Tristetraprolin affects invasion-associated genes expression and cell motility in triple-negative breast cancer model.

Tristetraprolin (TTP) is an RNA-binding protein that negatively regulates its target mRNAs and has been shown to inhibit tumor progression and invasion. Tumor invasion requires precise regulation of cytoskeletal components, and dysregulation of cytoskeleton-associated genes can significantly alter cell motility and invasive capability. Several genes, including SH3PXD2A, SH3PXD2B, CTTN, WIPF1, and WASL, are crucial components of the cytoskeleton reorganization machinery and are essential for adequate cell motility. These genes are also involved in invasion processes, with SH3PXD2A, SH3PXD2B, WIPF1, and CTTN being key components of invadopodia-specialized structures that facilitate invasion. However, the regulation of these genes is not well understood. This study demonstrates that ectopic expression of TTP in MDA-MB-231 cells leads to decreased mRNA levels of CTTN and SH3PXD2A, as well as defects in cell motility and actin filament organization. Additionally, doxorubicin significantly increases TTP expression and reduces the mRNA levels of cytoskeleton-associated genes, enhancing our understanding of how doxorubicin may affect the transcriptional profile of cells. However, doxorubicin affects target mRNAs differently than TTP ectopic expression, suggesting it may not be the primary mechanism of doxorubicin in breast cancer (BC) treatment. High TTP expression is considered as a positive prognostic marker in multiple cancers, including BC. Given that doxorubicin is a commonly used drug for treating triple-negative BC, using TTP as a prognostic marker in this cohort of patients might be limited since it might be challenging to understand if high TTP expression occurred due to the favorable physiological state of the patient or as a consequence of treatment.

Tristetraprolin mediates immune evasion of mycobacterial infection in macrophages.

Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA-binding proteins controlling the stability of targeted mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in intracellular mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by the mTOR inhibitor rapamycin. Rapamycin and AZD8055 specifically blocked 4EBP1 phosphorylation in infected macrophages and suppressed intracellular BCG growth. Rapamycin promoted TTP protein degradation through the ubiquitination pathway, whereas the proteasome inhibitor MG-132 blocked rapamycin function and thus stabilized TTP protein. TTP induction suppressed the expression of iNOS/TNF-α/IL-12/IL-23, and weakened protective immune responses in macrophages, whereas rapamycin enhanced the bactericidal effects through TTP inhibition. Moreover, blocking TTP binding increased the expression of TNF-α and iNOS and suppressed intracellular mycobacterial growth. Overall, our study reveals a novel role for RNA-binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host-directed therapy against tuberculosis (TB).

Hydroxychloroquine attenuates double-stranded RNA-stimulated hyper-phosphorylation of tristetraprolin/ZFP36 and AU-rich mRNA stabilization.

The human innate immune system recognizes dsRNA as a pathogen-associated molecular pattern that induces a potent inflammatory response. The primary source of pathogenic dsRNA is cells infected with replicating viruses, but can also be released from uninfected necrotic cells. Here, we show that the dsRNA poly(I:C) challenge in human macrophages activates the p38 MAPK-MK2 signalling pathway and subsequently the phosphorylation of tristetraprolin (TTP/ZFP36). The latter is an mRNA decay-promoting protein that controls the stability of AU-rich mRNAs (AREs) that code for many inflammatory mediators. Hydroxychloroquine (HCQ), a common anti-malaria drug, is used to treat inflammatory and autoimmune disorders and, controversially, during acute COVID-19 disease. We found that HCQ reduced the dsRNA-dependent phosphorylation of p38 MAPK and its downstream kinase MK2. Subsequently, HCQ reduced the abundance and protein stability of the inactive (phosphorylated) form of TTP. HCQ reduced the levels and the mRNA stability of poly (I:C)-induced cytokines and inflammatory mRNAs like TNF, IL-6, COX-2, and IL-8 in THP-1 and primary blood monocytes. Our results demonstrate a new mechanism of the anti-inflammatory role of HCQ at post-transcriptional level (TTP phosphorylation) in a model of dsRNA activation, which usually occurs in viral infections or RNA release from necrotic tissue.

