Discriminating between HuR and TTP binding sites using the k-spectrum kernel method.

Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cdelta (PKCdelta)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCdelta mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D(1), and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions.

Post-transcriptional processing of mRNA transcripts plays a critical role in establishing the gene expression profile of a cell. Such processing events are mediated by a host of factors, including RNA-binding proteins and microRNAs. A number of critical cellular pathways are subject to regulation at multiple levels that allow fine-tuning of key biological responses. Programmed cell death 4 (PDCD4) is a tumor suppressor and an important modulator of mRNA translation that is regulated by a number of mechanisms, most notably as a target of the oncomiR, miR-21. Here, we provide evidence for post-transcriptional regulation of PDCD4 by the RNA-binding proteins, HuR and TIA1. Complementary approaches reveal binding of both HuR and TIA1 to the PDCD4 transcript. Consistent with a model where RNA-binding proteins modulate the PDCD4 transcript, knockdown of HuR and/or TIA1 results in a significant decrease in steady-state PDCD4 mRNA and protein levels. However, fractionation experiments suggest that the mode of regulation of the PDCD4 transcript likely differs in the cytoplasm and the nucleus as the pool of PDCD4 mRNA present in the cytoplasm is more stable than the nuclear pool of PDCD4 transcript. We observe a competitive mode of binding between HuR and TIA1 on the PDCD4 transcript in the cytoplasm, suggesting that these two factors dynamically interact with one another as well as the PDCD4 transcript to maintain tight control of PDCD4 levels. Overall, this study reveals an additional set of regulatory interactions that modulate the expression of PDCD4, a key pro-apoptotic factor, and also reveals new insights into how HuR and TIA1 functions are integrated to achieve such regulation.

Inflammation plays a prominent role in the events following traumatic injury to the central nervous system (CNS). The initial inflammatory response is driven by mediators such as tumor necrosis factor α and interleukin 1β, which are produced by activated astrocytes and microglia at the site of injury. These factors are regulated post-transcriptionally by RNA binding proteins (RBP) that interact with adenylate and uridylate-rich elements (ARE) in the 3'-untranslated region of the messenger RNA (mRNA). Human antigen R (HuR) is one of these RBPs and generally functions as a positive regulator of ARE-containing mRNAs. Here, we hypothesized that HuR plays an important role in the induction of cytokine and chemokines in astrocytes following traumatic injury. Using a mouse model of spinal cord injury, we found HuR to be extensively translocated to the cytoplasm in astrocytes at the level of injury, consistent with its activation. In an in vitro stretch injury model of CNS trauma, we observed a similar cytoplasmic shift of HuR in astrocytes and an attenuation of cytokine induction with HuR knockdown. RNA kinetics and luciferase assays suggested that the effect was more related to transcription than RNA destabilization. A small molecule inhibitor of HuR suppressed cytokine induction of injured astrocytes and reduced chemoattraction for neutrophils and microglia. In summary, HuR is activated in astrocytes in the early stages of CNS trauma and positively regulates the molecular response of key inflammatory mediators in astrocytes. Our findings suggest that HuR may be a therapeutic target in acute CNS trauma for blunting secondary tissue injury triggered by the inflammatory response.

