The Protective Role of TLR2 Mediates Impaired Autophagic Flux by Activating the mTOR Pathway During Neospora caninum Infection in Mice

Autophagy has been shown to play an essential role in defending against intracellular bacteria, viruses, and parasites. Mounting evidence suggests that autophagy plays different roles in the infection process of different pathogens. Until now, there has been no conclusive evidence regarding whether host autophagy is involved in Neospora caninum infection. In the current study, we first monitored the activation of autophagy by N. caninum, which occurred mainly in the early stages of infection, and examined the role of host autophagy in N. caninum infection. Here, we presented evidence that N. caninum induced an increase in autophagic vesicles with double-membrane structures in macrophages at the early stage of infection. LC3-II expression peaked and decreased as infection continued. However, the expression of P62/SQSTM1 showed significant accumulation within 12 h of infection, indicating that autophagic flux was blocked. A tandem fluorescence protein mCherry-GFP-LC3 construct was used to corroborate the impaired autophagic flux. Subsequently, we found that N. caninum infection induced the activation of the TLR2–AKT–mTOR pathways. Further investigation revealed that TLR2–mTOR, accompanied by the blockade of autophagic flux, was responsible for impaired autophagy but was not associated with AKT. In vitro and in vivo, N. caninum replication was strongly blocked by the kinase inhibitor 3-methyladenine (3-MA, autophagy inhibitor). In contrast, rapamycin (Rapa, an autophagy inducer) was able to promote intracellular proliferation and reduce the survival rate of N. caninum-infected mice. On the other hand, the accumulation of autophagosomes facilitated the proliferation of N. caninum. Collectively, our findings suggest that activation of host autophagy facilitates N. caninum replication and may counteract the innate immune response of the host. In short, inhibition of the early stages of autophagy could potentially be a strategy for neosporosis control.

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CMTM7 as a novel molecule of ATG14L-Beclin1-VPS34 complex enhances autophagy by Rab5 to regulate tumorigenicity

Cell Communication and Signaling ◽

10.1186/s12964-021-00720-3 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Baocai Liu ◽

Yinliang Lu ◽

Tingting Zhang ◽

Xinyue Yu ◽

Qian Wang ◽

...

Keyword(s):

Early Stage ◽

Xenograft Model ◽

Vital Role ◽

Autophagic Flux ◽

In Vivo Experiments ◽

Transmission Electron ◽

Autophagy Induction

Abstract Background CMTM7 is a tumor suppressor that positively regulates EGFR degradation by promoting Rab5 activation, and plays a vital role in tumor progression. Rab5 forms complexes with Beclin1 and VPS34, and acts in the early stage of autophagy. However, the affects of CMTM7 on autophagy and its mechanism are still unclear. Methods The effect of CMTM7 on autophagy induction was confirmed by western blotting, confocal microscopy and transmission electron microscopy. Co-immunoprecipitation was used to analyse the interaction of CMTM7 with autophagy initiation complex and Rab5. The xenograft model in nude mice was used to elucidate the function of CMTM7 in tumorigenicity and autophagy in vivo. Results In this study, we first demonstrated that CMTM7 facilitated the initiation of autophagosome formation, which consequently promoted the subsequent multistage process of autophagic flux, i.e. from autophagosome assembly till autolysosome formation and degradation. Confocal and co-immunoprecipitation showed that CMTM7 interacted with Rab5, VPS34, Beclin1, and ATG14L, but not with ULK1, UVRAG and LC3B. CMTM7 also increased the activity of ATG14L-linked VPS34 complex and its association with Rab5. Both in vitro and in vivo experiments demonstrated that knockdown of CMTM7 enhanced tumor growth by impairing autophagy. Conclusion These findings highlighted the role of CMTM7 in the regulation of autophagy and tumorigenicity, revealing it as a novel molecule that is associated with the interaction of Rab5 and ATG14L-Beclin1-VPS34 complex.

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CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage

Stroke ◽

10.1161/strokeaha.120.032749 ◽

2021 ◽

Author(s):

Yueyang Liu ◽

Xiaohang Che ◽

Haotian Zhang ◽

Xiaoxiao Fu ◽

Yang Yao ◽

...

Keyword(s):

Cerebral Ischemia ◽

Kinase Inhibitor ◽

Neuronal Damage ◽

Neuronal Cell ◽

In Vivo Model ◽

Autophagic Flux ◽

Autophagosome Formation

Background and Purpose: CAPN1 (calpain1)—an intracellular Ca 2+ -regulated cysteine protease—can be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined. Methods: In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucose–deprived primary neuron in vitro model. Results: CAPN1 was activated in the cortex of permanent middle cerebral occlusion–operated rats and oxygen-glucose deprivation–exposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage. Conclusions: The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemia–mediated autophagy-lysosomal pathway defects and neuronal damage.

