Inhibition Of Autophagic Degradation Research Articles

Effect of Rapamycin on Endothelial Function in Systemic Lupus Erythematosus (SLE)

Mitophagy is regulated by multiple molecular pathways, including the mechanistic target of rapamycin (mTOR), which inhibits mitophagy. Elevated activation of mTOR complex 1 (mTORC1) is reported in humans and mice with SLE, suggesting that impaired mitophagy may contribute to the pathogenesis of SLE. As previously studied in genetically modified mice, mTORC1 also is proposed to be a critical regulator of endothelial function. However, it is unclear if pathologically elevated mTORC1, as in SLE, causes impaired mitophagy and endothelial dysfunction. Therefore, the study aimed to determine the effect of inhibition of mTORC1 on mitophagy, endothelial function, and inflammatory responses in SLE. We hypothesize that inhibiting mTORC1 with rapamycin will increase mitophagy and attenuate endothelial dysfunction and inflammatory responses in SLE. Nine-week-old female lupus-prone (MRL/lpr) and healthy control (MRL/MpJ) mice were randomly assigned into one of two groups, rapamycin treatment (lpr_Rapa and MpJ_Rapa) or control (lpr_Con and MpJ_Con). Rapamycin was injected i.p. three days a week for 8 weeks. After 8 weeks of treatment, endothelium-dependent vasorelaxation to acetylcholine (Ach) and endothelium-independent vasorelaxation to sodium nitroprusside (SNP) were measured in thoracic aortas using a wire myograph. Maximal responses to Ach were significantly impaired in lpr_Con (51.7 ± 6.6%, n = 11) compared to MpJ_Con (86.7 ± 3.6%, n = 11) (p < 0.0001). Rapamycin prevented endothelial dysfunction in the thoracic aorta from lupus mice (lpr_Rapa) (79.6 ± 4.2%, n = 10) compared to lpr_Con (p = 0.002). Maximal responses to SNP were not significantly different across the groups. Thoracic aortas were used to measure the content of mitophagy, inflammation, and endothelial function related proteins (n = 4-6/group). We assessed markers downstream of mTORC1 to confirm the inhibition of the mTORC1 activity. As expected, inhibition of mTORC1 activity decreased the phosphorylation of s6rp (p-s6rp/s6rp) in lpr_Rapa (0.39 ± 0.07) compared to lpr_Con (1.47 ± 0.16) (p < 0.0001). Phosphorylation of endothelial nitric oxide synthase (p-eNOS/eNOS) was 42% lower in lpr_Con (0.69 ± 0.14) than MpJ_Con (1.00 ± 0.12). Phosphorylated eNOS was 46% higher in lpr_Rapa (1.28 ± 0.15) than lpr_Con. Vascular cell adhesion protein 1 (Vcam 1), is an indicator of inflammation and is associated with atherosclerosis susceptibility. Protein content of Vcam 1 was significantly higher in lpr_Con (1.36 ± 0.06) compared to MpJ_Con (1.00 ± 0.01) (p = 0.001). The protein content of LC3II was measured as a marker of mitophagy. Though it was not significant, LC3II level was 2-fold higher in lpr_Con (2.12 ± 1.13) compared to MpJ_Con (1.00 ± 0.17), which may imply the accumulation of autophagosome resulted from inhibition of autophagic degradation. Collectively, these results demonstrate the beneficial effects of inhibiting mTORC1 on endothelial function in SLE mice and suggest inflammation and altered mitophagy contributing to endothelial dysfunction in SLE. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Viruses and autophagy: bend, but don't break.