Lipopolysaccharide regulation of antiinflammatory tristetraprolin family and proinflammatory gene expression in mouse macrophages.

OBJECTIVE: Tristetraprolin (TTP/ZFP36) family proteins exhibit antiinflammatory effects by destabilizing proinflammatory mRNAs. Previous studies showed that bacterial endotoxin lipopolysaccharides (LPS) stimulated TTP and tumor necrosis factor (TNF) gene expression, but less was known about LPS effects on TTP homologues and other proinflammatory gene expression in macrophages. The objective was to investigate LPS regulation of TTP family gene and TTP-targeted gene expression in mouse RAW264.7 macrophages using much higher concentrations of LPS and much longer treatment time than previous studies. RESULTS: MTT assay showed that LPS was not toxic to the cells under LPS treatment up to 1000 ng/mL for 2-24 h. LPS mildly affected the soluble protein content in the cells. qPCR assay showed that LPS stimulated TTP mRNA rapidly but not sustainably with 40, 10, and 3 fold of the DMSO control after 2, 8 and 24 h treatment, respectively. Immunoblotting confirmed qPCR results on LPS stimulation of TTP gene expression in the mouse macrophages. LPS exhibited minimal effects on ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels. LPS increased mRNA levels of TNF, COX2, GM-CSF, INFγ and IL12b up to 311, 418, 11, 9 and 4 fold, respectively. This study demonstrated that LPS did not affect macrophage viability, dramatically increased antiinflammatory TTP gene expression as well as proinflammatory TNF and COX2 gene expression but had only mild effects on TTP homologues and other proinflammatory cytokine gene expression in the mouse macrophages.

circ_HIPK3 regulates function and morphology of Aβ induced hippocampal neurons by targeting miR-381-3p/ZNF217 axis / 天津医药

Objective To analyze the influence of cyclic RNA homologous domain interacting protein kinase 3(circ_HIPK3)on function and morphology of myloid β-protein(Aβ)induced hippocampal neurons by targeting miR-381-3p/zinc finger protein 217(ZNF217)axis.Methods Hippocampal neurons of neonatal rats were prepared and divided into the control group,the Aβ group,the si NC1 group,the si HIPK3 group,the si HIPK3+inhibitor NC group,the si HIPK3+miR-381-3p inhibitor group,the si HIPK3+miR-381-3p inhibitor+si NC2 group and the si HIPK3+miR-381-3p inhibitor+si ZNF217 group.Except the control group,all the other groups were modeled by 40 μmol/L Aβ1～42.qRT-PCR was used to determine the circ of hippocampal neurons circ_HIPK3,miR-381-3p and ZNF217 mRNA levels.Cell morphology was observed by transmission electron microscope,and the survival rate of hippocampal neurons was measured by CCK-8 method.Hochesst 33342 method was used to measure apoptosis of hippocampal neurons.The intracellular Ca2+ fluorescence intensity of hippocampal neurons was detected by flow cytometry.The expression levels of P-Tau,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Caspase-3 and ZNF217 proteins in hippocampal neurons were measured by Western blot assay.Double luciferase reporter genes were used to analyze the targeting relationship between miR-381-3p and circ_HIPK3,ZNF217.Results In the control group,the structure of hippocampal neurons was normal,the morphology of nucleus was normal,and there were no pathological changes in mitochondria and endoplasmic reticulum.In the Aβ group,hippocampal neurons showed degenerative changes,abnormal nuclear morphology,membrane invagination,a large number of mitochondria swelling and a large number of lipid droplets vacuoles in cytoplasm.Compared with the Aβ group,the hippocampal neuronal structure was partially restored in the si HIPK3 group.Compared with the si HIPK3 group,the hippocampal neuronal structure was severely damaged in the si HIPK3+miR-381-3p inhibitor group.Compared with the si HIPK3+miR-381-3p inhibitor group,the damage of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor+si ZNF217 group was reduced.Compared with the control group,the circ_HIPK3,ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax,Caspase-3 protein expression of hippocampal neurons were increased in the Aβ group,and the miR-381-3p level,survival rate and Bcl-2 protein expression decreased(P＜0.05).Compared with the Aβ group,the circ_HIPK3,ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax and Caspase-3 protein expression of hippocampal neurons were decreased in the si HIPK3 group,and miR-381-3p level,survival rate and Bcl-2 protein expression increased(P＜0.05).Compared with the si HIPK3 group,the circ_HIPK3,ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax and Caspase-3 protein expression of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor group were increased,and the miR-381-3p level,survival rate and Bcl-2 protein expression decreased(P＜0.05).Compared with the si HIPK3+miR-381-3p inhibitor group,the ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax and Caspase-3 protein expression of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor+si ZNF217 group were decreased,and the survival rate and Bcl-2 protein expression increased(P＜0.05).miR-381-3p targeted and combined with HIPK3 and ZNF217.Conclusion circ_HIPK3 silencing may ameliorate Aβ-induced damage of hippocampal neuronal structure and function by regulating miR-381-3p/ZNF217 axis.

