The Class III PI3K/Beclin-1 Autophagic Pathway Participates in the mmLDL-Induced Upregulation of ETA Receptor in Mouse Mesenteric Arteries

Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. The current study explored the effect of mmLDL on the endothelin type A (ETA) receptor in mouse mesenteric arteries in vivo, as well as the role of autophagy in this process. mmLDL was injected via the caudal vein, and the Class III PI3K autophagic pathway inhibitor 3-methyladenine (3-MA) was injected intraperitoneally. The animals were divided into physiological saline (NS), mmLDL, and mmLDL + 3-MA groups. The dose-effect curve of endothelin-1- (ET-1-) induced mesenteric artery contraction was measured using myography, while ETA receptor mRNA expression was detected using real-time polymerase chain reactions, and the protein levels of the ETA receptor, class III PI3K, Beclin-1, LC3 II/I, p62, NF-κB, and p-NF-κB were observed using Western blot analysis. mmLDL significantly strengthened ET-1-induced contraction (the Emax value increased from 184.87 ± 7.46% in the NS group to 319.91 ± 20.31% in the mmLDL group (P<0.001), and the pEC50 value increased from 8.05 ± 0.05 to 9.11 ± 0.09 (P<0.01). In addition to upregulating the protein levels of Class III PI3K, Beclin-1, and LC3 II/I and downregulating that of p62, mmLDL significantly increased the mRNA expression and protein level of the ETA receptor and increased the protein level of p-NF-κB. However, these effects were significantly inhibited by 3-MA. mmLDL activates autophagy via the Class III PI3K/Beclin-1 pathway and upregulates the ETA receptor via the downstream NF-κB pathway. Understanding the effect of mmLDL on the ETA receptor and the underlying mechanisms may provide a new idea for the prevention and treatment of cardiovascular diseases.

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Effects of chronic portal hypertension on small heat-shock proteins in mesenteric arteries

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00439.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. G616-G620 ◽

Cited By ~ 4

Author(s):

Xuesong Chen ◽

Hai-Ying Zhang ◽

Kristin Pavlish ◽

Joseph N. Benoit

Keyword(s):

Portal Hypertension ◽

Heat Shock ◽

Heat Shock Proteins ◽

Mrna Expression ◽

Protein Level ◽

Small Heat Shock Proteins ◽

Mesenteric Arteries ◽

Protein Levels ◽

Small Hsps ◽

Shock Proteins

Previous studies have shown that impaired vasoconstrictor function in chronic portal hypertension is mediated via cAMP-dependent events. Recent data have implicated two small heat-shock proteins (HSP), namely HSP20 and HSP27, in the regulation of vascular tone. Phosphorylation of HSP20 is associated with vasorelaxation, whereas phosphorylation of HSP27 is associated with vasoconstriction. We hypothesized that alterations in the expression and/or phosphorylation of small HSPs may play a role in impaired vasoconstriction in portal hypertension. A rat model of prehepatic chronic portal hypertension was used. Studies were conducted in small mesenteric arteries isolated from normal and portal hypertensive rats. Protein levels of HSP20 and HSP27 were detected by Western blot analysis. Protein phosphorylation was analyzed by isoelectric focusing. HSP20 mRNA expression was determined by RT-PCR. To examine the role of cAMP in the regulation of small HSP phosphorylation and expression, we treated both normal and portal hypertensive vessels with a PKA inhibitor Rp-cAMPS. We found both an increased HSP20 phosphorylation and a decreased HPS20 protein level in portal hypertension, both of which were restored to normal by PKA inhibition. However, PKA did not change HSP20 mRNA expression. We conclude that decreased HSP20 protein level is mediated by cAMP-dependent pathway and that impaired vasoconstrictor function in portal hypertension may be partially explained by decreased expression of HSP20. We also suggest that the phosphorylation of HSP20 by PKA may alter HSP20 turnover.

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Prodigiosin-Emerged PI3K/Beclin-1-Independent Pathway Elicits Autophagic Cell Death in Doxorubicin-Sensitive and -Resistant Lung Cancer

Journal of Clinical Medicine ◽

10.3390/jcm7100321 ◽

2018 ◽

Vol 7 (10) ◽

pp. 321 ◽

Cited By ~ 12

Author(s):

Wei-Jun Chiu ◽

Shian-Ren Lin ◽

Yu-Hsin Chen ◽

May-Jwan Tsai ◽

Max Leong ◽

...