COVID-19 has rapidly spread through the world, and is caused by SARS-CoV-2. Patients with chronic obstructive pulmonary disease (COPD) face an increased risk for severe illness from COVID-19 because of angiotensin-converting enzyme 2 (ACE2) upregulation, an entry receptor for SARS-CoV-2. Our group recently revealed that COPD-derived lung fibroblasts express higher level of ACE2, and provide direct evidence that chronic cigarette smoke (CS) exposure significantly increases pulmonary ACE2 protein. CS is the primary risk factor for COPD. ACE2 expression may be regulated at the transcriptional, post-transcriptional and translational levels. Changes in protein expression can be controlled by RNA-binding proteins (RBPs). One of the best studied RBPs is human antigen R (HuR), a ubiquitously expressed RBP, that regulates the stability, localization and/or translation of target mRNAs. To stabilize target mRNA, HuR translocates from the nucleus to the cytoplasm. CS may increases HuR translocation to the cytoplasm, thereby indirectly augmenting cellular protein levels. Therefore, we hypothesized that HuR regulates ACE2 expression in response to CS, thereby accounting for higher ACE2 in COPD-derived cells. Aims: 1)- Evaluate HuR expression and localization in Normal, Smoker and COPD lung tissues;2) Establish that HuR induces ACE2 expression in primary human lung fibroblasts (HLF);3)- Determine if HuR controls ACE2 expression by regulating mRNA stability. Methods: 1)- Multiplex Immunohistochemistry was performed on lung tissue from Normal, Smoker and COPD subjects. The localization of HuR was assessed in HLF exposed to 2% cigarette smoke extract (CSE) for 4h by subcellular fractionation-western blot. 2)- Normal, Smoker and COPD HLF were transfected with either small interfering RNA (siRNA) against HuR or Control siRNA. ACE2 protein was evaluated by western blot. 3)- RNA-binding protein immunoprecipitation-qPCR was used to assess the association of HuR to ACE2 mRNA in Normal, Smoker and COPD HLF. The effect of HuR on the stability of ACE2 mRNA was assessed by using actinomycin D pulse-chase followed by qPCR. Results: 1)- HuR cytoplasmic localization is higher in Smoker and COPD lung tissues. There was an increase in cytoplasmic HuR in HLF exposed to 2%CSE for 4h. 2)- Knockdown of HuR slightly increases ACE2 protein. 3)- HuR associates with ACE2 mRNA in Normal and Smoker HLF. Significance: Our work is the first to highlight the association between ACE2 and HuR in COPD. The regulation of ACE2 by HuR could provide the basis to understand the upregulation of ACE2 expression in COPD HLF and the potential consequence towards COVID-19.

Background:Cachexia is defined by chronic loss of fat and muscle, is a frequent complication of pancreatic ductal adenocarcinoma (PDAC), and negatively impacts patient outcomes. Nutritional supplementation cannot fully reverse tissue wasting, and the mechanisms underlying this phenotype are unclear. This work aims to define the relative contributions of catabolism and anabolism to adipose wasting in PDAC-bearing mice. Human antigen R (HuR) is an RNA-binding protein recently shown to suppress adipogenesis. We hypothesize that fat wasting results from a loss of adipose anabolism driven by increased HuR activity in adipocytes of PDAC-bearing mice.Methods:Adult C57BL/6J mice received orthotopic PDAC cell (KrasG12D; p53R172H/+; Pdx1-cre) (PDAC) or PBS (sham) injections. Mice exhibiting moderate cachexia (9 days after injection) were fasted for 24h, or fasted 24h and refed 24h before euthanasia. A separate cohort of PDAC mice were treated with an established HuR inhibitor (KH-3, 100 mg/kg) and subjected to the fast/refeed paradigm. We analyzed body mass, gross fat pad mass, and adipose tissue mRNA expression. We quantified lipolytic rate as the normalized quantity of glycerol released from 3T3-L1 adipocytes in vitro, and gonadal fat pads (gWAT) ex vivo.Results:3T3-L1 adipocytes treated with PDAC cell conditioned media (CM) liberated less triglyceride into the culture media than control-treated adipocytes (−28.1%) and had lower expression of lipolysis and lipogenesis genes than control cells. PDAC gWAT cultured ex vivo displayed decreased lipolysis compared to sham gWAT (−54.7%). PDAC and sham mice lost equivalent fat mass after a 24h fast, however, PDAC mice could not restore inguinal fat pads (iWAT) (−40.5%) or gWAT (−31.8%) mass after refeeding. RNAseq revealed 572 differentially expressed genes in gWAT from PDAC compared to sham mice. Downregulated genes (n=126) were associated with adipogenesis (adj p=0.05), and expression of adipogenesis master regulators Pparg and Cebpa were reduced in gWAT from PDAC mice. Immunohistochemistry revealed increased HuR staining in gWAT (+74.9%) and iWAT (+41.2%) from PDAC mice. Inhibiting HuR binding restored lipogenesis in refed animals with a concomitant increase in iWAT mass (+131.7%) and genes regulating adipogenesis (Pparγ, Cebpa, Retn, Adipoq, Fasn).Conclusions:Our work highlights deficient adipose anabolism as a driver of lipid-droplet loss in 3T3-L1 adipocytes treated with PDAC conditioned media and PDAC mice. The small molecule KH-3, which disrupts HuR binding, restored adipose anabolism in PDAC mice. This highlights HuR as a potentially targetable regulatory node for adipose anabolism in cancer cachexia.

