NRSN2 promotes the malignant behavior of HPV-transfected laryngeal carcinoma cells through AMPK/ULK1 pathway mediated autophagy activation.

Neurensin-2 (NRSN2) performs a pro-carcinogenic function in multiple cancers. However, the function of NRSN2 in HPV-infected laryngeal carcinoma (LC) remains unclear. HPV transfection was performed in LC cells. The mRNA and protein levels were monitored using RT-qPCR, immunoblotting, and IF. Cell viability and proliferation were found using the CCK-8 assay and Edu staining. Cell invasion, migration, and apoptosis were probed using the Transwell, wound healing, and flow cytometry, respectively. The autophagosome was observed using TEM. NRSN2 was overexpressed in HPV-transfected LC cells. Inhibition of NRSN2 restrained the autophagy and malignant behavior of HPV-transfected LC cells. Meanwhile, the inhibition of AMPK/ULK1 pathway limited the increased autophagy of HPV-transfected LC cells caused by NRSN2 overexpression. Furthermore, NRSN2 knockdown inhibits autophagy by suppressing AMPK/ULK1 pathway, thereby restraining the malignant behavior of HPV-transfected LC cells. Our research confirmed that HPV transfection increased the autophagy and malignant behavior of LC cells by regulating the NRSN2-mediated activation of the AMPK/ULK1 pathway, offering a new target for cure of LC.

Sirt3 Protects Retinal Pigment Epithelial Cells From High Glucose-Induced Injury by Promoting Mitophagy Through the AMPK/mTOR/ULK1 Pathway.

Purpose: The regulation of mitophagy by Sirt3 has rarely been studied in ocular diseases. In the present study, we determined the effects of Sirt3 on AMPK/mTOR/ULK1 signaling pathway-mediated mitophagy in retinal pigment epithelial (RPE) cells in a high glucose environment. Methods: The mRNA expression levels of Sirt3, AMPK, mTOR, ULK1, and LC3B in RPE cells under varying glucose conditions were measured by real-time polymerase chain reaction (RT-PCR). The expressions of Sirt3, mitophagy protein, and AMPK/mTOR/ULK1 signaling pathway-related proteins were detected by Western blotting. Lentivirus (LV) transfection mediated the stable overexpression of Sirt3 in cell lines. The experimental groups were NG (5.5 mM glucose), hypertonic, HG (30 mM glucose), HG + LV-GFP, and HG + LV-Sirt3. Western blotting was performed to detect the expressions of mitophagy proteins and AMPK/mTOR/ULK1-related proteins in a high glucose environment during the overexpression of Sirt3. Reactive oxygen species (ROS) production in a high glucose environment was measured by DCFH-DA staining. Mitophagy was detected by labeling mitochondria and lysosomes with MitoTracker and LysoTracker probes, respectively. Apoptosis was detected by flow cytometry. Results: Sirt3 expression was reduced in the high glucose group, inhibiting the AMPK/mTOR/ULK1 pathway, with diminished mitophagy and increased intracellular ROS production. The overexpression of Sirt3, increased expression of p-AMPK/AMPK and p-ULK1/ULK1, and decreased expression of p-mTOR/mTOR inhibited cell apoptosis and enhanced mitophagy. Conclusions: Sirt3 protected RPE cells from high glucose-induced injury by activating the AMPK/mTOR/ULK1 signaling pathway. Translational Relevance: By identifying new targets of action, we aimed to establish effective therapeutic targets for diabetic retinopathy treatment.

The mechanism of UNC-51-like kinase 1 and the applications of small molecule modulators in cancer treatment.

Autophagy is a process of self-renewal in cells, which not only provides the necessary nutrients for cells, but also clears necrotic organelles. Autophagy disorders are closely related to diseases such as cancer. UNC-51-like kinase 1 (ULK1) is a serine/threonine protein kinase that plays a crucial role in receiving input from energy and nutrient sensors, activating autophagy to maintain cellular homeostasis under stressful conditions. In recent years, targeting ULK1 has become a highly promising strategy for cancer treatment. This review introduces the regulatory mechanism of ULK1 in autophagy through the AMPK/mTOR/ULK1 pathway and reviews the research progress of ULK1 activators and inhibitors and their applications in cancer treatment. In addition, we analyze the binding modes between ULK1 and modulators through virtual molecular docking, which will provide a reliable basis and theoretical guidance for the design and development of new therapeutic drugs targeting ULK1.