Autophagy is a constitutive cellular process of degradation required to maintain homeostasis and turn over spent organelles and aggregated proteins. For some viruses, the process can be antiviral, degrading viral proteins or virions themselves. For many other viruses, the induction of the autophagic process provides a benefit and promotes viral replication. In this Review, we survey the roles that the autophagic pathway plays in the replication of viruses. Most viruses that benefit from autophagic induction block autophagic degradation, which is a 'bend, but don't break' strategy initiating but limiting a potentially antiviral response. In almost all cases, it is other effects of the redirected autophagic machinery that benefit these viruses. This rapid mechanism to generate small double-membraned vesicles can be usurped to shape membranes for viral genome replication and virion maturation. However, data suggest that autophagic maintenance of cellular homeostasis is crucial for the initiation of infection, as viruses have evolved to replicate in normal, healthy cells. Inhibition of autophagic degradation is important once infection has initiated. Although true degradative autophagy is probably a negative for most viruses, initiating nondegradative autophagic membranes benefits a wide variety of viruses.

Swainsonine-induced vacuolar degeneration is regulated by mTOR-mediated autophagy in HT22 cells

The indolizidine alkaloid, swainsonine (SW), is the main toxic component of locoweed, which can cause locoism in animals with characteristic neurological dysfunction. Pathological manifestations at cellular level include extensive vacuolar degeneration. Studies have shown that SW can induces autophagy, but the role and mechanism of autophagy in SW-induced vacuolar degeneration is unclear. In this study, we analyzed the role of autophagy in SW-induced cell injury in mouse hippocampal neurons cell line (HT22) using western blotting, qRT-PCR, transmission electron microscopy and immunofluorescence microscopy. The results showed that the expressions of LC3-II, ATG5, Beclin1 and p62 proteins and their mRNAs in HT22 cells were induced by SW treatment. The SW treatment increased the number of autophagosomes with enhanced fluorescence intensity of monodansylcadaverine (MDC) and LC3-II in a time-dose dependent manner. The results of lysosome staining showed that SW could increase the number of lysosomes, increase the intraluminal pH. Transmission electron microscopy results indicate that SW induced autophagosomes, and Baf A1 could effectively alleviate SW-induced vacuolar degeneration. At the molecular level, SW treatment inhibited the expression of p-PI3K, p-AKT, p-ERK, p-AMPK, p-mTOR, p-p70S6K and p-4EBP1 and promoted the expression of p53. Our results collectively suggest, PI3K/AKT/mTOR, ERK/mTOR and p53/mTOR signaling pathways are involved in the regulation of SW-induced autophagy in HT22 cells, while the AMPK/mTOR signaling pathway is not involved in this regulation. Inhibition of autophagic degradation can effectively alleviate SW-induced vacuolar degeneration.

Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells

Tributyltin (TBT) is an environmental pollutant that remains in marine sediments and is toxic to mammals. For example, TBT elicits neurotoxic and immunosuppressive effects on rats. However, it is not entirely understood how TBT causes toxicity. Autophagy plays a pivotal role in protein quality control and eliminates aggregated proteins and damaged organelles. We previously reported that TBT dephosphorylates mammalian target of rapamycin (mTOR), which may be involved in enhancement of autophagosome synthesis, in primary cultures of cortical neurons. Autophagosomes can accumulate due to enhancement of autophagosome synthesis or inhibition of autophagic degradation, and we did not clarify whether TBT alters autophagic flux. Here, we investigated the mechanism by which TBT causes accumulation of autophagosomes in SH-SY5Y cells. TBT inhibited autophagy without affecting autophagosome–lysosome fusion before it caused cell death. TBT dramatically decreased the acidity of lysosomes without affecting lysosomal membrane integrity. TBT decreased the mature protein level of cathepsin B, and this may be related to the decrease in lysosomal acidity. These results suggest that TBT inhibits autophagic degradation by decreasing lysosomal acidity. Autophagy impairment may be involved in the mechanism underlying neuronal death and/or T-cell-dependent thymus atrophy induced by TBT.

Saikosaponin D inhibits autophagosome‑lysosome fusion and induces autophagy‑independent apoptosis in MDA‑MB‑231 breast cancer cells.