Objective: Lineage tracing studies have shown that advanced atherosclerosis causes vascular smooth muscle cells (VSMCs) to become macrophage-like (MLCs), which increases inflammation. We investigated whether the RNA binding protein human antigen R (HuR) and the inflammatory receptor protease activated receptor 2 (PAR2) accelerate VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Downregulation of VSMC migration and cytokine release in Par2 -/- mice reduces atherosclerotic lesion area compared to proficient controls. To examine the role of PAR2 in the VSMC-MLC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10μg/mL methyl-β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers and upregulated macrophage markers, Par2 -/- VSMCs remained stable. When comparing 101 mice strains with induced atherosclerosis, PAR2 was significantly correlated with Krüppel-like factor 4 (KLF4), a gene that regulates VSMC dedifferentiation. Also, Par2-/- VSMCs had lower basal KLF4 expression. Previous studies have demonstrated PAR2 mRNA is bound and stabilized by the RNA binding protein HuR. Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression, suggesting a role of HuR in PAR2 mRNA stability. Our studies have also demonstrated a dampening of HuR activation in Par2 -/- VSMCs, suggesting a potential feedforward mechanism between HuRs stabilizing capabilities of PAR2 mRNA and PAR2s presence necessary for HuR activation. To delineate the role of HuR in VSMC dedifferentiation and atherosclerosis, a HuR flox mouse line has been bred on to a SM22 Cre as well as a Myh11 Cre lineage tracing reporter line. To date, we have determined male and female Ldlr -/- HuR flox/flox SM22 Cre+ mice have attenuated atherosclerosis compared to their littermate Cre - controls at both 12 and 24 weeks. Conclusions: These results suggest that VSMC PAR2 activation mediates the dedifferentiation of VSMCs via KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. We also have established a definitive role of VSMC HuR in the development of atherosclerosis. Future studies will continue to elucidate the specific role of HuR in VSMC dedifferentiation.

Homeostasis of the intestinal epithelium is tightly regulated by numerous extracellular and intracellular factors including vitamin D and the vitamin D receptor (VDR). VDR is highly expressed in the intestinal epithelium and is implicated in many aspects of gut mucosal pathophysiology, but the exact mechanism that controls VDR expression remains largely unknown. The RNA-binding protein human antigen R (HuR) regulates the stability and translation of target mRNAs and thus modulates various cellular processes and functions. Here we report a novel role of HuR in the posttranscriptional control of VDR expression in the intestinal epithelium. The levels of VDR in the intestinal mucosa decreased significantly in mice with ablated HuR, compared with control mice. HuR silencing in cultured intestinal epithelial cells (IECs) also reduced VDR levels, whereas HuR overexpression increased VDR abundance; neither intervention changed cellular Vdr mRNA content. Mechanistically, HuR bound to Vdr mRNA via its 3'-untranslated region (UTR) and enhanced VDR translation in IECs. Moreover, VDR silencing not only inhibited IEC migration over the wounded area in control cells but also prevented the increased migration in cells overexpressing HuR, although it did not alter IEC proliferation in vitro and growth of intestinal organoids ex vivo. The human intestinal mucosa from patients with inflammatory bowel diseases exhibited decreased levels of both HuR and VDR. These results indicate that HuR enhances VDR translation by directly interacting with its mRNA via 3'-UTR and that induced VDR by HuR is crucial for rapid intestinal epithelial restitution after wounding.

Interplay between RNA-binding protein HuR and Nox4 as a novel therapeutic target in diabetic kidney disease

Endometriosis, a major reproductive pathology affecting 8–10% of women is characterized by chronic inflammation and immune dysfunction. Human antigen R (HuR) and Tristetraprolin (TTP) are RNA binding proteins that competitively bind to cytokines involved in inflammation including: tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6) among others, and stabilize and destabilize them, respectively. The aim of this study was to examine RNA binding protein (RNABP) HuR/TTP axis in endometriosis patients compared to menstrual stage matched healthy fertile controls in hopes of better understanding their contribution to the pathogenesis of endometriosis. Additionally, using a targeted in vitro siRNA approach, we examined whether knock-down of TTP can play a functional role on other RNABPs that competitively bind to inflammatory targets of TTP in both endometriotic and endometrial epithelial cell lines. Our results suggest that RNABPs TTP and HuR are dysregulated in endometriotic lesions compared to matched eutopic patient samples as well endometrium from healthy controls. Silencing of TTP in endometriotic and endometrial epithelial cells revealed differential response to inflammatory cytokines and other RNABPs. Our results suggest potential involvement of HuR/TTP RNA binding protein axis in regulation of inflammation in endometriosis.

