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Significance Tamoxifen is beneficial in treating estrogen receptor–positive breast cancer, but resistance to this treatment eventually ensues. A method to identify mechanisms of tamoxifen resistance identified the histone deacetylase ZIP, leading to the finding that increased expression of the tyrosine kinase JAK2 is one important factor. As a result of this discovery, it may be possible to use an inhibitor of JAK2 to block the aberrant activation of STAT3 caused by ZIP deficiency to help overcome or prevent tamoxifen resistance., Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example is JAK2. By binding to two specific sequences in the promoter, ZIP suppresses JAK2 expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ER-positive breast cancer.

Dynamic regulation of transcription is crucial for cellular response to various environmental or developmental cues. Gdown1 is a ubiquitously expressed, RNA polymerase II (Pol II) interacting protein, essential for embryonic development. It tightly binds Pol II in vitro and competitively blocks binding of TFIIF and other transcriptional regulatory factors, yet its cellular functions and regulatory circuits remain unclear. Here, we show that Gdown1 strictly localizes in the cytoplasm of mammalian somatic cells and exhibits potent resistance to the imposed driving force for nuclear localization. Combined with genetic and microscope-based approaches, two types of functionally coupled and evolutionally conserved localization regulatory motifs are identified, including the CRM1-dependent nucleus export signal (NES) and a novel Cytoplasm Anchoring Signal (CAS) which mediates nuclear pore retention. Mutagenesis of CAS alleviates the cytoplasmic retention activity thus unlocks its nucleocytoplasmic shuttling properties, and increased nuclear import of Gdown1 causes drastic reduction of Pol II levels and global transcription. Importantly, nuclear translocation of Gdown1 occurs in a stress-responsive manner and ablation of GDOWN1 significantly weakens cellular tolerance. Collectively, our work uncovers the molecular basis of the localization of Gdown1 and highlights that its controlled nuclear translocation serves as a key strategy in modulating global transcription and stress-adaptation.

Detection and degradation of foreign nucleic acids is an ancient form of host defense. However, the underlying mechanisms are not completely clear. MCPIP1 is an endoribonuclease and an important regulator in both innate and adaptive immunity by targeting inflammatory mRNA degradation. Here we report that MCPIP1 RNase can also selectively detect and degrade the mRNAs encoded by transfected plasmids. In transient transfection, MCPIP1 expression potently degraded the mRNA from exogenously transfected vectors, which is independent on the vector, genes and cell types used. Conversely, the expression of transfected plasmids in MCPIP1-null cells is significantly higher than that in wild-type cells. Interestingly, overexpression of MCPIP1 or MCPIP1 deficiency does not affect the expression of the exogenous genes incorporated into the host genome in a stable cell line or the global gene expression of host genome. This ability is not associated with PKR/RNase L system, as PKR inhibitors does not block MCPIP1-mediated mRNA degradation of exogenously transfected genes. Lastly, expression of MCPIP1 suppressed replication of Zika virus in infected cells. The study may provide a model for understanding the antiviral mechanisms of MCPIP1, and a putative tool to increase the expression of transfected exogenous genes.

HIV-1 primarily infects activated CD4+ T cells and macrophages. Quiescent CD4+ T cells, however, possess cellular factors that limit HIV-1 infection at different postentry steps of the viral life cycle. Here, we show that the previously reported immune regulator monocyte chemotactic protein-induced protein 1 (MCPIP1) restricts HIV-1 production in CD4+ T cells. While the ectopic expression of MCPIP1 in cell lines abolished the production of HIV-1, silencing of MCPIP1 enhanced HIV-1 production. Subsequent analysis indicated that MCPIP1 imposes its restriction by decreasing the steady levels of viral mRNA species through its RNase domain. Remarkably, common T-cell stimuli induced the rapid degradation of MCPIP1 in both T-cell lines and quiescent human CD4+ T cells. Lastly, blocking the proteosomal degradation of MCPIP1 by MG132 abrogated HIV-1 production in phorbol 12-myristate 13-acetate/ionomycin-stimulated human CD4+ T cells isolated from healthy donors. Overall, MCPIP1 poses a potent barrier against HIV-1 infection at a posttranscriptional stage. Although the observed HIV restriction conferred by MCPIP1 does not seem to be overcome by any viral protein, it is removed during cellular stimulation. These findings provide insights into the mechanisms of cellular activation-mediated HIV-1 production in CD4+ T cells.

As a component of NURD histone deacetylase complex, ZIP serves as a tumor suppressor gene in the development of breast tumors. However, whether it takes part in chemotherapy resistance remains poorly defined. In the present study, we reported that ZIP enhanced the response to SERM chemotherapy in ER-negative cells. Overexpression of ZIP suppressed EGFR expression level and restored ERalpha protein level in cells resistant to Tamoxifen. In vivo data confirmed those in vitro findings.

