The Δ40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation

The naturally occurring Δ40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed Δ40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing Δ40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. Δ40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed Δ40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that Δ40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for Δ40p53. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription.

CCR2 Deficiency Impairs Ly6Clo and Ly6Chi Monocyte Responses in Orientia tsutsugamushi Infection

Orientia (O.) tsutsugamushi, the causative agent of scrub typhus, is a neglected, obligate intracellular bacterium that has a prominent tropism for monocytes and macrophages. Complications often involve the lung, where interstitial pneumonia is a typical finding. The severity of scrub typhus in humans has been linked to altered plasma concentrations of chemokines which are known to act as chemoattractants for myeloid cells. The trafficking and function of monocyte responses is critically regulated by interaction of the CC chemokine ligand 2 (CCL2) and its CC chemokine receptor CCR2. In a self-healing mouse model of intradermal infection with the human-pathogenic Karp strain of O. tsutsugamushi, we investigated the role of CCR2 on bacterial dissemination, development of symptoms, lung histology and monocyte subsets in blood and lungs. CCR2-deficient mice showed a delayed onset of disease and resolution of symptoms, higher concentrations and impaired clearance of bacteria in the lung and the liver, accompanied by a slow infiltration of interstitial macrophages into the lungs. In the blood, we found an induction of circulating monocytes that depended on CCR2, while only a small increase in Ly6Chi monocytes was observed in CCR2-/- mice. In the lung, significantly higher numbers of Ly6Chi and Ly6Clo monocytes were found in the C57BL/6 mice compared to CCR2-/- mice. Both wildtype and CCR2-deficient mice developed an inflammatory milieu as shown by cytokine and inos/arg1 mRNA induction in the lung, but with delayed kinetics in CCR2-deficient mice. Histopathology revealed that infiltration of macrophages to the parenchyma, but not into the peribronchial tissue, depended on CCR2. In sum, our data suggest that in Orientia infection, CCR2 drives blood monocytosis and the influx and activation of Ly6Chi and Ly6Clo monocytes into the lung, thereby accelerating bacterial replication and development of interstitial pulmonary inflammation.

Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes

Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron stimulates expression of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells, which in turn binds to BMP receptors on hepatocytes and induces the SMAD signaling cascade for transcriptional activation of the hepcidin-encoding HAMP mRNA. SMAD signaling is also essential for inflammatory HAMP mRNA induction by the IL-6/STAT3 pathway. Herein, we utilized human Huh7 hepatoma cells and primary murine hepatocytes to assess the effects of iron perturbations on signaling to hepcidin. Iron chelation appeared to slightly impair signaling to hepcidin. Subsequent iron supplementation not only failed to reverse these effects, but drastically reduced basal HAMP mRNA and inhibited HAMP mRNA induction by BMP6 and/or IL-6. Thus, treatment of cells with excess iron inhibited basal and BMP6-mediated SMAD5 phosphorylation and induction of HAMP, ID1 and SMAD7 mRNAs in a dose-dependent manner. Iron also inhibited IL-6-mediated STAT3 phosphorylation and induction of HAMP and SOCS3 mRNAs. These responses were accompanied by induction of GCLC and HMOX1 mRNAs, known markers of oxidative stress. We conclude that hepatocellular iron overload suppresses hepcidin by inhibiting the SMAD and STAT3 signaling pathways downstream of their respective ligands.

Activation of the β-adrenergic receptor exacerbates lipopolysaccharide-induced wasting of skeletal muscle cells by increasing interleukin-6 production