RNF135 promotes cell proliferation and autophagy in lung adenocarcinoma by promoting the phosphorylation of ULK1.

OBJECTIVE: To detect the expression of RING finger protein 135 (RNF135) in lung adenocarcinoma tissues and explore its role in the progression of lung adenocarcinoma. METHODS: Bioinformation analysis, quantitative polymerase chain reaction, and immunoblotting technique discovered the expression of RNF135 in lung adenocarcinoma tissues. Cell counting kit-8 and colony formation, immunostaining, and immunoblot assays examined the effects of RNF135 on cell growth and autophagy. Co-immunoprecipitation (Co-IP), immunostaining, and immuoblotting were conducted to confirm the mechanism. RESULTS: RNF135 was highly expressed in lung adenocarcinoma. In addition, RNF135 promoted lung adenocarcinoma cell growth. Further, data confirmed that RNF135 promoted autophagy in lung adenocarcinoma cells. Mechanically, RNF135 directly interacted with Unc-51-like autophagy activating kinase 1 (ULK1) to promote its phosphorylation level. CONCLUSION: RNF135 promoted cell growth and autophagy in lung adenocarcinoma by promoting the phosphorylation of ULK1.

Trifluoperazine activates AMPK / mTOR / ULK1 signaling pathway to induce mitophagy in osteosarcoma cells.

Osteosarcoma is a prevalent kind of primary bone malignancy. Trifluoperazine, as an antipsychotic drug, has anti-tumor activity against a variety of cancers. Nevertheless, the impact of trifluoperazine on osteosarcoma is unclear. Our investigation aimed to explore the mechanism of trifluoperazine's effect on osteosarcoma. We found that trifluoperazine inhibited 143B and U2-OS osteosarcoma cell proliferation in a method based on the dose. Furthermore, it was shown that trifluoperazine induced the accumulation of reactive oxygen species (ROS) to cause mitochondrial damage and induced mitophagy in osteosarcoma cells. Finally, combined with RNA-seq results, we first demonstrated the AMPK/mTOR/ULK1 signaling pathway as a potential mechanism of trifluoperazine-mediated mitophagy in osteosarcoma cells and can be suppressed by AMPK inhibitor Compound C.

DDIT3 deficiency accelerates bone remodeling during bone healing by enhancing osteoblast and osteoclast differentiation through ULK1-mediated autophagy.

The coordination of osteoblasts and osteoclasts is essential for bone remodeling. DNA damage inducible script 3 (DDIT3) is an important regulator of bone and participates in cell differentiation, proliferation, autophagy, and apoptosis. However, its role in bone remodeling remains unexplored. Here, we found that Ddit3 knockout (Ddit3-KO) enhanced both bone formation and resorption. The increased new bone formation and woven bone resorption, i.e., enhanced bone remodeling capacity, was found to accelerate bone defect healing in Ddit3-KO mice. In vitro experiments showed that DDIT3 inhibited both osteoblast differentiation and Raw264.7 cell differentiation by regulating autophagy. Cell coculture assay showed that Ddit3-KO decreased the ratio of receptor activator of nuclear factor-κß ligand (RANKL) to osteoprotegerin (OPG) in osteoblasts, and Ddit3-KO osteoblasts inhibited osteoclast differentiation. Meanwhile, DDIT3 knockdown (DDIT3-sh) increased receptor activator of nuclear factor-κß (RANK) expression in Raw264.7 cells, and DDIT3-sh Raw264.7 cells promoted osteoblast differentiation, whereas, DDIT3 overexpression had the opposite effect. Mechanistically, DDIT3 promoted autophagy partly by increasing ULK1 phosphorylation at serine555 (pULK1-S555) and decreasing ULK1 phosphorylation at serine757 (pULK1-S757) in osteoblasts, thereby inhibiting osteoblast differentiation. DDIT3 inhibited autophagy partly by decreasing pULK1-S555 in Raw264.7 cells, thereby suppressing osteoclastic differentiation. Taken together, our data indicate that DDIT3 is one of the elements regulating bone remodeling and bone healing, which may become a potential target in bone defect treatment.

Abnormal expression of CUX1 influences autophagy activation in paroxysmal nocturnal hemoglobinuria.