Tristetraprolin overexpression drives hematopoietic changes in young and middle-aged mice generating dominant mitigating effects on induced inflammation in murine models.

Tristetraprolin (TTP), encoded by Zfp36 in mice, is one of the best-characterized tandem zinc-finger mRNA binding proteins involved in mRNA deadenylation and decay. TTPΔARE mice lack an AU-rich motif in the 3'-untranslated regions of TTP mRNA, leading to increased TTP mRNA stability and more TTP protein, resulting in elevated mRNA decay rates of TTP targets. We examined the effect of TTP overexpression on the hematopoietic system in both young and middle-aged mice using TTPΔARE mice and found alterations in blood cell frequencies, with loss of platelets and B220 cells and gains of eosinophils and T cells. TTPΔARE mice also have skewed primitive populations in the bone marrow, with increases in myeloid-biased hematopoietic stem cells (HSCs) but decreases in granulocyte/macrophage-biased multipotent progenitors (MPP3) in both young and middle-aged mice. Changes in the primitive cells' frequencies were associated with transcriptional alterations in the TTP overexpression cells specific to age as well as cell type. Regardless of age, there was a consistent elevation of transcripts regulated by TNFα and TGFß signaling pathways in both the stem and multipotent progenitor populations. HSCs with TTP overexpression had decreased reconstitution potential in murine transplants but generated hematopoietic environments that mitigated the inflammatory response to the collagen antibody-induced arthritis (CAIA) challenge, which models rheumatoid arthritis and other autoimmune disorders. This dampening of the inflammatory response was even present when there was only a small frequency of TTP overexpressing cells present in the middle-aged mice. We provide an analysis of the early hematopoietic compartments with elevated TTP expression in both young and middle-aged mice which inhibits the reconstitution potential of the HSCs but generates a hematopoietic system that provides dominant repression of induced inflammation.

Tristetraprolin regulates the skeletal phenotype and osteoclastogenic potential through monocytic myeloid-derived suppressor cells.

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, in vitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype.

Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin.

Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3' UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

The RNA binding proteins ZFP36L1 and ZFP36L2 are dysregulated in airway epithelium in human and a murine model of asthma.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma. Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma. Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2. Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.

ZIKV induction of tristetraprolin in endothelial and Sertoli cells post-transcriptionally inhibits IFNß/λ expression and promotes ZIKV persistence.