Toll-like receptors (TLR) are pivotal in macrophage activation. The molecular mechanisms controlling TLR signaling and macrophage activation are not completely understood. Zc3h12d is originally identified as a possible tumor suppressor gene. However, its function remains unknown. We here report that Zc3h12d negatively regulates TLR signaling and macrophage activation. Zc3h12d was enriched in spleen, lung and lymph node. In macrophages, the expression of Zc3h12d was remarkably induced by TLR ligands through JNK and NF-κB signal pathways. On the other hand, overexpression of Zc3h12d significantly inhibited TLR2 and TLR4 activation-induced JNK, ERK and NF-κB signaling as well as macrophage inflammation. Similar to Zc3h12a/MCPIP1, Zc3h12d also decreased the global cellular protein ubiquitination. These findings suggest that Zc3h12d is a novel negative feedback regulator of TLR signaling and macrophage activation and thus may play a role in host immunity and inflammatory diseases.

It is unclear how stress granule (SG) formation and cellular apoptosis are coordinately regulated. MCPIP1 (monocyte chemotactic protein-induced protein 1), also known as Zc3h12a, is a critical regulator of the inflammatory response and immune homeostasis. However, the role of MCPIP1 in stress response remains unknown. Here, we report that overexpression of MCPIP1 inhibited the assembly of SGs in response to various stresses. Conversely, MCPIP1-deficient splenocytes developed more SGs even without stress. On the other hand, overexpression of MCPIP1 sensitized RAW 264.7 cells to apoptosis under stress, whereas MCPIP1-deficient cells were resistant to stress-induced apoptosis. Mutagenesis study showed that the ability of MCPIP1 to repress SG formation is dependent on its deubiquitinating activity. Consistently, MCPIP1 negatively regulated stress-induced phosphorylation of eIF2α and thus released stress-induced inhibition of protein translation. However, MCPIP1 also inhibited 15-deoxy-Δ(12,14)-prostaglandin J(2)-induced SG formation, which was reported to be independent of eIF2α phosphorylation. Taken together, these results suggest that MCPIP1 coordinates SG formation and apoptosis during cellular stress and may play a critical role in immune homeostasis and resolution of macrophage inflammation.

Ferrocene compounds are a class of biologically active compounds that has antitumour and antifungal properties. This study investigated the induction of apoptosis in human fibrosarcoma cells (HT1080) after treatment with a series of 6-ferrocenyl-3-subsituted7H-1,2,4-triazolo[3,4-b]- 1,3,4-thiadiazine (FTFs). We found that FTFs could suppress the viability of HT1080 cells. Cell cycle analysis showed that proliferative inhibition of HT1080 cells occurred through apoptosis, as the cells were blocked in G1 phase. Moreover, mitochondrial membrane staining assay demonstrated that FTFs exposure significantly decreased mitochondrial membrane potential. Finally, under the stress of FTFs, Bax/Bcl-2 ratio in HT1080 cells was significantly increased. These results suggested that FTFs-induced apoptosis in HT1080 cells may work dependent on a Bax/Bcl-2 pathway.

We evaluated the inhibitory effect of 3,7-dinitrodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay. The degree of DNA damage in K562 cells treated with cBr, was detected by isotopic tracer method (3H-TdR). The morphological changes of these K562 cells were examined by fluorescence and electron microscopy. Biochemical characteristics of K562 cells were detected by flow cytometry and 3H-thymidine incorporation assay. Findings indicated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells. cBr is a new type of immunostimulant and can induce cell apoptosis.

Podophyllotoxin is a well known anti-tumor chemical, but because of its strong side effects much effort has been paid to reduce cytotoxicity by modifying its structure. Here, we evaluate the anti-tumor activity of a new isolated derivative of podophyllotoxin, 4'-demethyl-4-dehydroxy-4-seleno-phenyl-beta-peltatin-epipodophyllotoxin (CPZ) and find that CPZ can suppress the proliferation of human hepatoma SMMC-7721 cells in a dose- and time-dependent manner. Phase-contrast microscope observation and flow cytometric analysis through PI stains showed that the reagents have strong inhibition of SMMC-7721 cell growth, as the cells were blocked in the G2/M period. Cell apoptosis induced by CPZ was further confirmed by staining with M30 Cytodeath antibody. Rh123 label testing revealed that the mitochondrial membrane potential had been decreased by CPZ treatment. Under the stress of CPZ, cytochrome c was secreted into the cytoplasm by mitochondria, and Bax in cytoplasm was translocated into the mitochondrial membrane. These results suggest that CPZ-induced apoptosis may work through a Bax-dependent pathway.