Keyword(s):

Lung Cancer ◽

Cell Death ◽

Mammalian Target Of Rapamycin ◽

Underlying Mechanism ◽

Beclin 1 ◽

Class Iii ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

Anti Tumor Activity

Prodigiosin (PG) belongs to a family of prodiginines isolated from gram-negative bacteria. It is a water insoluble red pigment and a potent proapoptotic compound. This study elucidates the anti-tumor activity and underlying mechanism of PG in doxorubicin-sensitive (Dox-S) and doxorubicin-resistant (Dox-R) lung cancer cells. The cytotoxicity and cell death characteristics of PG in two cells were measured by MTT assay, cell cycle analysis, and apoptosis/autophagic marker analysis. Then, the potential mechanism of PG-induced cell death was evaluated through the phosphatidylinositol-4,5-bisphosphate 3-kinase-p85/Protein kinase B /mammalian target of rapamycin (PI3K-p85/Akt/mTOR) and Beclin-1/phosphatidylinositol-4,5-bisphosphate 3-kinase-Class III (Beclin-1/PI3K-Class III) signaling. Finally, in vivo efficacy was examined by intratracheal inoculation and treatment. There was similar cytotoxicity with PG in both Dox-S and Dox-R cells, where the half maximal inhibitory concentrations (IC50) were all in 10 μM. Based on a non-significant increase in the sub-G1 phase with an increase of microtubule-associated proteins 1A/1B light chain 3B-phosphatidylethanolamine conjugate (LC3-II), the cell death of both cells was categorized to achieve autophagy. Interestingly, an increase in cleaved-poly ADP ribose polymerase (cleaved-PARP) also showed the existence of an apoptosis-sensitive subpopulation. In both Dox-S and Dox-R cells, PI3K-p85/Akt/mTOR signaling pathways were reduced, which inhibited autophagy initiation. However, Beclin-1/PI3K-Class III downregulation implicated non-canonical autophagy pathways were involved in PG-induced autophagy. At completion of the PG regimen, tumors accumulated in the mice trachea and were attenuated by PG treatment, which indicated the efficacy of PG for both Dox-S and Dox-R lung cancer. All the above results concluded that PG is a potential chemotherapeutic agent for lung cancer regimens regardless of doxorubicin resistance.

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Effect of dietary protein level on in vivo and in vitro vitamin A esterase activity in the chick

British Journal Of Nutrition ◽

10.1079/bjn19670060 ◽

1967 ◽

Vol 21 (3) ◽

pp. 565-581 ◽

Cited By ~ 4

Author(s):

I. Nir ◽

I. Bruckental ◽

I. Ascarelli ◽

A. Bondi

Keyword(s):

Vitamin A ◽

Oral Dose ◽

Dietary Protein ◽

Protein Level ◽

Blood Stream ◽

Duodenal Mucosa ◽

Protein Levels ◽

Temporary Decrease

1. The efficiency of absorption of and liver storage from a single oral dose of 10000 i.u. vitamin A palmitate decreased in chicks reared on a diet containing 10% protein as compared to the efficiency in chicks reared on a diet in which the protein level was adequate. When the chicks were given orally an equivalent dose of vitamin A alcohol, the absorption was equally efficient at both dietary protein levels.2. The vitamin A alcohol content of this intestine, plasma and liver of chicks dosed with vitamin A palmitate was decreased by protein restriction. The physiological change responsible for this decrease seems to be the lowering of the hydrolysing activity for vitamin A palmitate in pancreas and in the duodenal mucosa.3. The importance of the enzymic step in the absorption of an oral dose of vitamin A palmitate is shown by the finding that protein malnutrition reduced only slightly the final liver stores when vitamin A in its different forms (palmitate, acetate or alcohol) was injected directly into the blood stream.4. The uptake of injected vitamin A from the blood was much delayed when the vitamin was injected as palmitate, i.e. the ester of a long-chain fatty acid, instead of the acetate ester of the free alcohol.5. When vitamin A was injected, the liver content did not rise continuously with time, but showed a temporary decrease after a certain period. The phenomenon was apparently due to changes in the rate of the two inverse processes of uptake of the vitamin by the liver and liberation from it.

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Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1

Biochemical Journal ◽

10.1042/bj20111424 ◽

2011 ◽

Vol 441 (1) ◽

pp. 399-406 ◽

Cited By ~ 90

Author(s):

Harald W. Platta ◽

Hilde Abrahamsen ◽

Sigrid B. Thoresen ◽

Harald Stenmark

Keyword(s):

Tumour Suppressor ◽

Beclin 1 ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Interacting Protein ◽

Polyubiquitin Chains ◽

Protein Levels ◽

Regulatory Link ◽

A Subunit ◽

The Stability

Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.