The RNA binding protein human antigen R (HuR) participates in the post transcriptional regulation of many mRNAs bearing AU rich and U rich elements by influencing stabilization and translation. Based on the types of mRNAs regulated by HuR, the cellular role of HuR can be identified as pro-survival and pro-inflammatory. Using the S49 thymoma cell line, we recently showed that cAMP treatment, which triggers apoptotic signaling events, leads to a decrease in the effectiveness of HuR function. In this work, we investigate if there is a relationship between cAMP mediated cell death and HuR activity by utilizing small interfering RNA-mediated suppression of HuR in S49 cells. We found that knockdown of HuR protein decreases normal cell growth, and increases cAMP mediated apoptosis. qPCR analysis of mRNAs important in inflammation, cell growth, and apoptosis reveal that knockdown of HuR downregulates TNF-α mRNA and upregulates IL-10 and Bim mRNAs in untreated cells, and upregulates p27, and Bim mRNAs upon treatment with cAMP. Furthermore, cAMP treatment causes a PKA dependent decrease in HuR cytoplasmic levels. These results suggest that a pro-inflammatory and pro-survival role for HuR in S49 thymoma cells exists and may be compromised upon the elevation of cAMP by decreasing cytoplasmic HuR protein and allowing the expression of apoptotic factors.

Diabetic kidney disease (DKD) is one of the most common complications of diabetes, and the most common cause of end-stage renal disease, for which no effective therapies are yet available. RNA-binding proteins (RBPs) play a pivotal role in epigenetic regulation; tristetraprolin (TTP) and human antigen R (HuR) competitively bind cytokine mRNAs, exert contrasting effects on RNA stability, and drive inflammation. However, RBPs’ roles in diabetes-related glomerulopathy are poorly understood. Herein, we investigated whether TTP and HuR are involved in post-transcriptional regulation of podocytopathic molecules and inflammatory cytokines in DKD. In DKD patients and db/db mice, TTP expression was significantly decreased and HuR expression was increased in glomerular podocytes, concurrent with podocyte injury, histological signs of DKD, and augmented glomerular expression of interleukin (IL)-17 and claudin-1, which are targets of TTP and HuR, as evidenced by RNA immunoprecipitation. In cultured podocytes, exposure to high ambient glucose amplified HuR expression and repressed TTP expression, upregulated IL-17 and claudin-1, and promoted podocyte injury. Thus, TTP hypoactivity or HuR hyperactivity is sufficient and essential to diabetic podocytopathy. Moreover, in silico analysis revealed that several kinases govern phosphorylation and activation of TTP and HuR, and glycogen synthase kinase (GSK)-3β activated both TTP and HuR, which harbor putative GSK-3β consensus phosphorylation motifs. Treatment of db/db mice with a small molecule inhibitor of GSK-3β abrogated the changes in TTP and HuR in glomeruli and mitigated the overexpression of their target genes (IL-17, claudin-1, B7-1, and MCP-1) thus also mitigating proteinuria and DKD pathology. Our study indicates that TTP and HuR are dysregulated in DKD via a GSK-3β-mediated mechanism and play crucial roles in podocyte injury through post-transcriptional regulation of diverse genes. It also provides novel insights into DKD’s pathophysiology and identifies potential therapeutic targets.

BackgroundMost of the microRNAs (MiRs) involved in myogenesis are transcriptional regulated. The role of MiR biogenesis in myogenesis has not been characterized yet. RNA‐binding protein Musashi 2 (Msi2) is considered to be one of the major drivers for oncogenesis and stem cell proliferation. The functions of Msi2 in myogenesis have not been explored yet. We sought to investigate Msi2‐regulated biogenesis of MiRs in myogenesis and muscle stem cell (MuSC) ageing.MethodsWe detected the expression of Msi2 in MuSCs and differentiated myotubes by quantitative reverse transcription PCR (RT‐qPCR) and western blot. Msi2‐binding partner human antigen R (HuR) was identified by immunoprecipitation followed by mass spectrometry analysis. The cooperative binding of Msi2 and HuR on MiR7a‐1 was analysed by RNA immunoprecipitation and electrophoresis mobility shift assays. The inhibition of the processing of pri‐MiR7a‐1 mediated by Msi2 and HuR was shown by Msi2 and HuR knockdown. Immunofluorescent staining, RT‐qPCR and immunoblotting were used to characterize the function of MiR7a‐1 in myogenesis. Msi2 and HuR up‐regulate cryptochrome circadian regulator 2 (Cry2) via MiR7a‐1 was confirmed by the luciferase assay and western blot. The post‐transcriptional regulatory cascade was further confirmed by RNAi and overexpressing of Msi2 and HuR in MuSCs, and the in vivo function was characterized by histopathological and molecular biological methods in Msi2 knockout mice.ResultsWe identified a post‐transcription regulatory cascade governed by a pair of RNA‐binding proteins Msi2 and HuR. Msi2 is enriched in differentiated muscle cells and promotes MuSC differentiation despite its pro‐proliferation functions in other cell types. Msi2 works synergistically with another RNA‐binding protein HuR to repress the biogenesis of MiR7a‐1 in an Msi2 dose‐dependent manner to regulate the translation of the key component of the circadian core oscillator complex Cry2. Down‐regulation of Cry2 (0.6‐fold, vs. control, P < 0.05) mediated by MiR7a‐1 represses MuSC differentiation. The disruption of this cascade leads to differentiation defects of MuSCs. In aged muscles, Msi2 (0.3‐fold, vs. control, P < 0.01) expression declined, and the Cry2 protein level also decreases (0.5‐fold, vs. control, P < 0.05), suggesting that the disruption of the Msi2‐mediated post‐transcriptional regulatory cascade could attribute to the declined ability of muscle regeneration in aged skeletal muscle.ConclusionsOur findings have identified a new post‐transcriptional cascade regulating myogenesis. The cascade is disrupted in skeletal muscle ageing, which leads to declined muscle regeneration ability.