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Enhanced phosphorylation of Na+–Cl− co-transporter in experimental metabolic syndrome: role of insulin

Clinical Science ◽

10.1042/cs20120003 ◽

2012 ◽

Vol 123 (11) ◽

pp. 635-647 ◽

Cited By ~ 38

Author(s):

Radko Komers ◽

Shaunessy Rogers ◽

Terry T. Oyama ◽

Bei Xu ◽

Chao-Ling Yang ◽

...

Keyword(s):

Metabolic Syndrome ◽

Time Course ◽

Kinase Inhibitor ◽

Endogenous Expression ◽

The Metabolic Syndrome ◽

Mammalian Cell Lines ◽

Phosphoinositide 3 Kinase

In the present study, we investigated the activity of the thiazide-sensitive NCC (Na+–Cl− co-transporter) in experimental metabolic syndrome and the role of insulin in NCC activation. Renal responses to the NCC inhibitor HCTZ (hydrochlorothiazide), as a measure of NCC activity in vivo, were studied in 12-week-old ZO (Zucker obese) rats, a model of the metabolic syndrome, and in ZL (Zucker lean) control animals, together with renal NCC expression and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were studied further in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinaemia, but no differences in plasma aldosterone, compared with ZL rats. In ZO rats, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL rats, and were associated with a decrease in BP (blood pressure). ZO rats displayed enhanced Thr53 NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK (Ste20-related proline/alanine-rich kinase) isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by a PI3K (phosphoinositide 3-kinase) inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinaemic rodents in conjunction with the SPAK expression profile consistent with NCC activation and reduced WNK4, as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinaemia is an important driving force of NCC activity in the metabolic syndrome with possible consequences for BP regulation.

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Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens

10.1101/556324 ◽

2019 ◽

Author(s):

Sara Pedron ◽

Gabrielle L. Wolter ◽

Jee-Wei E. Chen ◽

Sarah E. Laken ◽

Jann N. Sarkaria ◽

...

Keyword(s):

Extracellular Matrix ◽

Hyaluronic Acid ◽

Tumor Microenvironment ◽

Kinase Inhibitor ◽

Egfr Mutations ◽

Primary Glioblastoma ◽

Stat3 Phosphorylation

AbstractTherapeutic options to treat primary glioblastoma (GBM) tumors are scarce. GBM tumors with epidermal growth factor receptor (EGFR) mutations, in particular a constitutively active EGFRvIII mutant, have extremely poor clinical outcomes. GBM tumors with concurrent EGFR amplification and active phosphatase and tensin homolog (PTEN) are sensitive to the tyrosine kinase inhibitor erlotinib, but the effect is not durable. A persistent challenge to improved treatment is the poorly understood role of cellular, metabolic, and biophysical signals from the GBM tumor microenvironment on therapeutic efficacy and acquired resistance. The intractable nature of studying GBM cell in vivo motivates tissue engineering approaches to replicate aspects of the complex GBM tumor microenvironment. Here, we profile the effect of erlotinib on two patient-derived GBM specimens: EGFR+ GBM12 and EGFRvIII GBM6. We use a three-dimensional gelatin hydrogel to present brain-mimetic hyaluronic acid (HA) and evaluate the coordinated influence of extracellular matrix signals and EGFR mutation status on GBM cell migration, survival and proliferation, as well as signaling pathway activation in response to cyclic erlotinib exposure. Comparable to results observed in vivo for xenograft tumors, erlotinib exposure is not cytotoxic for GBM6 EGFRvIII specimens. We also identify a role of extracellular HA (via CD44) in altering the effect of erlotinib in GBM EGFR+ cells by modifying STAT3 phosphorylation status. Taken together, we report an in vitro tissue engineered platform to monitor signaling associated with poor response to targeted inhibitors in GBM.