The present study aimed to explore the effect of Saikosaponin D (SSD) and its underlying mechanism on apoptosis and autophagy in human breast cancer MDA-MB-231 cells. MTT assay, flow cytometry, western blotting and confocal fluorescence microscopy detection were employed. SSD, a kind of triterpenoid saponins extracted from Radix bupleuri, has been demonstrated to have the effects of anti-inflammatory, antioxidative and anticancer effects and can regulate autophagy. The present study revealed that SSD induced apoptosis through the activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in human breast cancer MDA-MB-231 cells. The administration of SSD promoted the phosphorylation/activation of p38 MAPK in MDA-MB-231 cells, whereas pretreatment with SB203580, an effective p38 MAPK inhibitor, attenuated SSD-mediated apoptosis, the cleavage of PARP and the activation of caspase-3. In addition, SSD blocked autophagic degradation by inhibiting autolysosome formation, resulting in the accumulation of autophagosomes. Mechanistically, the results of the present study revealed that SSD inhibited the formation of autophagosomes by inhibiting autophagosome-lysosome fusion, rather than by damaging lysosome function. Furthermore, blocking autophagy degradation was not associated with SSD-mediated apoptosis. The genetic knockdown of autophagy-related protein 5 markedly reduced SSD-mediated LC3B-II accumulation; however, it did not affect the SSD-mediated phosphorylation/activation of p38, cleavage of PARP, activation of caspase-3 or apoptosis. In conclusion, the findings of the present study suggest that SSD may induce apoptosis and block autophagic degradation, which provides further evidence of the association between the inhibition of autophagic degradation and cell death.

Autophagic degradation of SQSTM1 inhibits ovarian cancer motility by decreasing DICER1 and AGO2 to induce MIRLET7A-3P

ABSTRACTThe relationship between macroautophagy/autophagy and miRNA in regulating cancer cell motility is not clearly delineated. Here, we found that induction of BECN1-dependent or -independent autophagy decreased ubiquitin-binding proteins SQSTM1/p62 and CALCOCO2/NDP52. Downregulation of SQSTM1 (but not CALCOCO2) led to a decrease of the miRNA-processing enzyme DICER1 and the miRNA effector AGO2. The autophagy-mediated reduction of levels of SQSTM1, DICER1 or AGO2 resulted in increased MIRLET7A-3P (but not MIRLET7A-5P or PRE-MIRLET7A miRNA) and suppressed ovarian cancer motility. The investigation of the MIRLET7A effects on cancer cell motility showed that synthetic MIRLET7A-3P (3 nM) inhibited, whereas MIRLET7A-5P (100 nM) increased cancer cell motility. Moreover, downregulation of MIRLET7A-3P with antisense of MIRLET7A-3P miRNA (MIRLET7A-3P inhibitor; 3 nM) reversed the nutrient depletion- and rVP1-mediated suppression of ovarian cancer cell motility. In addition, restoring SQSTM1, DICER1 and AGO2 with inhibition of autophagic degradation or overexpression of DICER1 and AGO2 reversed the autophagy-associated enhancement of MIRLET7A-3P and inhibition of motility. Examination of ovarian cancer tissue microarray further showed that the levels of SQSTM1, DICER1 and AGO2 in the tumor were higher than those in the non-tumor cells and negatively correlated with the levels of autophagy and MIRLET7A-3P. Our results demonstrated that induction of autophagy to decrease SQSTM1, DICER1 and AGO2 and increase MIRLET7A-3P is a potential therapeutic strategy for suppressing ovarian cancer cell motility.Abbreviations: ACTB: actin beta; AGO2: argonaute 2, RISC catalytic component; ATG: autophagy related; BCIP/NBT: 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium; BECN1: beclin 1, autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; DICER1: dicer 1, ribonuclease III; EBSS: Earle balanced salt solution; FBS: fetal bovine serum; HGF: hepatocyte growth factor; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MIRLET7A: microRNA LET-7A: MIR16: microRNA 16; MIR29C: microRNA 29C; miRNA: microRNA; MMP: matrix metallopeptidase; PRE-MIRNA: precursor microRNA; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RISC: RNA-induced silencing complex; rVP1: recombinant foot-and-mouth disease virus capsid protein VP1; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; WIPI: WD repeat domain, phosphoinositide interacting.

Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers.

Tight junction dysfunction plays a vital role in some chronic inflammatory diseases. Pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), act as important factors in intestinal epithelial tight junction dysfunction during inflammatory conditions. Autophagy has also been shown to be crucial in tight junction function and claudin-2 expression, but whether autophagy has an effect on the change of claudin-2 expression and tight junction function induced by TNF-α is still unknown. To answer this question, we examined the expression of claudin-2 protein, transepithelial electrical resistance (TER), and permeability of cell monolayers, autophagy flux change, and lysosomal pH after TNF-α with or without PP242 treatment. Our study showed that claudin-2 expression, intestinal permeability, microtubule-associated protein 1 light chain 3B II (LC3B-II) and sequestosome 1 (P62) expression largely increased while TER values decreased in TNF-α treated cell monolayers. Further research using 3-methyladenine (3-MA), bafilomycin A1, and ad-mCherry-GFP-LC3B adenovirus demonstrated that LC3B-II increase induced by TNF-α was attributed to the inhibition of autophagic degradation. Moreover, both qualitative and quantitative method confirmed the increase of lysosomal pH, and mammalian target of rapamycin (mTOR) inhibitor PP242 treatment relieved this elevation. Moreover, PP242 treatment also alleviated the change of autophagy flux, TER, and claudin-2 expression induced by TNF-α. Therefore, we conclude that increase of claudin-2 levels and intestinal epithelial tight junction dysfunction are partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers.

Role of subcellular calcium redistribution in regulating apoptosis and autophagy in cadmium-exposed primary rat proximal tubular cells

Ca2+ signaling plays a vital role in regulating apoptosis and autophagy. We previously proved that cytosolic Ca2+ overload is involved in cadmium (Cd)-induced apoptosis in rat proximal tubular (rPT) cells, but the source of elevated cytosolic Ca2+ concentration ([Ca2+]c) and the effect of potential subcellular Ca2+ redistribution on apoptosis and autophagy remain to be elucidated. Firstly, data showed that Cd-induced elevation of [Ca2+]c was primarily generated intracellularly. Moreover, elevations of [Ca2+]c and mitochondrial Ca2+ concentration ([Ca2+]mit) with depletion of endoplasmic reticulum (ER) Ca2+ levels ([Ca2+]ER) were revealed in Cd-treated rPT cells, but this subcellular Ca2+ redistribution was significantly suppressed by 2-Aminoethoxydiphenyl borate (2-APB). Elevated inositol 1,4,5-trisphosphate (IP3) levels with up-regulated IP3 receptor (IP3R) protein levels were shown in Cd-exposed cells, confirming that IP3R-mediated ER Ca2+ release results in the elevation of [Ca2+]c. Up-regulated sequestosome 1 (p62) protein levels and autophagic flux assay demonstrated that Cd impaired autophagic degradation, while N-acetylcysteine (NAC) markedly attenuated Cd-induced p62 and microtubule-associated protein 1 light chain 3-II (LC3-II) accumulation, implying that the inhibition of autophagic flux was due to oxidative stress. Furthermore, pharmacological modulation of [Ca2+]c with 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and 2-APB alleviated Cd-mediated apoptosis, inhibition of autophagic degradation and subsequent cytotoxicity, while thapsigargin (TG) had the opposite regulatory effect on them. In summary, cytosolic calcium overload originated from IP3R-mediated ER Ca2+ release has a negative impact on Cd nephrotoxicity through its promotion of apoptosis and inhibition of autophagic flux.

A role of autophagy in PTP4A3-driven cancer progression

Autophagy, a “self-eating” cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.