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Cryptotanshinone Regulates Androgen Synthesis through the ERK/c-Fos/CYP17 Pathway in Porcine Granulosa Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/5985703 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Danfeng Ye ◽

Meifang Li ◽

Yuehui Zhang ◽

Xinhua Wang ◽

Hua Liu ◽

...

Keyword(s):

Western Blot ◽

Mrna Expression ◽

Molecular Mechanism ◽

Granulosa Cells ◽

Protein Level ◽

Salvia Miltiorrhiza ◽

Androgen Excess ◽

Protein Levels ◽

Androgen Synthesis ◽

Porcine Granulosa Cells

The aim of the study is to investigate the molecular mechanism behind androgen reduction in porcine granulosa cells (pGCs) withSalvia miltiorrhizaBunge extract cryptotanshinone. PGCs were isolated from porcine ovaries and identified. Androgen excess model of the pGCs was induced with the MAPK inhibitor PD98059 and then treated with cryptotanshinone. The testosterone level was measured by radioimmunoassay in the culture media. The protein levels of P-ERK1/2, c-Fos, and CYP17 in the cells were measured by western blot. Cryptotanshinone decreased the concentration of testosterone and the protein level of CYP17 and increased the protein levels of P-ERK1/2 and c-Fos in the androgen excess mode. After the c-Fos gene was silenced by infection with c-Fos shRNA lentivirus, we measured the mRNA expression by quantitative RT-PCR and protein level by western blot of P-ERK1/2, c-Fos, and CYP17. This showed that the mRNA expression and protein level of P-ERK1/2 and c-Fos were significantly reduced in the shRNA–c-Fos group compared to the scrambled group, while those of CYP17 were significantly increased. So we concluded that cryptotanshinone can significantly reduce the androgen excess induced by PD98059 in pGCs. The possible molecular mechanism for this activity is regulating the ERK/c-Fos/CYP17 pathway.

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Suppression of TCAB1 Expression Induced Cellular Senescence by Lessening Proteasomal Degradation of p21 in Cancer Cells

10.21203/rs.3.rs-66104/v1 ◽

2020 ◽

Author(s):

Jing Niu ◽

Rui-Qi Gao ◽

Meng-Tian Cui ◽

Chen-Guang Zhang ◽

Shen-Tao Li ◽

...

Keyword(s):

Mrna Expression ◽

Cancer Cells ◽

Cellular Senescence ◽

Proteasomal Degradation ◽

Cell Senescence ◽

Rapid Progression ◽

Telomere Elongation ◽

Protein Levels ◽

Treatment Regimens

Abstract Background: TCAB1, a.k.a. WRAP53β or WDR79, is an important molecule for the maintenance of Cajal bodies and critically involved in telomere elongation and DNA repair. Upregulation of TCAB1 were discovered in a variety types of cancers. However, the function of TCAB1 in tumor cell senescence remains absent. Methods: The TCAB1 knockdown cell lines were constructed. The expression levels of TCAB1, p21, p16 and p53 were detected by qRT-PCR and western blotting. Staining of senescence-associated β-galactosidase was used to detect senescent cells. The ubiquitination of the p21 was analysed by immunoprecipitatation and in vivo ubiquitination assay. TCGA databases were employed to perform in silico analyses for the mRNA expression of TCAB1, p21, p16 and p53. Results: Here, we discovered that knockdown of TCAB1 induced rapid progression of cellular senescence in A549, H1299 and HeLa cells. In exploiting the mechanism underlining the role of TCAB1 on senescence, we found a significant increase of p21 at the protein levels upon TCAB1 depletion, whereas the p21 mRNA expression was not altered. We verified that TCAB1 knockdown was able to shunt p21 from proteasomal degradation by regulating the ubiquitination of p21. In rescue assays, it was demonstrated that decreasing the expression of p21 was able to attenuate the cellular senescence process induced by TCAB1 silencing. Conclusions: This study revealed the importance of TCAB1 for its biological functions in the regulation of cell senescence. Our results will be helpful to understand the mechanisms of senescence in cancer cells, which could provide clues for designing novel strategies for developing effective treatment regimens.

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Protective effects of piperine on the retina of mice with streptozotocin-induced diabetes by suppressing HIF-1/VEGFA pathway and promoting PEDF expression

International Journal of Ophthalmology ◽

10.18240/ijo.2021.05.04 ◽

2021 ◽

Vol 14 (5) ◽

pp. 656-665

Author(s):

Pu Zhang ◽

◽

Yao Tan ◽

Ling Gao ◽

◽

...

