Ganoderic acid D prevents oxidative stress‐induced senescence by targeting 14‐3‐3ε to activate CaM/CaMKII/NRF2 signaling pathway in mesenchymal stem cells

Stem cell senescence is an important cause of aging. Delaying senescence may present a novel way to combat aging and age‐associated diseases. This study provided a mechanistic insight into the protective effect of ganoderic acid D (GA‐D) against human amniotic mesenchymal stem cell (hAMSCs) senescence. GA‐D, a Ganoderma lucidum‐derived triterpenoid, markedly prevented hAMSCs senescence via activating the Ca²⁺ calmodulin (CaM)/CaM‐dependent protein kinase II (CaMKII)/nuclear erythroid 2‐related factor 2 (Nrf2) axis, and 14‐3‐3ε was identified as a target of GA‐D. 14‐3‐3ε‐encoding gene (YWHAE) knockdown in hAMSCs reversed the activation of the CaM/CaMKII/Nrf2 signals to attenuate the GA‐D anti‐aging effect and increase senescence‐associated β‐galactosidase (SA‐β‐gal), p16 and p21 expression levels, including reactive oxygen species (ROS) production, thereby promoting cell cycle arrest and decreasing differentiation potential. YWHAE overexpression maintained or slightly enhanced the GA‐D anti‐aging effect. GA‐D prevented d‐galactose‐caused aging in mice by significantly increasing the total antioxidant capacity, as well as superoxide dismutase and glutathione peroxidase activity, and reducing the formation of malondialdehyde, advanced glycation end products, and receptor of advanced glycation end products. Consistent with the protective mechanism of GA‐D against hAMSCs senescence, GA‐D delayed the senescence of bone‐marrow mesenchymal stem cells in this aging model in vivo, reduced SA‐β‐gal and ROS production, alleviated cell cycle arrest, and enhanced cell viability and differentiation via regulating 14‐3‐3ε and CaM/CaMKII/Nrf2 axis. Therefore, GA‐D retards hAMSCs senescence by targeting 14‐3‐3ε to activate the CaM/CaMKII/Nrf2 signaling pathway. Furthermore, the in vivo GA‐D anti‐aging effect may involve the regulation of stem cell senescence via the same signal axis.     

TNF‐α/IFN‐γ synergy amplifies senescence‐associated inflammation and SARS‐CoV‐2 receptor expression via hyper‐activated JAK/STAT1

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.     

Anti‐hsa‐miR‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

Hutchinson‐Gilford progeria syndrome (HGPS) is a lethal premature aging disorder without an effective therapeutic regimen. Because of their targetability and influence on gene expression, microRNAs (miRNAs) are attractive therapeutic tools to treat diseases. Here we identified that hsa‐miR‐59 (miR‐59) was markedly upregulated in HGPS patient cells and in multiple tissues of an HGPS mouse model (Lmna ^G609G/G609G ), which disturbed the interaction between RNAPII and TFIIH, resulting in abnormal expression of cell cycle genes by targeting high‐mobility group A family HMGA1 and HMGA2. Functional inhibition of miR‐59 alleviated the cellular senescence phenotype of HGPS cells. Treatment with AAV9‐mediated anti‐miR‐59 reduced fibrosis in the quadriceps muscle, heart, and aorta, suppressed epidermal thinning and dermal fat loss, and yielded a 25.5% increase in longevity of Lmna ^G609G/G609G mice. These results identify a new strategy for the treatment of HGPS and provide insight into the etiology of HGPS disease.     

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (K_D) of 2.98 × 10^–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.     

NLRP3 activation contributes to endothelin‐1‐induced erectile dysfunction

In the present study, we hypothesized that endothelin (ET) receptors (ET_A and ET_B) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain‐Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3^−/− and caspase^−/− mice in the presence of ET‐1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET‐1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET‐1 effect. ET‐1 decreased CC ACh‐, sodium nitroprusside (SNP)‐induced relaxation, and increased caspase‐1 expression. BQ123 an ET_A receptor antagonist reversed the effect. The ET_B receptor antagonist BQ788 also reversed ET‐1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET‐1‐induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase‐1 expression, while BQ788 increased caspase‐1 and IL‐1β levels in a concentration‐dependent manner (100 nM–10 μM). Furthermore, tiron and BAPTA AM prevented ET‐1‐induced increase in caspase‐1. In addition, BAPTA AM blocked ET‐1‐induced ROS generation. In conclusion, ET‐1‐induced erectile dysfunction depends on ET_A‐ and ET_B‐mediated activation of NLRP3 in mouse CC via Ca²⁺‐dependent ROS generation.     

p16INK4A Positively Regulates p21WAF1 Expression by suppressing AUF1-dependent mRNA decay

Background

p16^INK4a and p21^WAF1 are two independent cyclin-dependent kinase inhibitors encoded by the CDKN2A and CDKN1A genes, respectively. p16^INK4a and p21^WAF1 are similarly involved in various anti-cancer processes, including the regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis. Therefore, we sought to elucidate the molecular mechanisms underlying the link between these two important tumor suppressor proteins.