Cumin seeds (Cuminum cyminumL.) have been commonly used in food flavoring and perfumery. In this study, cumin essential oil (CuEO) extracted from seeds was employed to investigate the anti-inflammatory effects in lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells and the underlying mechanisms. A total of 26 volatile constituents were identified in CuEO by GC-MS, and the most abundant constituent was cuminaldehyde (48.773%). Mitochondrial-respiration-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay demonstrated that CuEO did not exhibit any cytotoxic effect at the employed concentrations (0.0005–0.01%). Real-time PCR tests showed that CuEO significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), interleukin- (IL-) 1, and IL-6. Moreover, western blotting analysis revealed that CuEO blocked LPS-induced transcriptional activation of nuclear factor-kappa B (NF-κB) and inhibited the phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). These results suggested that CuEO exerted anti-inflammatory effects in LPS-stimulated RAW 264.7 cells via inhibition of NF-κB and mitogen-activated protein kinases ERK and JNK signaling; the chemical could be used as a source of anti-inflammatory agents as well as dietary complement for health promotion.

Previous studies using MCP-induced protein 1 (MCPIP1)/Zc3h12a-deficient mice suggest that MCPIP1 is an important regulator of inflammation and immune homeostasis. However, the characterization of the immunological phenotype of MCPIP1-deficient mice has not been detailed. In this study, we performed evaluation through histological, flow cytometric, enzyme-linked immunosorbent assay and real-time PCR analysis and found that targeted disruption of MCPIP1 gene leads to fatal, highly aggressive and widespread immune-related lesions. In addition to previously observed growth retardation, splenomegaly, lymphoadenopathy, severe anemia and premature death, MCPIP1-deficient mice showed disorganization of lymphoid organs, including spleen, lymph nodes and thymus, and massive infiltration of lymphocytes, macrophages and neutrophils into many other non-lymphoid organs, primarily in lungs and liver. Flow cytometric analysis found significant increase in activated and differentiated T cells in peripheral blood and spleen of MCPIP1-deficient mice. Moreover, heightened production of inflammatory cytokines from activated macrophages and T cells were observed in MCPIP1-deficient mice. Interestingly, treatment of MCPIP1-deficient mice with antibiotics resulted in significant improvement of life span and a decrease in inflammatory syndrome. Taken together, these results suggest a prominent role for MCPIP1 in the control of inflammation and immune homeostasis.

Objective: MCPIP1 is a newly identified protein that profoundly impacts immunity and inflammation. We aim to test if MCPIP1 deficiency in hematopoietic cells results in systemic inflammation and accelerates atherogenesis in mice. Approach and Results: After lethally irradiated, LDLR 2/2 mice were transplanted with bone marrow cells from either wildtype or MCPIP1 2/2 mice. These chimeric mice were fed a western-type diet for 7 weeks. We found that bone marrow MCPIP1 2/2 mice displayed a phenotype similar to that of whole body MCPIP1 2/2 mice, with severe systemic and multiorgan inflammation. However, MCPIP1 2/2 bone marrow recipients developed .10-fold less atherosclerotic lesions in the proximal aorta than WT bone marrow recipients, and essentially no lesions in en face aorta. The diminishment in atherosclerosis in bone marrow MCPIP1 2/2 mice may be partially attributed to the slight decrease in their plasma lipids. Flow cytometric analysis of splenocytes showed that bone marrow MCPIP1 2/2 mice contained reduced numbers of T cells and B cells, but increased numbers of regulatory T cells, Th17 cells, CD11b+/Gr1+ cells and CD11b+/Ly6C low cells. This overall anti-atherogenic leukocyte profile may also contribute to the reduced atherogenesis. We also examined the cholesterol efflux capability of MCPIP1 deficient macrophages, and found that MCPIP1deficiency increased cholesterol efflux to apoAI and HDL, due to increased protein levels of ABCA1 and ABCG1. Conclusions: Hematopoietic deficiency of MCPIP1 resulted in severe systemic and multi-organ inflammation but paradoxically diminished atherogenesis in mice. The reduced atheroegensis may be explained by the decreased plasma cholesterol levels, the anti-atherogenic leukocyte profile, as well as enhanced cholesterol efflux capability. This study suggests that, while atherosclerosis is a chronic inflammatory disease, the mechanisms underlying atherogenesis-associated inflammation in arterial wall versus the inflammation in solid organs may be substantially different.

Ferrocene compounds are a class of biologically active compounds that has antitumour and antifungal properties. This study investigated the induction of apoptosis in human fibrosarcoma cells (HT1080) after treatment with a series of 6-ferrocenyl-3-subsituted7H-1,2,4-triazolo[3,4-b]- 1,3,4-thiadiazine (FTFs). We found that FTFs could suppress the viability of HT1080 cells. Cell cycle analysis showed that proliferative inhibition of HT1080 cells occurred through apoptosis, as the cells were blocked in G1 phase. Moreover, mitochondrial membrane staining assay demonstrated that FTFs exposure significantly decreased mitochondrial membrane potential. Finally, under the stress of FTFs, Bax/Bcl-2 ratio in HT1080 cells was significantly increased. These results suggested that FTFs-induced apoptosis in HT1080 cells may work dependent on a Bax/Bcl-2 pathway.