Keyword(s):

Protein Level ◽

Pigment Epithelium ◽

Hypoxia Inducible Factor ◽

Protective Effects ◽

Protein Levels ◽

Chemical Hypoxia ◽

Diabetic Retina ◽

Streptozotocin Induced Diabetes

AIM: To evaluate the protective mechanisms of piperine in the retina of mice with streptozotocin-induced diabetes. METHODS: In experiments in vitro, stimulation by chemical hypoxia was established in ARPE-19 cells. Then, the expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor A (VEGFA), and pigment epithelium-derived factor (PEDF) was assessed at the mRNA and protein levels. In experiments in vivo, diabetes mellitus was established by intraperitoneally injecting 150 mg/kg streptozotocin once. After 3wk of the onset of diabetes, 15 mg/kg piperine was intraperitoneally injected once daily for 1 or 3wk. Then, the retinal morphology and mRNA and protein expression were assessed. RESULTS: In hypoxia, 1-100 μmol/L piperine significantly decreased the expression of VEGFA mRNA and increased the expression of PEDF mRNA without affecting HIF-1α mRNA. Meanwhile, 100 μmol/L piperine substantially decreased the protein level of VEGFA and increased the protein level of PEDF. The HIF-1α protein level was also hampered by piperine. In the diabetic retina of mice, the morphological damage was alleviated by piperine. Likewise, the retinal vascular leakage was substantially decreased by piperine. Further, the protein levels of HIF-1α and VEGFA were significantly reduced by piperine. Moreover, the level of the antiangiogenic factor of PEDF dramatically increased by piperine. CONCLUSION: Piperine may exert protective effects on the retina of mice with diabetes via regulating the pro-antiangiogenic homeostasis composed of HIF-1/VEGFA and PEDF.

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SETD3 is regulated by a couple of microRNAs and plays opposing roles in proliferation and metastasis of hepatocellular carcinoma

Clinical Science ◽

10.1042/cs20190666 ◽

2019 ◽

Vol 133 (20) ◽

pp. 2085-2105 ◽

Cited By ~ 3

Author(s):

Liangliang Xu ◽

Peng Wang ◽

Xinfu Feng ◽

Jianwei Tang ◽

Lian Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Mrna Expression ◽

Protein Level ◽

Downstream Target ◽

In Vivo Experiments ◽

Pathological Differentiation ◽

Important Pathway ◽

Proliferation And Metastasis

Abstract A previous study reported that histone methyltransferase SETD3 is up-regulated in tumor tissues of hepatocellular carcinoma (HCC) and is associated with the growth of HCC. However, the clinical significance and the effect of SETD3 on HCC metastasis remain unclear. In the present study, both the protein and mRNA expression levels of SETD3 were measured in a larger cohort of HCC patients. The results showed that the protein level of SETD3 in HCC tissues was significantly higher than that in non-tumorous tissues, which was inconsistent with the mRNA expression level of SETD3. The high protein level of SETD3 in HCC tissues was significantly associated with male gender, poor pathological differentiation, liver cirrhosis and unfavorable prognosis of HCC patients. Subsequently, we demonstrated that SETD3 could be regulated at post-transcriptional step by a couple of miRNAs (miR-16, miR-195 and miR-497). Additionally, in vitro and in vivo experiments revealed that SETD3 played opposing roles in proliferation and metastasis of HCC: promoting proliferation but inhibiting metastasis. Mechanistic experiments revealed that doublecortin-like kinase 1 (DCLK1) was a downstream target of SETD3. SETD3 could increase the DNA methylation level of DCLK1 promoter to inhibit the transcription of DCLK1. Further study revealed that DCLK1/PI3K/matrix metalloproteinase (MMP) 2 (MMP-2) was an important pathway that mediated the effect of SETD3 on HCC metastasis. In conclusion, the present study revealed that SETD3 is associated with tumorigenesis and is a promising biomarker for predicting the prognosis of HCC patients after surgical resection. In addition, SETD3 plays inhibitory role in HCC metastasis partly through DCLK1/PI3K/MMP-2 pathway.

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Abstract TP268: Induction Profile of Mesencephalic Astrocyte-derived Neurotrophic Factor by Cerebral Ischemia and its Implication for Neuroprotection

Stroke ◽

10.1161/str.48.suppl_1.tp268 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Ludmila Belayev ◽

Sung-Ha Hong ◽

Pranab K Mukherjee ◽

Hemant Menghani ◽

Larissa Khoutorova ◽

...