The mechanism underlying autophagy in paroxysmal nocturnal hemoglobinuria (PNH) remains largely unknown. We previously sequenced the entire genome exon of the CD59- cells from 13 patients with PNH and found genes such as CUX1 encoding Cut-like homeobox 1. Peripheral blood samples from 9 patients with PNH and 7 healthy control subjects were obtained to measure CUX1 expression. The correlation between CUX1 messenger RNA expression and PNH clinical indicators was analyzed. To simulate CUX1 expression in patients with PNH, we generated a panel of PNH cell lines by knocking out PIGA in K562 cell lines and transfected lentivirus with CUX1. CCK-8 and EDU assay assessed cell proliferation. Western blotting was used to detect Beclin-1, LC3A, LC3B, ULK1, PI3K, AKT, p-AKT, mTOR, and p-mTOR protein levels. Autophagosomes were observed with transmission electron microscopy. Chloroquine was used to observe CUX1 expression in PNH after autophagy inhibition. Leukocytes from patients with PNH had lower levels of CUX1 messenger RNA expression and protein content than healthy control subjects. The lactose dehydrogenase level and the percentage of PNH clones were negatively correlated with CUX1 relative expression. We reduced CUX1 expression in a PIGA knockout K562 cell line, leading to increased cell proliferation. Levels of autophagy markers Beclin-1, LC3B, LC3A, and ULK1 increased, and autophagosomes increased. Furthermore, PI3K/AKT/mTOR protein phosphorylation levels were lower. CUX1 expression did not change and cell proliferation decreased in CUX1 knocked down PNH cells after inhibition of autophagy by chloroquine. In brief, CUX1 loss-of-function mutation resulted in stronger autophagy in PNH.

Regulation of ULK1 by WTAP/IGF2BP3 axis enhances mitophagy and progression in epithelial ovarian cancer.

There is a pressing need for innovative therapeutic strategies for patients with epithelial ovarian cancer (EOC). Previous studies have shown that UNC-51-like kinase 1 (ULK1), a serine/threonine kinase, is crucial in regulating cellular autophagy and mitophagy across various tumor types. However, the clinical implications, biological functions, and potential mechanisms of ULK1 in EOC remain poorly understood. This study demonstrates that ULK1 expression is upregulated in EOC tissue samples and EOC cell lines, with increased ULK1 expression correlating with poor prognosis. Functionally, overexpressed ULK1 enhances the proliferation and migration abilities of EOC cells both in vitro and in vivo. Mechanistically, ULK1 was identified as an m6A target of WTAP. WTAP-mediated m6A modification of ULK1 enhanced its mRNA stability in an IGF2BP3-dependent manner, leading to elevated ULK1 expression and enhanced mitophagy in EOC. In summary, our research reveals that the WTAP/IGF2BP3-ULK1 axis significantly influences protective mitophagy in EOC, contributing to its progression. Therefore, the regulatory mechanisms and biological function of ULK1 identify it as a potential molecular target for therapeutic intervention in EOC.

SPHK1 potentiates colorectal cancer progression and metastasis via regulating autophagy mediated by TRAF6-induced ULK1 ubiquitination.

A sphingolipid metabolite regulator, sphingosine kinase 1 (SPHK1), plays a critical role in the development of colorectal cancer (CRC). Studies have demonstrated that invasion and metastasis of CRC are promoted by SPHK1-driven autophagy. However, the exact mechanism of SPHK1 drives autophagy to promote tumor progression remains unclear. Here, immunohistochemical detection showed the expression of SPHK1 and tumor necrosis factor receptor-associated factor-6 (TRAF6) in human CRC tissues was stronger than in adjacent normal tissues, they were both associated with distance metastasis. It was discovered that knockdown of SPHK1 reduced the expression of TRAF6, inhibited autophagy, and inhibited the growth and metastasis of CRC cells in vitro. Moreover, the effects of SPHK1-downregulating were reversed by overexpression of TRAF6 in CRC cells transfected by double-gene. Overexpression of SPHK1 and TRAF6 promoted the expression of autophagy protein LC3 and Vimentin, while downregulated the expression of autophagy protein P62 and E-cadherin. The expression of autophagy-related ubiquitination protein ULK1 and Ubiquitin protein were significantly upregulated in TRAF6-overexpressed CRC cells. In addition, autophagy inhibitor 3-methyladenine (3MA) significantly inhibited the metastasis-promoting effect of SPHK1 and TRAF6, suppressed the expression of LC3 and Vimentin, and promoted the expression of P62 and E-cadherin, in CRC cells. Immunofluorescence staining showed SPHK1 and TRAF6 were co-localized in HT29 CRC cell membrane and cytoplasm. Immunoprecipitation detection showed SPHK1 was efficiently combined with the endogenous TRAF6, and the interaction was also detected reciprocally. Additionally, proteasome inhibitor MG132 treatment upregulated the expression of TRAF6 and the level of Ubiquitin protein, in SPHK1-downregulating CRC cells. These results reveal that SPHK1 potentiates CRC progression and metastasis via regulating autophagy mediated by TRAF6-induced ULK1 ubiquitination. SPHK1-TRAF6-ULK1 signaling axis is critical to the progression of CRC and provides a new strategy for the therapeutic control of CRC.