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New insights on microRNAs in diabetic nephropathy: potential biomarkers for diagnosis and therapeutic targets

Diabetes Mellitus ◽

10.14341/dm8237 ◽

2017 ◽

Vol 20 (1) ◽

pp. 42-50 ◽

Cited By ~ 2

Author(s):

Elena Sergeevna Kamyshova ◽

Irina Nikolaevna Bobkova ◽

Irina Mikhailovna Kutyrina

Keyword(s):

Diabetic Nephropathy ◽

Early Detection ◽

Early Stage ◽

Biological Fluids ◽

Renal Tissue ◽

In Vivo Models ◽

New Biomarkers

Diabetic nephropathy (DN) is a severe complication of diabetes mellitus associated with the progressive deterioration of renal function. Although microalbuminuria is considered as a gold standard for DN diagnosis, it has limited predictive powers and specificity as a diagnostic tool for the early stage of DN. Therefore, new biomarkers are required for the early detection of DN. Studies using in vitro and in vivo models of DN have revealed an important role of microRNAs (miRNAs), short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression, in DN development. Recent studies have shown that the dysregulation of miRNAs, which is associated with the key features of DN, such as the mesangial expansion and accumulation of extracellular matrix proteins, is related to fibrosis and glomerular dysfunction. Thus, the up- and downregulation of miRNA expression in the renal tissue or biological fluids, including urine, may represent new biomarkers for the diagnosis and monitoring of DN progression. In this review, we highlight the significance of miRNAs as biomarkers for the early detection of DN and emphasise their potential role as a therapeutic target.

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The p21 dependent G2 arrest of the cell cycle in epithelial tubular cells links to the early stage of renal fibrosis

10.1101/560078 ◽

2019 ◽

Author(s):

Takayuki Koyano ◽

Masumi Namba ◽

Tomoe Kobayashi ◽

Kyomi Nakakuni ◽

Daisuke Nakano ◽

...

Keyword(s):

Cell Cycle ◽

Renal Fibrosis ◽

Kinase Inhibitor ◽

Early Stage ◽

G2 Arrest ◽

Tubular Cells ◽

Initial Stage ◽

Deficient Mice

AbstractRenal fibrosis is accompanied with the progression of chronic kidney disease (CKD). Despite a number of past and ongoing studies, our understanding of the underlying mechanisms remains elusive. Here we explored the progression of renal fibrosis by using a mouse model, unilateral ureter obstruction (UUO). We found that in the initial stage of the progression where extracellular matrix did not deposit yet, the proximal tubular cells arrested at the G2 of the cell cycle. This G2 arrest was induced prior to activation of both DNA damage checkpoint and Wnt/β-Catenin pathway. Further analyses in vivo and in vitro indicated the cyclin dependent kinase inhibitor p21 is involved in the G2 arrest after the damage. The newly produced monoclonal antibody against p21 revealed that the p21 levels were sharply upregulated in response to the damage during the initial stage, but dropped down toward the later stage. To examine the function of p21 in the progression of renal fibrosis, we constructed the novel p21 deficient mice by i-GONAD. Compared with wild-type mice, p21 deficient mice showed the exacerbation of the fibrosis. Thus we propose that during the initial stage of the fibrosis following the renal damage, tubular cells arrest in the G2 phase depending on p21, thereby safeguarding the kidney functions.

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Interleukin-37b inhibits the growth of murine endometriosis-like lesions by regulating proliferation, invasion, angiogenesis and inflammation

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa014 ◽

2020 ◽

Vol 26 (4) ◽

pp. 240-255

Author(s):

Yongpei He ◽

Ting Xiong ◽

Fang Guo ◽

Zhenzhen Du ◽

Yixian Fan ◽

...

Keyword(s):

Early Stage ◽

Inflammatory Factors ◽

Epithelial Cell Line ◽

Vascular Endothelial ◽

Gynecological Disease ◽

Endothelial Growth Factor ◽

In Vitro Treatment

Abstract Endometriosis is a gynecological disease with abnormal expression of interleukin (IL)-37 which can suppress inflammation and the immune system. Here we investigated the role of the IL-37b splice variant in endometriosis in vivo and in vitro. In a murine model of endometriosis, in vivo administration of IL-37b significantly inhibited the development of lesions judged by the number (P = 0.0213), size (P = 0.0130) and weight (P = 0.0152) of lesions. IL-37b had no effect on the early stage of lesion formation, however administration in the growth stage of lesions decreased the number (P = 0.0158), size (P = 0.0158) and weight (P = 0.0258) of lesions compared with PBS control, an effect that was not reversed by macrophage depletion. Expressions of inflammatory factors, matrix metalloproteinases and vascular endothelial growth factor-A mRNA/protein were significantly inhibited in ectopic lesions following IL-37b administration, and in uterine segments treated in vitro. In vitro treatment of uterine segments with IL-37b inhibited phosphorylation of Akt and Erk1/2 in uterine segments. Isolated mouse endometrial stromal treated with IL-37b and transfected with pIL-37b plasmid got suppressed cell proliferation, invasion, angiogenesis and the expression of inflammatory factors. In addition, transfection with pIL-37b significantly decreased the phosphorylation of Akt and Erk1/2. IL-37b also inhibited proliferation and the expression of inflammatory and angiogenesis factors in epithelial cell line RL95–2. These findings suggest that IL-37b may inhibit the growth of lesions by regulating proliferation, invasion, angiogenesis and inflammation through Akt and Erk1/2 signaling pathway.