Methodology/Principal Findings

We have shown here that the p16^INK4a protein positively controls the expression of p21^WAF1 in both human and mouse cells. p16^INK4a stabilizes the CDKN1A mRNA through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by quantitative RT-PCR indicated that endogenous AUF1 binds to the CDKN1A mRNA in a p16^INK4A-dependent manner. Furthermore, while AUF1 down-regulation increased the expression level of the CDKN1A mRNA, the concurrent knockdown of AUF1 and CDKN2A, using specific silencing RNAs, restored the normal expression of the gene. Moreover, we used EGFP reporter fused to the CDKN2A AU-rich element (ARE) to demonstrate that p16^INK4A regulation of the CDKN1A mRNA is AUF1- and ARE-dependent. Furthermore, ectopic expression of p16^INK4A in p16^INK4A-deficient breast epithelial MCF-10A cells significantly increased the level of p21^WAF1, with no effect on cell proliferation. In addition, we have shown direct correlation between p16^INK4a and p21^WAF1 levels in various cancer cell lines.

Conclusion/Significance

These findings show that p16^INK4a stabilizes the CDKN1A mRNA in an AUF1-dependent manner, and further confirm the presence of a direct link between the 2 important cancer-related pathways, pRB/p16^INK4A and p14^ARF/p53/p21^WAF1.     

A circular intronic RNA ciPVT1 delays endothelial cell senescence by regulating the miR‐24‐3p/CDK4/pRb axis

Circular RNAs (circRNAs) have been established to be involved in numerous processes in the human genome, but their function in vascular aging remains largely unknown. In this study, we aimed to characterize and analyze the function of a circular intronic RNA, ciPVT1, in endothelial cell senescence. We observed significant downregulation of ciPVT1 in senescent endothelial cells. In proliferating endothelial cells, ciPVT1 knockdown induced a premature senescence‐like phenotype, inhibited proliferation, and led to an impairment in angiogenesis. An in vivo angiogenic plug assay revealed that ciPVT1 silencing significantly inhibited endothelial tube formation and decreased hemoglobin content. Conversely, overexpression of ciPVT1 in old endothelial cells delayed senescence, promoted proliferation, and increased angiogenic activity. Mechanistic studies revealed that ciPVT1 can sponge miR‐24‐3p to upregulate the expression of CDK4, resulting in enhanced Rb phosphorylation. Moreover, enforced expression of ciPVT1 reversed the senescence induction effect of miR‐24‐3p in endothelial cells. In summary, the present study reveals a pivotal role for ciPVT1 in regulating endothelial cell senescence and may have important implications in the search of strategies to counteract the development of age‐associated vascular pathologies.     

The PAX5‐JAK2 translocation acts as dual‐hit mutation that promotes aggressive B‐cell leukemia via nuclear STAT5 activation

While PAX5 is an important tumor suppressor gene in B‐cell acute lymphoblastic leukemia (B‐ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5‐JAK2 encodes a protein consisting of the PAX5 DNA‐binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5‐JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5 ^Jak2/+ mice rapidly developed an aggressive B‐ALL in the absence of another cooperating exogenous gene mutation. The DNA‐binding function and kinase activity of Pax5‐Jak2 as well as IL‐7 signaling contributed to leukemia development. Interestingly, all Pax5 ^Jak2/+ tumors lost the remaining wild‐type Pax5 allele, allowing efficient DNA‐binding of Pax5‐Jak2. While we could not find evidence for a nuclear role of Pax5‐Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5 ^Jak2/+ B‐ALL tumors, implying that nuclear Pax5‐Jak2 phosphorylates STAT5. Together, these data reveal Pax5‐Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.     

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2^LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2^LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2^LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.     