ULK1 confers neuroprotection by regulating microglial/macrophages activation after ischemic stroke.

Microglial activation and autophagy play a critical role in the progression of ischemic stroke and contribute to the regulation of neuroinflammation. Unc-51-like kinase 1 (ULK1) is the primary autophagy kinase involved in autophagosome formation. However, the impact of ULK1 on neuroprotection and microglial activation after ischemic stroke remains unclear. In this study, we established a photothrombotic stroke model, and administered SBI-0206965 (SBI), an ULK1 inhibitor, and LYN-1604 hydrochloride (LYN), an ULK1 agonist, to modulate ULK1 activity in vivo. We assessed sensorimotor deficits, neuronal apoptosis, and microglial/macrophage activation to evaluate the neurofunctional outcome. Immunofluorescence results revealed ULK1 was primarily localized in the microglia of the infarct area following ischemia. Upregulating ULK1 through LYN treatment significantly reduced infarct volume, improved motor function, promoted the increase of anti-inflammatory microglia. In conclusion, ULK1 facilitated neuronal repair and promoted the formation of anti-inflammatory microglia pathway after ischemic injury.

Polystyrene nanoplastic exposure induces excessive mitophagy by activating AMPK/ULK1 pathway in differentiated SH-SY5Y cells and dopaminergic neurons in vivo.

BACKGROUND: Microplastics and nanoplastics (MNPs) are emerging environmental contaminants detected in human samples, and have raised concerns regarding their potential risks to human health, particularly neurotoxicity. This study aimed to investigate the deleterious effects of polystyrene nanoplastics (PS-NPs, 50 nm) and understand their mechanisms in inducing Parkinson's disease (PD)-like neurodegeneration, along with exploring preventive strategies. METHODS: Following exposure to PS-NPs (0.5-500 µg/mL), we assessed cytotoxicity, mitochondrial integrity, ATP levels, and mitochondrial respiration in dopaminergic-differentiated SH-SY5Y cells. Molecular docking and dynamic simulations explored PS-NPs' interactions with mitochondrial complexes. We further probed mitophagy's pivotal role in PS-NP-induced mitochondrial damage and examined melatonin's ameliorative potential in vitro. We validated melatonin's intervention (intraperitoneal, 10 mg/kg/d) in C57BL/6 J mice exposed to 250 mg/kg/d of PS-NPs for 28 days. RESULTS: In our in vitro experiments, we observed PS-NP accumulation in cells, including mitochondria, leading to cell toxicity and reduced viability. Notably, antioxidant treatment failed to fully rescue viability, suggesting reactive oxygen species (ROS)-independent cytotoxicity. PS-NPs caused significant mitochondrial damage, characterized by altered morphology, reduced mitochondrial membrane potential, and decreased ATP production. Subsequent investigations pointed to PS-NP-induced disruption of mitochondrial respiration, potentially through interference with complex I (CI), a concept supported by molecular docking studies highlighting the influence of PS-NPs on CI. Rescue experiments using an AMPK pathway inhibitor (compound C) and an autophagy inhibitor (3-methyladenine) revealed that excessive mitophagy was induced through AMPK/ULK1 pathway activation, worsening mitochondrial damage and subsequent cell death in differentiated SH-SY5Y cells. Notably, we identified melatonin as a potential protective agent, capable of alleviating PS-NP-induced mitochondrial dysfunction. Lastly, our in vivo experiments demonstrated that melatonin could mitigate dopaminergic neuron loss and motor impairments by restoring mitophagy regulation in mice. CONCLUSIONS: Our study demonstrated that PS-NPs disrupt mitochondrial function by affecting CI, leading to excessive mitophagy through the AMPK/ULK1 pathway, causing dopaminergic neuron death. Melatonin can counteract PS-NP-induced mitochondrial dysfunction and motor impairments by regulating mitochondrial autophagy. These findings offer novel insights into the MNP-induced PD-like neurodegenerative mechanisms, and highlight melatonin's protective potential in mitigating the MNP's environmental risk.