The skeletal muscle mass has been shown to be affected by catecholamines, such as epinephrine (Epi), norepinephrine (NE), and isoproterenol (ISO). On the other hand, lipopolysaccharide (LPS), one of the causative substances of sepsis, induces muscle wasting via toll-like receptors expressed in skeletal muscle. Although catecholamines are frequently administered to critically ill patients, it is still incompletely understood how these drugs affect skeletal muscle during critical illness, including sepsis. Herein, we examined the direct effects of catecholamines on LPS-induced skeletal muscle wasting using the C2C12 myoblast cell line. Muscle wasting induced by catecholamines and/or LPS was analyzed by the use of the differentiated C2C12 myotubes, and its underlying mechanism was explored by immunoblotting analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and the TransAM kit for p-65 NF-κB. Epi augmented myosin heavy chain (MHC) protein loss and reduction of the myotube diameter induced by LPS. LPS induced C/EBPδ protein, Atrogin-1 and inteleukin-6 (IL-6), and these responses were potentiated by Epi. An IL-6 inhibitor, LMT28, suppressed the potentiating effect of Epi on the LPS-induced responses. NF-κB activity was induced by LPS, but was not affected by Epi and recombinant IL-6, and the NF-κB inhibitor, Bay 11–7082, abolished Atrogin-1 mRNA expression induced by LPS with or without Epi. NE and ISO also potentiated LPS-induced IL-6 and Atroign-1 mRNA expression. Carvedilol, a nonselective β-adrenergic receptor antagonist, suppressed the facilitating effects of Epi on the Atrogin-1 mRNA induction by LPS, and abolished the effects of Epi on the MHC protein loss in the presence of LPS. It was concluded that Epi activates the β-adrenergic receptors in C2C12 myotubes and the IL-6-STAT3 pathway, leading to the augmentation of LPS-induced activation of the NF-κB- C/EBPδ-Atrogin-1 pathway and to the exacerbation of myotube wasting.

Cold-induction of afadin in brown fat supports its thermogenic capacity

The profound energy-expending nature of brown adipose tissue (BAT) thermogenesis makes it an attractive target tissue to combat obesity-associated metabolic disorders. While cold exposure is the strongest inducer of BAT activity, the temporal mechanisms tuning BAT adaptation during this activation process are incompletely understood. Here we show that the scaffold protein Afadin is dynamically regulated by cold in BAT, and participates in cold acclimation. Cold exposure acutely increases Afadin protein levels and its phosphorylation in BAT. Knockdown of Afadin in brown pre-adipocytes does not alter adipogenesis but restricts β3-adrenegic induction of thermogenic genes expression and HSL phosphorylation in mature brown adipocytes. Consistent with a defect in thermogenesis, an impaired cold tolerance was observed in fat-specific Afadin knockout mice. However, while Afadin depletion led to reduced Ucp1 mRNA induction by cold, stimulation of Ucp1 protein was conserved. Transcriptomic analysis revealed that fat-specific ablation of Afadin led to decreased functional enrichment of gene sets controlling essential metabolic functions at thermoneutrality in BAT, whereas it led to an altered reprogramming in response to cold, with enhanced enrichment of different pathways related to metabolism and remodeling. Collectively, we demonstrate a role for Afadin in supporting the adrenergic response in brown adipocytes and BAT function.

Health Effects and Life Stage Sensitivities in Zebrafish Exposed to an Estrogenic Wastewater Treatment Works Effluent

A wide range of health effects in fish have been reported for exposure to wastewater treatment work (WwTW) effluents including feminized responses in males. Most of these exposure studies, however, have assessed acute health effects and chronic exposure effects are less well established. Using an Estrogen Responsive Element-Green Fluorescent Protein (ERE-GFP)-Casper transgenic zebrafish, we investigated chronic health effects and life stage sensitivities for exposure to an estrogenic WwTW effluent and the synthetic estrogen 17α-ethinylestradiol (EE2). Exposure to the WwTW effluent (at full strength;100%) and to 10 ng/L (nominal) EE2 delayed testis maturation in male fish but accelerated ovary development in females. Exposure to 50% and 100% effluent, and to 10 ng/L EE2, also resulted in skewed sex ratios in favor of females. Differing patterns of green fluorescent protein (GFP) expression, in terms of target tissues and developmental life stages occurred in the ERE-GFP- zebrafish chronically exposed to 100% effluent and reflected the estrogenic content of the effluent. gfp and vitellogenin (vtg) mRNA induction were positively correlated with measured levels of steroidal estrogens in the effluent throughout the study. Our findings illustrate the importance of a fish’s developmental stage for estrogen exposure effects and demonstrate the utility of the ERE-GFP zebrafish for integrative health analysis for exposure to estrogenic chemical mixtures.

