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Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.Cell Reports Apr 2024The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis....
The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of growth arrest and DNA damage-inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We establish that the 3' untranslated region of PPP1R15A mRNA contains an active AU-rich element (ARE) recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a component of the transient ISR memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.
Topics: Animals; Humans; Mice; 3' Untranslated Regions; Adaptation, Physiological; AU Rich Elements; HEK293 Cells; Protein Phosphatase 1; RNA Stability; RNA, Messenger; Stress, Physiological; Tristetraprolin
PubMed: 38602876
DOI: 10.1016/j.celrep.2024.114069 -
BMC Research Notes Mar 2024Tristetraprolin (TTP/ZFP36) family proteins exhibit antiinflammatory effects by destabilizing proinflammatory mRNAs. Previous studies showed that bacterial endotoxin...
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.
Topics: Mice; Animals; Lipopolysaccharides; Tristetraprolin; Cyclooxygenase 2; Macrophages; Anti-Inflammatory Agents; Gene Expression; RNA, Messenger; Tumor Necrosis Factor-alpha; Gene Expression Regulation
PubMed: 38504371
DOI: 10.1186/s13104-024-06743-6 -
Inflammation and Regeneration Mar 2024Inflammation orchestrates a finely balanced process crucial for microorganism elimination and tissue injury protection. A multitude of immune and non-immune cells,... (Review)
Review
Inflammation orchestrates a finely balanced process crucial for microorganism elimination and tissue injury protection. A multitude of immune and non-immune cells, alongside various proinflammatory cytokines and chemokines, collectively regulate this response. Central to this regulation is post-transcriptional control, governing gene expression at the mRNA level. RNA-binding proteins such as tristetraprolin, Roquin, and the Regnase family, along with RNA modifications, intricately dictate the mRNA decay of pivotal mediators and regulators in the inflammatory response. Dysregulated activity of these factors has been implicated in numerous human inflammatory diseases, underscoring the significance of post-transcriptional regulation. The increasing focus on targeting these mechanisms presents a promising therapeutic strategy for inflammatory and autoimmune diseases. This review offers an extensive overview of post-transcriptional regulation mechanisms during inflammatory responses, delving into recent advancements, their implications in human diseases, and the strides made in therapeutic exploitation.
PubMed: 38491500
DOI: 10.1186/s41232-024-00326-5 -
Scientific Reports Feb 2024Loss of inflammatory effector function, such as cytokine production and proliferation, is a fundamental driver of failure in T cell therapies against solid tumors. Here,...
Loss of inflammatory effector function, such as cytokine production and proliferation, is a fundamental driver of failure in T cell therapies against solid tumors. Here, we used CRISPR/Cas9 to genetically disrupt ZFP36, an RNA binding protein that regulates the stability of mRNAs involved in T cell inflammatory function, such as the cytokines IL2 and IFNγ, in human T cells engineered with a clinical-stage mesothelin-targeting CAR to determine whether its disruption could enhance antitumor responses. ZFP36 disruption slightly increased antigen-independent activation and cytokine responses but did not enhance overall performance in vitro or in vivo in a xenograft tumor model with NSG mice. While ZFP36 disruption does not reduce the function of CAR-T cells, these results suggest that singular disruption of ZFP36 is not sufficient to improve their function and may benefit from a multiplexed approach.
Topics: Humans; Animals; Mice; Mesothelin; Immunotherapy, Adoptive; T-Lymphocytes; Immunity; Cytokines; Disease Models, Animal; Xenograft Model Antitumor Assays; Cell Line, Tumor; Tristetraprolin
PubMed: 38326511
DOI: 10.1038/s41598-024-53769-8 -
Frontiers in Cell and Developmental... 2023Tristetraprolin (TTP) is an RNA binding protein that destabilizes mRNAs of factors involved in proliferation, invasiveness, and inflammation. Disruption of the gene that...
Tristetraprolin (TTP) is an RNA binding protein that destabilizes mRNAs of factors involved in proliferation, invasiveness, and inflammation. Disruption of the gene that codes for TTP () led to severe arthritis, autoimmunity, cachexia and dermatitis in mice. It has been shown that these phenotypes were mostly due to excessive TNFα levels in the affected tissues. We have previously reported that TTP expression is required for lactation maintenance. Our results indicated that conditional MG TTP-KO female mice displayed early involution due to the untimely induction of pro-inflammatory pathways led mostly by TNFα overexpression. Here we show that reducing TTP levels not only affects the fully differentiated mammary gland, but also harms morphogenesis of this tissue by impairing the progenitor cell population. We found that expression is linked to mammary stemness in human and mice. In addition, diminishing TTP expression and activity induced apoptosis of stem-like mouse mammary cells, reduced its ability to form mammospheres in culture and to develop into complete glands when implanted into cleared mammary fat pads . Our results show that survival of the stem-like cells is compromised by increased levels of inflammatory cytokines and stimulation of signaling cascades involving NFκB, STAT3 and MAPK-p38 activation. Moreover, TNFα overexpression and the consequent p38 phosphorylation would be the leading cause of progenitor cell death upon TTP expression restriction. Taken together, our results reveal the relevance of TTP for the maintenance of the mammary progenitor cell compartment by maintaining local TNFα levels at bay.