IMPORTANCE: Our findings define a novel role for ZIKV-induced TTP expression in regulating IFNß/IFNλ production in primary hBMECs and Sertoli cells. These cells comprise key physiological barriers subverted by ZIKV to access brain and testicular compartments and serve as reservoirs for persistent replication and dissemination. We demonstrate for the first time that the ARE-binding protein TTP is virally induced and post-transcriptionally regulates IFNß/IFNλ secretion. In ZIKV-infected hBMEC and Sertoli cells, TTP knockout increased IFNß/IFNλ secretion, while TTP expression blocked IFNß/IFNλ secretion. The TTP-directed blockade of IFN secretion permits ZIKV spread and persistence in hBMECs and Sertoli cells and may similarly augment ZIKV spread across IFNλ-protected placental barriers. Our work highlights the importance of post-transcriptional ZIKV regulation of IFN expression and secretion in cells that regulate viral access to protected compartments and defines a novel mechanism of ZIKV-regulated IFN responses which may facilitate neurovirulence and sexual transmission.

The 3' Untranslated Regions of Ebola Virus mRNAs Contain AU-Rich Elements Involved in Posttranscriptional Stabilization and Decay.

The 3' untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3' UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3' UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3' UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3' UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity.

Degradation of MK2 with natural compound andrographolide: A new modality for anti-inflammatory therapy.

The p38MAPK-MK2 signaling axis functions as an initiator of inflammation. Targeting the p38MAPK-MK2 signaling axis represents a direct therapeutic intervention of inflammatory diseases. We described here a novel role of andrographolide (AG), a small-molecule ent-labdane natural compound, as an inhibitor of p38MAPK-MK2 axis via MK2 degradation. AG was found to bind to the activation loop of MK2, located at the interface of the p38MAPK-MK2 biomolecular complex. This interaction disrupted the complex formation and predisposed MK2 to proteasome-mediated degradation. We showed that AG induced MK2 degradation in a concentration- and time-dependent manner and exerted its anti-inflammatory effects by enhancing the mRNA-destabilizing activity of tristetraprolin, thereby inhibiting pro-inflammatory mediator production (e.g., TNF-α, MCP-1). Administration of AG via intratracheal (i.t.) route to mice induced MK2 downregulation in lung alveolar macrophages, but not lung tissues, and prevented macrophage activation. Our study also demonstrated that the anti-inflammatory effects achieved by AG via MK2 degradation were more durable and sustained than that achieved by the conventional MK2 kinase inhibitors (e.g., PF-3644022). Taken together, our findings illustrated a novel mode of action of AG by modulating the p38MAPK-MK2 signaling axis and would pave the way for the development of a novel class of anti-inflammatory agents targeting MK2 for degradation by harnessing the privileged scaffold of AG.

Plant Polyphenol Gossypol Induced Cell Death and Its Association with Gene Expression in Mouse Macrophages.

Gossypol is a complex plant polyphenol reported to be cytotoxic and anti-inflammatory, but little is known about its effect on gene expression in macrophages. The objective of this study was to explore gossypol's toxicity and its effect on gene expression involved in the inflammatory response, glucose transport and insulin signaling pathways in mouse macrophages. Mouse RAW264.7 macrophages were treated with multiple concentrations of gossypol for 2-24 h. Gossypol toxicity was estimated by MTT assay and soluble protein content. qPCR analyzed the expression of anti-inflammatory tristetraprolin family (TTP/ZFP36), proinflammatory cytokine, glucose transporter (GLUT) and insulin signaling genes. Cell viability was greatly reduced by gossypol, accompanied with a dramatic reduction in soluble protein content in the cells. Gossypol treatment resulted in an increase in TTP mRNA level by 6-20-fold and increased ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels by 26-69-fold. Gossypol increased proinflammatory cytokine TNF, COX2, GM-CSF, INFγ and IL12b mRNA levels up to 39-458-fold. Gossypol treatment upregulated mRNA levels of GLUT1, GLUT3 and GLUT4 genes as well as INSR, AKT1, PIK3R1 and LEPR, but not APP genes. This study demonstrated that gossypol induced macrophage death and reduced soluble protein content, which was accompanied with the massive stimulation of anti-inflammatory TTP family and proinflammatory cytokine gene expression, as well as the elevation of gene expression involved in glucose transport and the insulin signaling pathway in mouse macrophages.

