway. Targeting HMGB1 and MCPIP1 with NGA could provide insights into the potential development of a therapeutic approach for alleviating the inflammation and fibrosis induced by SiO2.

Endogenous RNase inhibitor contributes to stability of RNA in crude cell lysates: Applicability to RT-qPCR.

Crude cell lysates are increasingly used as input for direct analysis by reverse transcription quantitative PCR (RT-qPCR), particularly for high-throughput applications. We previously demonstrated that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercial cell-lysis reagents for the preparation of RT-qPCR-ready cell lysates; addition of an exogenous RNase inhibitor (RI) to the lysis buffer was found to be unnecessary to maintain RNA stability in cell lysates either freshly prepared or previously stored frozen at -80 °C. In the present study, we have demonstrated that the stability of RNA observed in our cell lysates is due to the presence of the endogenous RI. Furthermore, we have established the generalizability and applicability of this phenomenon by evaluating lysates prepared from cell lines commonly used in virology (A549, HeLa, MDCK, and Vero). Awareness of the mechanism underlying RNA stability may engender greater confidence in generating cell lysates for RT-qPCR without relying on addition of exogenous RI (a substantial cost-saving benefit) and encourage appropriate practices for handling and storage of samples.

A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity.

In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in post-transcriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease-exonuclease-phosphatase domain. Both domains require the presence of two Mg2+ ions in the active site. To facilitate the biochemical analysis of deadenylase enzymes, we have developed a fluorescence-based deadenylase assay. The assay is based on end-point measurement, suitable for quantitative analysis and can be adapted for 96- and 384-well microplate formats. We demonstrate the utility of the assay by screening a chemical compound library, resulting in the identification of non-nucleoside inhibitors of the Caf1/CNOT7 enzyme, a catalytic subunit of the Ccr4-Not deadenylase complex. These compounds may be useful tools for the biochemical analysis of the Caf1/CNOT7 deadenylase subunit of the Ccr4-Not complex and indicate the feasibility of developing selective inhibitors of deadenylase enzymes using the fluorescence-based assay.

Arid5a controls IL-6 mRNA stability, which contributes to elevation of IL-6 level in vivo.

Posttranscriptional regulation of IL-6 has been largely uncharacterized, with the exception of the ribonuclease Regnase-1, which prevents autoimmunity by destabilizing IL-6 mRNA. Here, we identified AT-rich interactive domain-containing protein 5A (Arid5a) as a unique RNA binding protein, which stabilizes IL-6 but not TNF-α mRNA through binding to the 3' untranslated region of IL-6 mRNA. Arid5a was enhanced in macrophages in response to LPS, IL-1ß, and IL-6. Arid5a deficiency inhibited elevation of IL-6 serum level in LPS-treated mice and suppressed IL-6 levels and the development of T(H)17 cells in experimental autoimmune encephalomyelitis. Importantly, Arid5a inhibited the destabilizing effect of Regnase-1 on IL-6 mRNA. These results indicate that Arid5a plays an important role in promotion of inflammatory processes and autoimmune diseases.

A Review of Ribonuclease 7's Structure, Regulation, and Contributions to Host Defense.

The Ribonuclease A Superfamily is composed of a group of structurally similar peptides that are secreted by immune cells and epithelial tissues. Several members of the Ribonuclease A Superfamily demonstrate antimicrobial activity, and it has been suggested that some of these ribonucleases play an essential role in host defense. Ribonuclease 7 (RNase 7) is an epithelial-derived secreted peptide with potent broad-spectrum antimicrobial activity. This review summarizes the published literature on RNase 7's antimicrobial properties, structure, regulation, and contributions to host defense. In doing so, we conclude by highlighting key knowledge gaps that must be investigated to completely understand the potential of developing RNase 7 as a novel therapeutic for human infectious diseases.

Mycobacterium tuberculosis 38-kDa antigen induces endoplasmic reticulum stress-mediated apoptosis via toll-like receptor 2/4.

Endoplasmic reticulum (ER) stress responses play critical roles in the pathogenesis of tuberculosis. To investigate the regulatory role of the ER stress response in 38-kDa antigen-induced apoptosis, we examined the relationship between the ER stress response and apoptosis in bone marrow-derived macrophages (BMDMs) stimulated with Mycobacterium tuberculosis antigen (38-kDa Ag). The expression of ER molecular chaperones, including C/EBP homologous protein (CHOP), glucose-regulated protein (Bip) and phosphorylated alpha subunit of eukaryotic initiation factor 2, was induced in BMDMs stimulated with the 38-kDa Ag. Interestingly, 38-kDa Ag-stimulation induced apoptosis via activation of caspase-12, -9 and -3. However, 38-kDa Ag-induced apoptosis was significantly reduced in TLR2- and TLR4-deficient macrophages. Because toll-like receptors (TLRs) initiate the activation of mitogen-activated protein kinase (MAPK) signaling cascades, we evaluated the effect of MAPK activation on ER stress. The 38-kDa Ag activated Jun N-terminal kinase, extracellular signal-regulated kinase and p38 phosphorylation. MAPK signaling induced the secretion of proinflammatory cytokines such as MCP-1, TNF-α and IL-6. The 38-kDa Ag-induced MCP-1 was especially associated with the induction of MCP-1-induced protein (MCPIP), which increased the generation of reactive oxygen species (ROS) and ER stress. To investigate the role of MCPIP in ROS-induced ER stress by 38-kDa Ag stimulation, we transfected MCPIP siRNA into RAW264.7 cells before 38-kDa Ag stimulation, and measured the generation of ROS and expression of ER molecular chaperones. ROS production and CHOP expression were decreased by the silencing of MCPIP induction. Our results demonstrate that the expression of MCPIP by 38-kDa Ag stimulation is increased through a TLR-MAPK-dependent signaling pathway, and leads to ER stress-induced apoptosis. In conclusion, MCPIP is important for host defense mechanisms in mycobacterial pathogenesis.