An ULK1/2-PXN mechanotransduction pathway suppresses breast cancer cell migration.

The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well-characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN-dependent mechanotransduction.

Exosomal circHIPK3 derived from umbilical cord-derived mesenchymal stem cells enhances skin fibroblast autophagy by blocking miR-20b-5p/ULK1/Atg13 axis.

BACKGROUND: Umbilical cord-derived mesenchymal stem cells (UCMSCs) could alleviate diabetes-induced injury. Hence, this investigation aimed to explore the role and mechanism of UCMSCs-derived exosomal circHIPK3 (exo-circHIPK3) in diabetes mellitus (DM). METHODS: HFF-1 cells were cultured in high glucose (HG) medium or normal medium, and treated with UCMSCs-derived exo-circHIPK3 or miR-20b-5p mimics or Unc-51-like autophagy activating kinase 1 (ULK1) overexpression vector. The surface markers of UCMSCs were analyzed using a flow cytometer. The differentiation potential of UCMSCs was evaluated using oil red O staining, alizarin red staining and alkaline phosphatase (ALP) staining. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The miRNA expressions were analyzed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR). Protein levels were quantified by western blot. An immunofluorescence staining was used for observing LC3 expression. The interaction between miR-20b-5p and circHIPK3, and between miR-20b-5b and ULK1 were identified by a RNA immunoprecipitation (RIP) assay and a luciferase reporter assay. RESULTS: Up-regulation of circHIPK3 was found in UCMSCs-derived exosomes. Exo-circHIPK3 decreased the miR-20b-5p level while increasing the contents of ULK1 and autophagy-related gene 13 (Atg13) in HG-induced fibroblasts. In addition, exo-circHIPK3 activated HG-induced fibroblast autophagy and proliferation. Overexpressed miR-20b-5p promoted fibroblast injury by inhibiting cell autophagy via the ULK1/Atg13 axis in HG conditions of high glucose. Moreover, exo-circHIPK3 enhanced autophagy and cell viability in HG-induced fibroblasts through the miR-20b-5p/ULK1/Atg13 axis. CONCLUSION: UCMSCs-derived exosomal circHIPK3 promoted cell autophagy and proliferation and accelerated the fibroblast injury repair by the miR-20b-5p/ULK1/Atg13 axis.

In vitro evaluation of autophagy and cell death induction in Panc1 pancreatic cancer by secretome of hAMSCs through downregulation of p-AKT/p-mTOR and upregulation of p-AMPK/ULK1 signal transduction pathways.

One of the main causes of cancer mortality in the world is pancreatic cancer. Therapies based on stem cells are currently thought to be a hopeful option in the treatment of cancer. Herein, we intend to evaluate the antitumor effects of secretome of human amniotic mesenchymal stromal cells (hAMSCs) on autophagy and cell death induction in Panc1 pancreatic cancer cells. We adopted a co-culture system using Transwell 6-well plates and after 72 h, hAMSCs-treated Panc1 cancer cells were analyzed using quantitative real time PCR (qRT-PCR), flow cytometry, western blot, MTT assay, and DAPI staining. Based on our results, the microtubule-associated protein 1 light chain 3 (LC3) conversion from LC3-I to LC3-II and the upregulation of autophagy-related proteins expression including Beclin1, Atg7, and Atg12 were detected in hAMSCs-treated Panc1 cells. Furthermore, the level of phosphorylated proteins such as Unc-51-like kinase 1 (ULK1), AMP activated protein kinase (AMPK), AKT, and mTOR changed. Apoptotic cell death was also induced via the elevation of Bax and Caspase 3 expression and inhibition of Bcl-2. Our findings showed that secretome of hAMSCs induces autophagy and cell death in Panc1 cancer cells. However, more experiments will be needed to identify more details about the associated mechanisms.

Starvation-inactivated MTOR triggers cell migration via a ULK1-SH3PXD2A/TKS5-MMP14 pathway in ovarian carcinoma.