PubMed: 38274271
DOI: 10.3389/fcell.2023.1265475 -
STAR Protocols Mar 2024Here, we present a protocol for modulating the function of the Cth2 mRNA-binding protein (RBP) in Saccharomyces cerevisiae. We describe steps to amplify and integrate...
Here, we present a protocol for modulating the function of the Cth2 mRNA-binding protein (RBP) in Saccharomyces cerevisiae. We describe steps to amplify and integrate mutations in Cth2 that affect its stability and function. Next, we detail the functional assay to verify the activity of the wild-type and mutant versions of Cth2 in yeast cells. This protocol can be adopted to modify the function of other RBPs with their respective functional mutations. For complete details on the use and execution of this protocol, please refer to Patnaik et al. (2022)..
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Carrier Proteins; RNA, Messenger; Tristetraprolin; Iron
PubMed: 38165801
DOI: 10.1016/j.xpro.2023.102807 -
Cell Reports Jan 2024Vascular morphogenesis requires a delicate gradient of Notch signaling controlled, in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (JAG1)...
Vascular morphogenesis requires a delicate gradient of Notch signaling controlled, in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (JAG1) expression is compartmentalized in the vascular plexus remains unclear. Here, we show that Jag1 mRNA is a direct target of zinc-finger protein 36 (ZFP36), an RNA-binding protein involved in mRNA decay that we find robustly induced by vascular endothelial growth factor (VEGF). Endothelial cells lacking ZFP36 display high levels of JAG1 and increase angiogenic sprouting in vitro. Furthermore, mice lacking Zfp36 in endothelial cells display mispatterned and increased levels of JAG1 in the developing retinal vascular plexus. Abnormal levels of JAG1 at the sprouting front alters NOTCH1 signaling, increasing the number of tip cells, a phenotype that is rescued by imposing haploinsufficiency of Jag1. Our findings reveal an important feedforward loop whereby VEGF stimulates ZFP36, consequently suppressing Jag1 to enable adequate levels of Notch signaling during sprouting angiogenesis.
Topics: Animals; Mice; Calcium-Binding Proteins; Cell Proliferation; Endothelial Cells; Jagged-1 Protein; Membrane Proteins; Neovascularization, Physiologic; Receptors, Notch; Signal Transduction; Vascular Endothelial Growth Factor A
PubMed: 38157296
DOI: 10.1016/j.celrep.2023.113627 -
RNA Biology Jan 2024Inhibition 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...
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.
Topics: Humans; Female; Tristetraprolin; Survivin; Breast Neoplasms; 3' Untranslated Regions; Apoptosis; RNA, Messenger; RNA-Binding Proteins; RNA Stability
PubMed: 38111129
DOI: 10.1080/15476286.2023.2286101 -
Frontiers in Cell and Developmental... 2023Asthma 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...
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. We analysed the levels of , and 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. We found and 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 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 , and . 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.
PubMed: 37928904
DOI: 10.3389/fcell.2023.1241008 -
Synergistic roles of tristetraprolin family members in myeloid cells in the control of inflammation.Life Science Alliance Jan 2024Members of the tristetraprolin (TTP) family of RNA-binding proteins can bind to and promote the decay of specific transcripts containing AU-rich motifs. ZFP36 (TTP) is...
Members of the tristetraprolin (TTP) family of RNA-binding proteins can bind to and promote the decay of specific transcripts containing AU-rich motifs. ZFP36 (TTP) is best known for regulating pro-inflammatory cytokine expression in myeloid cells; however, its mammalian paralogues ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. We knocked out these genes in myeloid cells in mice, singly and together. Single-gene myeloid-specific knockouts resulted in almost no spontaneous phenotypes. In contrast, mice with myeloid cell deficiency of all three genes developed severe inflammation, with a median survival of 8 wk. Macrophages from these mice expressed many more stabilized transcripts than cells from myeloid-specific TTP knockout mice; many of these encoded pro-inflammatory cytokines and chemokines. The failure of weight gain, arthritis, and early death could be prevented completely by two normal alleles of any of the three paralogues, and even one normal allele of or was enough to prevent the inflammatory phenotype. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function.
Topics: Mice; Animals; Tristetraprolin; Inflammation; Myeloid Cells; Macrophages; Mice, Knockout; Cytokines; Mammals
PubMed: 37903626
DOI: 10.26508/lsa.202302222