Qualifying Osteogenic Potency Assay Metrics for Human Multipotent Stromal Cells: TGF-β2 a Telling Eligible Biomarker

Potency assays are critical for regenerative medicine, addressing the known challenge of functional heterogeneity among human multipotent stromal cells (hMSC). Necessary laboratory cell expansion allows analysis before implantation in the patient. Levels of induction of five signature gene biomarkers, ALPL, COL1A2, DCN, ELN and RUNX2, constituted a previously reported proof-of-principle osteogenic potency assay. We tested assay modification to enhance reproducibility using six consistent bone marrow derived hBM-MSC and explored applicability to three adipose tissue derived hAT-MSC. Using a potent proprietary osteogenic induction factor, the GUSB/YWAHZ reference gene pair provided real time PCR consistency. The novel assay conditions supported the concept that genes encoding extracellular matrix proteins one week after osteogenic induction were informative. Nonetheless, relatively low induction of COL1A2 and ELN encouraged search for additional biomarkers. TGFB2 mRNA induction, important for osteogenic commitment, was readily quantifiable in both hBM-MSC and hAT-MSC. Combined with DCN, TGFB2 mRNA induction data provided discriminatory power for resolving donor-specific heterogeneity. Histomorphometric decorin and TGF-β2 protein expression patterns in eight-week heterotopic bone implants also discriminated the two non-bone-forming hMSC. We highlight progress towards prompt osteogenic potency assays, needed by current clinical trials to accelerate improved intervention with enhanced stem cell therapy for serious bone fractures.

Rice Bran and Vitamin B6 Suppress Pathological Neovascularization in a Murine Model of Age-Related Macular Degeneration as Novel HIF Inhibitors

Pathological neovascularization in the eye is a leading cause of blindness in all age groups from retinopathy of prematurity (ROP) in children to age-related macular degeneration (AMD) in the elderly. Inhibiting neovascularization via antivascular endothelial growth factor (VEGF) drugs has been used for the effective treatment. However, anti-VEGF therapies may cause development of chorioretinal atrophy as they affect a physiological amount of VEGF essential for retinal homeostasis. Furthermore, anti-VEGF therapies are still ineffective in some cases, especially in patients with AMD. Hypoxia-inducible factor (HIF) is a strong regulator of VEGF induction under hypoxic and other stress conditions. Our previous reports have indicated that HIF is associated with pathological retinal neovascularization in murine models of ROP and AMD, and HIF inhibition suppresses neovascularization by reducing an abnormal increase in VEGF expression. Along with this, we attempted to find novel effective HIF inhibitors from natural foods of our daily lives. Food ingredients were screened for prospective HIF inhibitors in ocular cell lines of 661W and ARPE-19, and a murine AMD model was utilized for examining suppressive effects of the ingredients on retinal neovascularization. As a result, rice bran and its component, vitamin B6 showed inhibitory effects on HIF activation and suppressed VEGF mRNA induction under a CoCl2-induced pseudo-hypoxic condition. Dietary supplement of these significantly suppressed retinal neovascularization in the AMD model. These data suggest that rice bran could have promising therapeutic values in the management of pathological ocular neovascularization.

Interaction of Hydroxychloroquine with Pharmacokinetically Important Drug Transporters

(1) Background: Hydroxychloroquine is used to treat malaria and autoimmune diseases, and its potential use against COVID-19 is currently under investigation. Thus far, information on interactions of hydroxychloroquine with drug transporters mediating drug-drug interactions is limited. We assessed the inhibition of important efflux (P-glycoprotein (P-gp), breast cancer resistance protein (BCRP)) and uptake transporters (organic anion transporting polypeptide (OATP)-1B1, OATP1B3, OATP2B1) by hydroxychloroquine, tested its P-gp and BCRP substrate characteristics, and evaluated the induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X (PXR) (CYP3A4, ABCB1) and aryl hydrocarbon receptor (AhR) (CYP1A1, CYP1A2). (2) Methods: Transporter inhibition was evaluated in transporter over-expressing cell lines using fluorescent probe substrates. P-gp and BCRP substrate characteristics were assessed by comparing growth inhibition of over-expressing and parental cell lines. Possible mRNA induction was analysed in LS180 cells by quantitative real-time PCR. (3) Results: Hydroxychloroquine did not inhibit BCRP or the OATPs tested but inhibited P-gp at concentrations exceeding 10 µM. P-gp overexpressing cells were 5.2-fold more resistant to hydroxychloroquine than control cells stressing its substrate characteristics. Hydroxychloroquine did not induce genes regulated by PXR or AhR. (4) Conclusions: This is the first evidence that hydroxychloroquine’s interaction potential with drug transporters is low, albeit bioavailability of simultaneously orally administered P-gp substrates might be increased by hydroxychloroquine.

Enhancer RNAs predict enhancer–gene regulatory links and are critical for enhancer function in neuronal systems

Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer–gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer–gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.