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Trehalose Alleviates Crystalline Silica-Induced Pulmonary Fibrosis via Activation of the TFEB-Mediated Autophagy-Lysosomal System in Alveolar Macrophages

Cells ◽

10.3390/cells9010122 ◽

2020 ◽

Vol 9 (1) ◽

pp. 122 ◽

Cited By ~ 1

Author(s):

Xiu He ◽

Shi Chen ◽

Chao Li ◽

Jiaqi Ban ◽

Yungeng Wei ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Alveolar Macrophages ◽

Nuclear Translocation ◽

Protective Role ◽

Crystalline Silica ◽

Autophagic Flux ◽

Lysosomal System

Silicosis is an occupational lung disease characterized by persistent inflammation and irreversible fibrosis. Crystalline silica (CS) particles are mainly phagocytized by alveolar macrophages (AMs), which trigger apoptosis, inflammation, and pulmonary fibrosis. Previously, we found that autophagy-lysosomal system dysfunction in AMs was involved in CS-induced inflammation and fibrosis. Induction of autophagy and lysosomal biogenesis by transcription factor EB (TFEB) nuclear translocation can rescue fibrotic diseases. However, the role of TFEB in silicosis is unknown. In this study, we found that CS induced TFEB nuclear localization and increased TFEB expression in macrophages both in vivo and in vitro. However, TFEB overexpression or treatment with the TFEB activator trehalose (Tre) alleviated lysosomal dysfunction and enhanced autophagic flux. It also reduced apoptosis, inflammatory cytokine levels, and fibrosis. Both pharmacologically inhibition of autophagy and TFEB knockdown in macrophages significantly abolished the antiapoptotic and anti-inflammatory effects elicited by either TFEB overexpression or Tre treatment. In conclusion, these results uncover a protective role of TFEB-mediated autophagy in silicosis. Our study suggests that restoration of autophagy-lysosomal function by Tre-induced TFEB activation may be a novel strategy for the treatment of silicosis.

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Study on the Role of Cytc in Response to BmNPV Infection in Silkworm, Bombyx mori (Lepidoptera)

International Journal of Molecular Sciences ◽

10.3390/ijms20184325 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4325 ◽

Cited By ~ 3

Author(s):

Xue-Yang Wang ◽

Kang-Hui Wu ◽

Hui-Lin Pang ◽

Ping-Zhen Xu ◽

Mu-Wang Li ◽

...

Keyword(s):

Bombyx Mori ◽

Early Stage ◽

Malpighian Tubule ◽

Infection Process ◽

Insect Vector ◽

Apoptosis Pathway ◽

Expression Levels ◽

Mitochondrial Apoptosis Pathway

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens of the silkworm. Cytochrome c (cytc) showed a significant response to BmNPV infection in our previous transcriptome study. However, little is known about the role of Bombyx mori cytc (Bmcytc) in resistance to BmNPV infection. In this study, the expression levels analysis of Bmcytc showed stable expression levels in selected tissues of the resistant strain AN following BmNPV infection, while there was downregulation in the susceptible strain p50, except in the malpighian tubule. To further study the role of Bmcytc in viral infection, Bmcytc was knocked down with siRNA in vitro, resulting in significant downregulation of selected downstream genes of the mitochondrial pathway, including Bmapaf, Bmcaspase-Nc, and Bmcaspase-1; this was also confirmed by overexpression of Bmcytc using the pIZT/V5-His-mCherry insect vector, except Bmcaspase-1. Moreover, knockdown of Bmcytc significantly promoted the infection process of BmNPV in vitro, while the infection was inhibited by overexpression of Bmcytc at the early stage and subsequently increased rapidly. Based on these results, we concluded that Bmcytc plays a vital role in BmNPV infection by regulating the mitochondrial apoptosis pathway. Our work provides valuable data for the clarification of the mechanism of silkworm resistance to BmNPV infection.

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