Abstract SY31-01: The role of autophagy in cancer progression and therapy

Abstract Autophagy is induced by metabolic stress and can promote cell survival by maintaining energy homeostasis and by damage mitigation. Autophagy is negatively regulated by activation of mammalian target of rapamycin (mTOR) that links nutrient and growth factor availability to cell growth and catabolism. mTOR inhibitors have shown efficacy in renal cell carcinoma (RCC) but the role of autophagy stimulation and potentially counterproductive promotion of tumor cell stress tolerance was not known. We have found that the mTOR inhibitor CCI-779 (temsirolimus) promoted autophagy-mediated stress tolerance and that inhibition of autophagic degradation with chloroquine enhanced kidney tumor cell death in vitro and in vivo. Thus, autophagy inhibition may be a useful strategy to enhance the anti-tumor activity of mTOR inhibitors in RCC. Although therapeutic inhibitors of the mTOR pathway have been demonstrated to slow progression of advanced RCC in patients, and were approved by the FDA as a standard therapy for RCC, this beneficial effect is only temporary. One possible mechanism of resistance may be due to autophagy as a survival response. Additionally, metabolic stress caused by insufficient angiogenesis and the resulting mTOR inhibition activate tumor cell survival by autophagy, and tumor cells in these hypoxic regions respond most poorly to treatment. We reveal a new therapeutic paradigm of coordinate mTOR and autophagy inhibition to block the survival advantage and enhance tumor cell death to maximize therapeutic benefit. Citation Format: Eileen P. White. The role of autophagy in cancer progression and therapy [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY31-01

Inhibition of protein synthesis separates autophagic sequestration from the delivery of lysosomal enzymes

To investigate the role of newly synthesized proteins during autophagic sequestration and degradation, the effects of protein synthesis inhibition on autophagic vacuole (AV) formation and degradation were analyzed. The inhibition of protein synthesis was found to separate autophagic sequestration from the delivery of lysosomal enzymes to (AVs). Pretreatment with cycloheximide for > or = 3 h caused a drastic inhibition of autophagy-induced degradation. Surprisingly, morphological analyses showed that the inhibition of protein synthesis for up to 12 h did not block the formation of nascent AVs; however, it did prevent their conversion into degradative AVs. Using immunoperoxidase cytochemistry with an antibody against cathepsin D and labeling of lysosomes with endocytosed colloidal gold, we found that the nascent AVs that formed during prolonged cycloheximide pretreatment had not received lysosomal markers. The inhibition of autophagic degradation and lysosomal enzyme delivery were rapidly reversed following the removal of cycloheximide. These results suggest that there is a fairly rapid turnover of protein(s) that are necessary for lysosomal fusion, but that the initial formation of AVs is independent of new protein synthesis for a long period of time.

Inhibited autophagic degradation during ACTH-stimulated growth of rat adrenal zona fasciculata.

Autophagic vacuoles (AVs) in adrenal parenchymal cells of the outer part of the zona fasciculata were investigated by means of electron microscopic morphometry in 40 adult male Sprague Dawley rats. Four test groups and four control groups received twice daily subcutaneous injections of ACTH (12.5 U/kg body weight) or physiological saline, respectively, for 3, 6, 9 and 12 days. At 12 days the total dry weight of the adrenals was increased in ACTH-treated animals from 17.2 to 71.0 mg (about fourfold) and the average volume of zona fasciculata cells from 1,500 to 5,100 microns3 (about threefold). The relatively larger increase in total mass reflects mitotic activity in the growing adrenal tissue. The calculated growth rate of total tissue expressed as the percentage increase in dry weight per day was 12% at day 3 and decreased to 4.6% at day 12 of ACTH stimulation. During the time interval investigated the cytoplasmic volume fraction of autophagic vacuoles was significantly reduced from 10 X 10(-5) in controls to 1 X 10(-5) in ACTH-treated animals (p less than 0.001). Assuming a half-life for AVs of 7.9 min (Papadopoulos and Pfeifer 1986), autophagic degradation of cytoplasm can be calculated as 1.3%/day in controls, but only 0.15%/day in ACTH-treated animals. Thus growth of 1.15%/day can be attributed to inhibition of autophagic degradation. Although this is only part of total growth, it is evident from the present results that anticatabolic growth induction is utilized nearly to its maximum in adrenocortical fasciculata cells under the influence of ACTH.