CO-Induced TTP Activation Alleviates Cellular Senescence and Age-Dependent Hepatic Steatosis via Downregulation of PAI-1.

Aging can increase the risk of various hepatic diseases, especially non-alcoholic fatty liver disease (NAFLD). Although the mechanisms underlying the pathogenesis of age-related disorders such as NAFLD remain incompletely understood, recent studies have implicated the accumulation of senescent cells as a contributing factor. Here, we show that tristetraprolin (TTP) deficiency accelerates NAFLD during aging by enhancing the senescence-associated secretory phenotype (SASP) as well as several hallmarks of senescence. The sequestration of plasminogen activator inhibitor (PAI)-1, a mediator of cellular senescence, in stress granules, (SGs) inhibits cellular senescence. In our previous report, we have shown that carbon monoxide (CO), a small gaseous mediator, can induce the assembly of SGs via an integrated stress response. Here, we show that CO treatment promotes the assembly of SGs which can sequester PAI-1, resulting in the inhibition of etoposide (ETO)-induced cellular senescence. Notably, CO-induced TTP activation enhances PAI-1 degradation, leading to protection against ETO-induced cellular senescence. CO-dependent Sirt1 activation promotes the inclusion of TTP into SGs, leading to decreased PAI-1 levels. Therefore, our findings highlight the importance of TTP as a therapeutic target in age-related NAFLD and offer a potential new strategy to reduce the detrimental effects of senescent cells in hepatic disorders.

HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation.

Tristetraprolin (TTP) is a critical negative immune regulator. It binds AU-rich elements in the untranslated-regions of many mRNAs encoding pro-inflammatory mediators, thereby accelerating their decay. A key but poorly understood mechanism of TTP regulation is its timely proteolytic removal: TTP is degraded by the proteasome through yet unidentified phosphorylation-controlled drivers. In this study, we set out to identify factors controlling TTP stability. Cellular assays showed that TTP is strongly lysine-ubiquitinated, which is required for its turnover. A genetic screen identified the ubiquitin E3 ligase HUWE1 as a strong regulator of TTP proteasomal degradation, which we found to control TTP stability indirectly by regulating its phosphorylation. Pharmacological assessment of multiple kinases revealed that HUWE1-regulated TTP phosphorylation and stability was independent of the previously characterized effects of MAPK-mediated S52/S178 phosphorylation. HUWE1 function was dependent on phosphatase and E3 ligase binding sites identified in the TTP C-terminus. Our findings indicate that while phosphorylation of S52/S178 is critical for TTP stabilization at earlier times after pro-inflammatory stimulation, phosphorylation of the TTP C-terminus controls its stability at later stages.

MAPKAPK2-centric transcriptome profiling reveals its major role in governing molecular crosstalk of IGFBP2, MUC4, and PRKAR2B during HNSCC pathogenesis.

Transcriptome analysis of head and neck squamous cell carcinoma (HNSCC) has been pivotal to comprehending the convoluted biology of HNSCC tumors. MAPKAPK2 or MK2 is a critical modulator of the mRNA turnover of crucial genes involved in HNSCC progression. However, MK2-centric transcriptome profiles of tumors are not well known. This study delves into HNSCC progression with MK2 at the nexus to delineate the biological relevance and intricate crosstalk of MK2 in the tumor milieu. We performed next-generation sequencing-based transcriptome profiling of HNSCC cells and xenograft tumors to ascertain mRNA expression profiles in MK2-wild type and MK2-knockdown conditions. The findings were validated using gene expression assays, immunohistochemistry, and transcript turnover studies. Here, we identified a pool of crucial MK2-regulated candidate genes by annotation and differential gene expression analyses. Regulatory network and pathway enrichment revealed their significance and involvement in the HNSCC pathogenesis. Additionally, 3'-UTR-based filtering recognized important MK2-regulated downstream target genes and validated them by nCounter gene expression assays. Finally, immunohistochemistry and transcript stability studies revealed the putative role of MK2 in regulating the transcript turnover of IGFBP2, MUC4, and PRKAR2B in HNSCC. Conclusively, MK2-regulated candidate genes were identified in this study, and their plausible involvement in HNSCC pathogenesis was elucidated. These genes possess investigative values as targets for diagnosis and therapeutic interventions for HNSCC.