ABBREVIATIONS: AMPK: AMP-activated protein kinase; CHX: cycloheximide; RAD001: everolimus; HBSS: Hanks' balanced salt solution; LC-MS/MS: liquid chromatography-mass spectrometry/mass spectrometry; MMP14: matrix metallopeptidase 14; MTOR: mechanistic target of rapamycin kinase; MAPK: mitogen-activated protein kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology; SH3: Src homology 3; SH3PXD2A/TKS5: SH3 and PX domains 2A; SH3PXD2A-[6A]: S112A S142A S146A S147A S175A S348A mutant; ULK1: unc-51 like autophagy activating kinase 1.

ULK1-mediated metabolic reprogramming regulates Vps34 lipid kinase activity by its lactylation.

Autophagy and glycolysis are highly conserved biological processes involved in both physiological and pathological cellular programs, but the interplay between these processes is poorly understood. Here, we show that the glycolytic enzyme lactate dehydrogenase A (LDHA) is activated upon UNC-51-like kinase 1 (ULK1) activation under nutrient deprivation. Specifically, ULK1 directly interacts with LDHA, phosphorylates serine-196 when nutrients are scarce and promotes lactate production. Lactate connects autophagy and glycolysis through Vps34 lactylation (at lysine-356 and lysine-781), which is mediated by the acyltransferase KAT5/TIP60. Vps34 lactylation enhances the association of Vps34 with Beclin1, Atg14L, and UVRAG, and then increases Vps34 lipid kinase activity. Vps34 lactylation promotes autophagic flux and endolysosomal trafficking. Vps34 lactylation in skeletal muscle during intense exercise maintains muscle cell homeostasis and correlates with cancer progress by inducing cell autophagy. Together, our findings describe autophagy regulation mechanism and then integrate cell autophagy and glycolysis.

Exosomal lncRNA GAS5 promotes M1 macrophage polarization in allergic rhinitis via restraining mTORC1/ULK1/ATG13-mediated autophagy and subsequently activating NF-кB signaling.

Macrophages are involved in the pathogenesis of allergic rhinitis (AR), but how these macrophages are polarized to M1 or M2 type is undetermined. Long non-coding RNA growth arrest specific transcript 5 (GAS5) is upregulated in exosomes isolated from nasal mucus of AR patients (AR-EXO) and aggravates nasal symptoms in AR mice. In the present study, we are aimed to elucidate the potential role of GAS5 in macrophage polarization during AR pathogenesis. An AR mice model was constructed. The potential function of GAS5 was evaluated by western blot, RNA immunoprecipitation (RIP), biotinylated RNA pull-down assay, co-immunoprecipitation (co-IP) assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA) assay, and immunohistochemistry (IHC) staining. We found that GAS5 is upregulated in ovalbumin-treated human nasal epithelial cells RPMI 2650 (OVA-EXO) and nasal mucus of AR mice. OVA-EXO treatment or forced GAS5 expression promoted M1 macrophage polarization of peripheral blood monocytes (PB monocytes) and THP-1 macrophages in vitro. GAS5 overexpression aggravated the allergic nasal symptoms induced by OVA in AR mice and facilitated M1 macrophage polarization and allergic inflammation, while knockdown of GAS5 exhibited opposite effects in vivo. GAS5 activated NF-кB signaling via suppressing autophagy-dependent degradation of IKKα/ß in macrophages. Furthermore, GAS5 acted as a scaffold to strengthen the interaction between mTORC1 and ULK1, thus impaired ULK1/ATG13-mediated autophagy via increasing mTORC1 activity. Finally, restored autophagy by ATG13 overexpression suppressed the effect of GAS5 on M1 macrophage polarization. In conclusion, these results suggested that exosomal transfer of GAS5 promoted M1 macrophage polarization via restraining mTORC1/ULK1/ATG13-mediated autophagy and subsequently activating NF-кB signaling in allergic rhinitis.

Loss of miR-144/451 alleviates ß-thalassemia by stimulating ULK1-mediated autophagy of free α-globin.