The 5′-flanking region of the E1 α form of the murine p16 (MTS1) gene

We have PCR-amplified and sequenced the immediate (841 bp) 5′-flanking region of murine p16^INK4a (MTS1, CDKN2) tumor suppressor gene. Comparing to recently published 5'-flanking region of the human α form of p16^INK4a, homologies were found in several regions of murine p16^INK4a-α putative promoter sequence.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

4‐phenylpyridine suppresses UVB‐induced skin inflammation by targeting c‐Src in vitro and in vivo

Acute or repetitive exposure to ultraviolet (UV) cause disruptions to the skin barrier and subsequent inflammatory skin disease. 4‐phenylpyridine (4‐PP) is a constituent of Brassica campestris L. ssp. Pekinensis and its effect on skin inflammation and molecular target remain unclear. The purpose of this study is to confirm the anti‐inflammatory efficacy of 4‐PP on UVB‐induced skin inflammation in human keratinocytes HaCaT and mouse skin and validation of its molecular target. 4‐PP also attenuated UVB‐induced phosphorylation of p38/mitogen‐activated protein kinase kinase (MKK) 3/6, c‐Jun N‐terminal kinase 1/2, MKK 4/7, extracellular‐signal‐regulated kinase 1/2, mitogen‐activated protein kinase 1/2. Additionally, 4‐PP inhibited UVB‐induced phosphorylation of epidermal growth factor receptor (EGFR) Y1068, Y1045 and 854 residues but not the proto‐oncogene tyrosine‐protein kinase c‐Src. Drug affinity responsive target stability assay revealed that 4‐PP directly binds to c‐Src and inhibits pronase c‐proteolysis. Knockdown of c‐Src inhibited UVB‐induced COX‐2 expression and phosphorylation of MAPKs and EGFR in HaCaT cells. Dorsal treatment of 4‐PP prevented UVB (0.5 J/cm²)‐induced skin thickness, phosphorylation of EGFR and COX‐2 expression in mouse skin. Our findings suggest that 4‐PP can be used as anti‐inflammatory agent with an effect of skin inflammation by inhibiting the COX‐2 expression via suppressing the c‐Src/EGFR/MAPKs signalling pathway.     

p38α‐MAPK‐deficient myeloid cells ameliorate symptoms and pathology of APP‐transgenic Alzheimer's disease mice

Alzheimer''s disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid‐β peptides (Aβ) and microglia‐dominated inflammatory activation in the brain. p38α‐MAPK is activated in both neurons and microglia. How p38α‐MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α‐MAPK in all myeloid cells or specifically in microglia of APP‐transgenic mice, and examined animals for AD‐associated pathologies (i.e., cognitive deficits, Aβ pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aβ internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α‐MAPK‐deficient myeloid cells were more effective than p38α‐MAPK‐deficient microglia in reducing cerebral Aβ and neuronal impairment in APP‐transgenic mice. Deficiency of p38α‐MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aβ. Interestingly, p38α‐MAPK‐deficient myeloid cells reduced IL‐17a‐expressing CD4‐positive lymphocytes in 9 but not 4‐month‐old APP‐transgenic mice. By cross‐breeding APP‐transgenic mice with Il‐17a‐knockout mice, we observed that IL‐17a deficiency potentially activated microglia and reduced Aβ deposition in the brain as shown in 9‐month‐old myeloid p38α‐MAPK‐deficient AD mice. Thus, p38α‐MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL‐17a‐expressing lymphocytes may partially mediate the pathogenic role of p38α‐MAPK in peripheral myeloid cells. Our study supports p38α‐MAPK as a therapeutic target for AD patients.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

MicroRNA‐148a‐3p suppresses epithelial‐to‐mesenchymal transition and stemness properties via Wnt1‐mediated Wnt/β‐catenin pathway in pancreatic cancer

Although miR‐148a‐3p has been reported to function as a tumour suppressor in various cancers, the molecular mechanism of miR‐148a‐3p in regulating epithelial‐to‐mesenchymal transition (EMT) and stemness properties of pancreatic cancer (PC) cells remains to be elucidated. In the present study, we demonstrated that miR‐148a‐3p expression was remarkably down‐regulated in PC tissues and cell lines. Moreover, low expression of miR‐148a‐3p was associated with poorer overall survival (OS) in patients with PC. In vitro, gain‐of‐function and loss‐of‐function experiments showed that miR‐148a‐3p suppressed EMT and stemness properties as well as the proliferation, migration and invasion of PC cells. A dual‐luciferase reporter assay demonstrated that Wnt1 was a direct target of miR‐148a‐3p, and its expression was inversely associated with miR‐148a‐3p in PC tissues. Furthermore, miR‐148a‐3p suppressed the Wnt/β‐catenin pathway via down‐regulation of Wnt1. The effects of ectopic miR‐148a‐3p were rescued by Wnt1 overexpression. These biological functions of miR‐148a‐3p in PC were also confirmed in a nude mouse xenograft model. Taken together, these findings suggest that miR‐148a‐3p suppresses PC cell proliferation, invasion, EMT and stemness properties via inhibiting Wnt1‐mediated Wnt/β‐catenin pathway and could be a potential prognostic biomarker as well as a therapeutic target in PC.