Metformin-induced TTP mediates communication between Kupffer cells and hepatocytes to alleviate hepatic steatosis by regulating lipophagy and necroptosis.

OBJECTIVE: Emerging evidence suggests that crosstalk between Kupffer cells (KCs) and hepatocytes protects against non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanisms that lead to the reduction of steatosis in NAFLD remain obscure. METHODS: Ttp+/+ and Ttp-/- mice were fed with a high-fat diet. Hepatic steatosis was analyzed by Nile Red staining and measurement of inflammatory cytokines. Lipid accumulation and cell death were evaluated in co-culture systems with primary hepatocytes and KCs derived from either Ttp+/+ or Ttp-/- mice. RESULTS: Tristetraprolin (TTP), an mRNA binding protein, was essential for the protective effects of metformin in NAFLD. Metformin activated TTP via the AMPK-Sirt1 pathway in hepatocytes and KCs. TTP inhibited TNF-α production in KCs, which in turn decreased hepatocyte necroptosis. Downregulation of Rheb expression by TTP promoted hepatocyte lipophagy via mTORC1 inhibition and increased nuclear translocation of transcription factor-EB (TFEB). Consistently, TTP-deficient NAFLD mice failed to respond to metformin with respect to alleviation of hepatic steatosis, protection of hepatocyte necroptosis, or induction of lipophagy. CONCLUSIONS: TTP, which is essential for the protective effects of metformin, may represent a novel primary therapeutic target in NAFLD.

Resveratrol Ameliorates LPS-induced Acute Lung Injury Mouse Model via Induction of Tristetraprolin.

AIM: In this study, we aimed to investigate whether resveratrol has anti-inflammatory effects on LPS-induced ALI via TTP enhancement. BACKGROUND: Acute lung injury (ALI) is a syndrome of diffuse infammatory lung injury with increased pulmonary edema and the rapid onset of hypoxemic respiratory failure. Resveratrol is a stilbenoid, a form of natural phenol, and a phytoalexin produced by a variety of plants in reaction to injury or when they are attacked by pathogens like bacteria or fungi. Resveratrol exhibits a potent antiinflammatory effect in LPS-induced ALI, while the underlying mechanisms remain elusive. OBJECTIVE: Tristetraprolin (TTP) is a RNA binding protein that is an important endogenous inhibitor of inflammation. The objective of the present study is to investigate whether resveratrol has anti- inflammatory effects on LPS-induced ALI via TTP enhancement. METHODS: Forty male C57BL/6 mice were randomly assigned to four groups and intratracheally instilled with 5 mg/kg lipopolysaccharide (LPS) to induce ALI. RESULTS: LPS-induced lung pathological damage, lung edema, and neutrophil infiltration were reduced by resveratrol pretreatment. Furthermore, resveratrol inhibited the LPS-induced rise in TNF- α, IL-1ß and IL-6 levels in BAL fluids. In the LPS-challenged mouse's lung tissue, resveratrol clearly boosted sirtuin1 (SIRT1) and TTP protein expression, while also increasing TTP expression while reducing proinflammatory cytokines. EX527, on the other hand, reversed resveratrol's effects. CONCLUSION: According to our findings, resveratrol attenuated pulmonary inflammation and lung injury in mice with LPS­induced ALI， at least partly correlated with promoting the activation of SIRT1/TTP signaling pathway, highlighting these pathways as potential targets for intervention in LPS -induced lung injury.