Most cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder ß-thalassemia, mutations in the ß-globin gene (HBB) lead to a reduction in the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by Unc-51-like autophagy activating kinase 1 (ULK1)-dependent autophagy and that stimulating this pathway by systemic mammalian target of rapamycin complex 1 (mTORC1) inhibition alleviates ß-thalassemia pathologies. We show here that disrupting the bicistronic microRNA gene miR-144/451 alleviates ß-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through 2 mechanisms. Loss of miR-451 upregulated its target messenger RNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor adenosine monophosphate-activated protein kinase (AMPK). The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. In addition, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1, causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates, and improve hematological indices in ß-thalassemia. The beneficial effects of miR-144/451 loss in ß-thalassemia were inhibited by the disruption of Cab39 or Ulk1 genes. Together, our findings link the severity of ß-thalassemia to a highly expressed erythroid microRNA locus and a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.

mTOR/ULK1 signaling axis mediated ultrashort wave regulation of autophagy to alleviate diabetic kidney disease.

BACKGROUND: Diabetic kidney disease (DKD) is closely related to autophagy and inflammation. The mTOR/unc-51 like autophagy activating kinase 1 (ULK1) signaling axis is involved in the regulation of autophagy. Ultrashort wave (USW) therapy has been extensively studied in inflammatory diseases. However, the therapeutic effect of USW on DKD and the role of the mTOR/ULK1 signaling axis in USW interventions remain uncertain. OBJECTIVES: This study aimed to explore the therapeutic effects of USW on DKD rats and the role of the mTOR/ULK1 signaling axis in USW interventions. MATERIAL AND METHODS: A DKD rat model was established using a high-fat diet (HFD)/sugar diet and streptozocin (STZ) induction. The optimal duration of USW intervention was determined using different USW treatments. The levels of metabolism, inflammation and fibrosis associated with kidney injury in rats were measured. Western blot analysis was performed on the related indexes of autophagy and the mTOR/ULK1 signaling axis. RESULTS: In DKD rats, microalbuminuria (MAU), glucose (GLU), creatinine (CRE), and blood urea nitrogen (BUN) levels decreased after the USW intervention. Levels of interleukin (IL)-1ß, inducible nitric oxide synthase (iNOS), immunoglobulin M (IgM), immunoglobulin G (IgG), IL-18, tumor necrosis factor alpha (TNF-α), and IL-6 decreased in the USW group compared to the model group. The IL-10 and arginase (Arg-1) levels were increased in the USW group. The content of fibrosis-related indexes (vascular endothelial growth factor (VEGF), fibronectin (FN), type IV collagen, and type I collagen) decreased in the urine of the DKD rats. After USW treatment, LC3B and Beclin1 levels increased, while the level of p62 decreased. The levels of nephrin, podocin and synaptopodin increased. Ultrashort wave could reduce p-mTOR/mTOR ratios and increase ULK1 expression. After the overexpression of ULK1, the levels of LC3B and Beclin1 were higher in the overexpression (oe)-ULK1 group than in the oe-negative control (NC) group, while the level of p62 decreased. After mTOR activation, LC3B and ULK1 expression decreased, while CRE, BUN, MAU, and GLU levels increased. CONCLUSIONS: Ultrashort wave alleviated kidney injury induced by the HFD/sugar diet and STZ. The USW intervention reversed the decreased autophagy levels in the DKD rats. The mTOR/ULK1 signaling axis mediated USW to promote autophagy.

Oscillation of Autophagy Induction under Cellular Stress and What Lies behind It, a Systems Biology Study.

One of the main inducers of autophagy-dependent self-cannibalism, called ULK1, is tightly regulated by the two sensor molecules of nutrient conditions and energy status, known as mTOR and AMPK kinases, respectively. Recently, we developed a freely available mathematical model to explore the oscillatory characteristic of the AMPK-mTOR-ULK1 regulatory triangle. Here, we introduce a systems biology analysis to explain in detail the dynamical features of the essential negative and double-negative feedback loops and also the periodic repeat of autophagy induction upon cellular stress. We propose an additional regulatory molecule in the autophagy control network that delays some of AMPK's effect on the system, making the model output more consistent with experimental results. Furthermore, a network analysis on AutophagyNet was carried out to identify which proteins could be the proposed regulatory components in the system. These regulatory proteins should satisfy the following rules: (1) they are induced by AMPK; (2) they promote ULK1; (3) they down-regulate mTOR upon cellular stress. We have found 16 such regulatory components that have been experimentally proven to satisfy at least two of the given rules. Identifying such critical regulators of autophagy induction could support anti-cancer- and ageing-related therapeutic efforts.