The RNA-binding protein human antigen R (HuR) participates in the posttranscriptional regulation of mRNAs bearing 3' AU-rich and U-rich elements, which HuR can stabilize under conditions of cellular stress. Using the LLC-PK(1) proximal tubule cell line model, we recently suggested a role for HuR in protecting kidney epithelia from injury during ischemic stress (Jeyaraj S, Dakhlallah D, Hill SR, Lee BS. J Biol Chem 280: 37957-37964, 2005; Jeyaraj SC, Dakhlallah D, Hill SR, Lee BS. Am J Physiol Renal Physiol 291: F1255-F1263, 2006). Here, we have extended this work to show that small interfering RNA-mediated suppression of HuR in LLC-PK(1) cells increased apoptosis during energy depletion, while overexpression of HuR diminished apoptosis. Suppression of HuR also resulted in diminished levels of key cell survival proteins such as Bcl-2 and Hsp70. Furthermore, rat kidneys were subjected in vivo to transient ischemia followed by varying periods of reperfusion. Ischemia and reperfusion (I/R) affected intensity and distribution of HuR in a nephron segment-specific manner. Cells of the proximal tubule, which are most sensitive to I/R injury, demonstrated a transient shift of HuR to the cytoplasm immediately following ischemia. Over a 14-day period following the onset of reperfusion, nuclear and total HuR protein gradually increased in cortical and medullary proximal tubules, but not in non-proximal tubule cells. HuR mRNA was expressed in two forms with alternate transcriptional start sites that increased over a 14-day I/R period, and in vitro studies suggest selective translatability of these two mRNAs. Baseline and I/R-stimulated levels of HuR mRNA did not parallel those of HuR protein, suggesting translational control of HuR expression, particularly in medullary proximal tubules. These findings suggest that alterations in distribution and expression of the antiaptotic protein HuR specifically in cells of the proximal tubule effect a protective mechanism during and following I/R injury in kidney.

RETRACTED: Insulin-like growth factor-I receptor is suppressed through transcriptional repression and mRNA destabilization by a novel energy restriction-mimetic agent

Tamoxifen-resistant (TAMR) estrogen receptor-positive (ER+) breast cancer is characterized by elevated Erb-B2 receptor tyrosine kinase 2 (ERBB2) expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the TAMR cells remain poorly understood. Herein, we reported that the ERBB2 expression is regulated at the post-transcriptional level by miR26a/b and the RNA-binding protein human antigen R (HuR), both of which associate with the 3'-UTR of the ERBB2 transcripts. We demonstrated that miR26a/b inhibits the translation of ERBB2 mRNA, whereas HuR enhances the stability of the ERBB2 mRNA. In TAMR ER+ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER+ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER+ breast cancer cells is attributable to an increase in the HuR mRNA isoform with shortened 3'-UTR, which exhibits increased translational activity. This shortening of the HuR mRNA 3'-UTR via alternative polyadenylation (APA) was observed to be dependent on cleavage stimulation factor subunit 2 (CSTF2/CstF-64), which is up-regulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR post-transcriptionally up-regulates ERBB2 expression in TAMR ER+ breast cancer cells.