Pb(II) coordination to the nonclassical zinc finger tristetraprolin: retained function with an altered fold.

Tristetraprolin (TTP) is a nonclassical CCCH zinc finger (ZF) that plays a crucial role in regulating inflammation. TTP regulates cytokine mRNAs by specific binding of its two conserved ZF domains (CysX8CysX5CysX3His) to adenylate-uridylate-rich sequences (AREs) at the 3'-untranslated region, leading to degradation of the RNA. Dysregulation of TTP in animal models has demonstrated several cytokine-related syndromes, including chronic inflammation and autoimmune disorders. Exposure to Pb(II), a prevalent environmental toxin, is known to contribute to similar pathologies, in part by disruption of and/or competition with cysteine-rich metalloproteins. TTP's role during stress as a ubiquitous translational regulator of cell signaling (and dysfunction), which may underpin various phenotypes of Pb(II) toxicity, highlights the importance of understanding the interaction between TTP and Pb(II). The impact of Pb(II) binding on TTP's fold and RNA-binding function was analyzed via UV-Vis spectroscopy, circular dichroism, X-ray absorption spectroscopy, nuclear magnetic resonance spectroscopy, and fluorescence anisotropy. A construct containing the two ZF domains of TTP (TTP-2D) bound to Pb(II) with nanomolar affinity and exhibited a different geometry and fold in comparison to Zn2-TTP-2D. Despite the altered secondary structure, Pb(II)-substituted TTP-2D bound a canonical ARE sequence more selectively than Zn2-TTP-2D. Taken together, these data suggest that Pb(II) may interfere with proper TTP regulation and hinder the cell's ability to respond to inflammation.

FOXO1 Is a Key Mediator of Glucocorticoid-Induced Expression of Tristetraprolin in MDA-MB-231 Breast Cancer Cells.

The mRNA destabilizing factor tristetraprolin (TTP) functions as a tumor suppressor by down-regulating cancer-associated genes. TTP expression is significantly reduced in various cancers, which contributes to cancer processes. Enforced expression of TTP impairs tumorigenesis and abolishes maintenance of the malignant state, emphasizing the need to identify a TTP inducer in cancer cells. To search for novel candidate agents for inducing TTP in cancer cells, we screened a library containing 1019 natural compounds using MCF-7 breast cancer cells transfected with a reporter vector containing the TTP promoter upstream of the luciferase gene. We identified one molecule, of which the enantiomers are betamethasone 21-phosphate (BTM-21-P) and dexamethasone 21-phosphate (BTM-21-P), as a potent inducer of TTP in cancer cells. We confirmed that BTM-21-P, DXM-21-P, and dexamethasone (DXM) induced the expression of TTP in MDA-MB-231 cells in a glucocorticoid receptor (GR)-dependent manner. To identify potential pathways linking BTM-21-P and DXM-21-P to TTP induction, we performed an RNA sequencing-based transcriptome analysis of MDA-MB-231 cells at 3 h after treatment with these compounds. A heat map analysis of FPKM expression showed a similar expression pattern between cells treated with the two compounds. The KEGG pathway analysis results revealed that the upregulated DEGs were strongly associated with several pathways, including the Hippo signaling pathway, PI3K-Akt signaling pathway, FOXO signaling pathway, NF-κB signaling pathway, and p53 signaling pathway. Inhibition of the FOXO pathway using a FOXO1 inhibitor blocked the effects of BTM-21-P and DXM-21-P on the induction of TTP in MDA-MB-231 cells. We found that DXM enhanced the binding of FOXO1 to the TTP promoter in a GR-dependent manner. In conclusion, we identified a natural compound of which the enantiomers are DXM-21-P and BTM-21-P as a potent inducer of TTP in breast cancer cells. We also present new insights into the role of FOXO1 in the DXM-21-P- and BTM-21-P-induced expression of TTP in cancer cells.