Keyword(s):

Cerebral Ischemia ◽

Dentate Gyrus ◽

Mrna Expression ◽

Neurotrophic Factor ◽

Focal Cerebral Ischemia ◽

Artery Occlusion ◽

Ischemic Penumbra ◽

Protein Levels

Introduction: Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been identified as a secretion protein, which biases immune cells toward an anti-inflammatory phenotype, thereby promoting tissue repair after various injuries to neurons in vivo or in vitro. However, the function of MANF during and after brain ischemia is still not known. The purpose of our study was to examine the characteristics and implication of MANF expression induced by focal cerebral ischemia. In addition we investigated if docosahexaenoic acid (DHA) potentiates MANF mRNA expression and provides additional neuroprotection. Methods: Male SD (280-320) rats were anesthetized with isoflurane and subjected to 2 h of middle cerebral artery occlusion (MCAo) by intraluminal suture. DHA (5 mg/kg; n=13) or vehicle (saline; n=8) was administered IV at 3 h after the onset of MCAo. Neurological function was evaluated during occlusion (60 min) and on days 1, 3 and 7 after MCAo. MANF mRNA expression, protein levels, and apoptosis were measured by immunohistochemistry and Western blotting. Results: Behavioral deficit was significantly improved by treatment with DHA compared to vehicle on days 1, 3 and 7. MANF was found to be extremely upregulated in the ischemic penumbra. The expression of MANF was neuronal in the cortex and dentate gyrus. DHA administration significantly increased the number of MANF + /NeuN + cells in the cortex (by 76.6 %) and dentate gyrus (by 20.5 %) compared to saline-treated animals. The number of MANF/NeuN-positive cells was not different in the subcortex, CA1 and CA3 regions between DHA- and saline-treated groups. Treatment with DHA increased MANF + /GFAP + cells in the subcortex (by 27.7 %) and dentate gyrus (by 38.0 %) compared to the vehicle-treated brains. Total and cortical infarct volumes were attenuated by DHA treatment by 48 % and by 73 % compared to vehicle treatment at 24 h after MCAo. Conclusion: MANF mRNA expression and protein levels are increased after focal cerebral ischemia. It was found to be extremely upregulated in the ischemic penumbra and dentate gyrus. The expression of MANF was mostly neuronal and astrocytic. DHA potentiates MANF expression and provides additional neuroprotection.

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Production of VEGF receptor 1 and 2 mRNA and protein during endochondral bone repair is differential and healing phase specific

Journal of Applied Physiology ◽

10.1152/japplphysiol.00839.2010 ◽

2010 ◽

Vol 109 (6) ◽

pp. 1930-1938 ◽

Cited By ~ 6

Author(s):

Marie K. Reumann ◽

Turya Nair ◽

Olga Strachna ◽

Adele L. Boskey ◽

Philipp Mayer-Kuckuk

Keyword(s):

Mrna Expression ◽

Protein Level ◽

Bone Repair ◽

Callus Formation ◽

Detection Sensitivity ◽

Vegf Receptor ◽

Vegf Mrna ◽

Endochondral Bone ◽

Protein Levels ◽

Healing Phase

Physiological disturbances, including temporary hypoxia, are expected to drive angiogenesis during bone repair. Evidence suggests that the angiogenic ligand vascular endothelial growth factor (VEGF)-A plays an important role in this process. We characterized the expression of two receptors that are essential for mediating VEGF signaling, VEGFR1/Flt-1 and VEGFR2/Flk-1/KDR, in a mouse rib fracture model. Their mRNA and protein levels were assessed in four healing phases, which were characterized histologically as hemorrhage formation on postfracture day (PFD) 1, inflammatory response on PFD 3, initiation of callus development on PFD 7, and the presence of a mature callus on PFD 14. Transcript was detected for VEGFR1 and VEGFR2, as well as VEGF. While mRNA expression of VEGFR1 was monophasic throughout all healing phases, VEGFR2 showed a biphasic profile with significantly increased mRNA expression during callus formation and maturation. Expression of VEGF mRNA was characterized by a more gradual increase during callus formation. The protein level for VEGFR1 was below detection sensitivity during the initial healing phase. It was then restored to a stable level, detectable through the subsequent healing phases. Hence, the VEGFR1 protein levels partially mirrored the transcript expression profile. In comparison, the protein level of VEGFR2 increased gradually during the healing phases and peaked at callus maturation. This correlated well with the transcriptional expression of VEGFR2. Intact bone from age-matched male mice had considerable protein levels of VEGFR1 and VEGF, but no detectable VEGFR2. Together, these findings uncovered expression signatures of the VEGF-VEGFR axis in endochondral bone repair.

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