KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

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Abstract 182: CTRP9 - A Novel Cardiac Derived Secreted Factor With Anti-hypertrophic Properties

Circulation Research ◽

10.1161/res.113.suppl_1.a182 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Astrid H Breitbart ◽

Florian Brandes ◽

Oliver Müller ◽

Natali Froese ◽

Mortimer Korf-Klingebiel ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Real Time ◽

Real Time Pcr ◽

Weight Ratio ◽

Neonatal Rat ◽

Heart Weight ◽

Cardiomyocyte Hypertrophy ◽

Quantitative Real Time Pcr ◽

Rat Cardiomyocytes ◽

Knock Out

Background: CTRP9 (also called C1qtnf9) is a newly discovered secreted protein and a paralog of adiponectin. The biological functions of CTRP9, however, are still largely unknown. Results: Although previous data from a semi-quantitative real-time PCR had suggested that CTRP9 is mainly secreted by adipose tissue, we found its mRNA to be predominantly expressed in the heart by quantitative real-time PCR. Interestingly, we identified CTRP9 mRNA as significantly upregulated in hypertrophied mouse hearts (after 2 weeks of aortic constriction, TAC) as well as in hypertrophied human hearts (24±4-fold versus healthy human myocardium; p<0.01). LacZ staining in myocardial sections of C1qtnf9 tm1(KOMP)Vlcg mice (knock-out for CTRP9, containing a lacZ cassette to replace exon 1-3 of the gene) revealed exclusive expression of CTRP9 in capillary and venous endothelial cells. Adenoviral overexpression of CTRP9 or recombinant CTRP9 strongly inhibited cardiomyocyte hypertrophy (assessed as cell size, protein/DNA-ratio, expression of skeletal α-actin) after stimulation with phenylephrine (PE). Accordingly, myocardial overexpression of CTRP9 via a cardioselective adeno-associated virus (AAV9-CTRP9) in mice dramatically reduced cardiac hypertrophy after two weeks of pressure overload (heart weight/body weight ratio, HW/BW in mg/g: AAV9-control 6.5±0.2 versus AAV9-CTRP9 5.6±0.2; p<0.01). In turn, downregulation of CTRP9 by a specific siRNA aggravated cardiomyocyte growth in response to PE in vitro and CTRP9 knock-out (KO) mice exerted an enhanced hypertrophic response after two weeks of TAC in vivo (% increase in HW/BW versus sham: wild-type 77±13, KO 106±9; p<0.05). Mechanistically, we found that CTRP9 binds to the adiponectin receptor 1 (AdipoR1) and inhibits prohypertrophic mTOR signalling in cardiac myocytes. SiRNA mediated downregulation of AdipoR1 or mTOR in neonatal rat cardiomyocytes abolished the anti-hypertrophic effect of CTRP9. Conclusion: Endothelial cell derived CTRP9 inhibits cardiac hypertrophy through binding to AdipoR1 and inhibition of the mTOR pathway in cardiomyocytes. Therefore, myocardial application of CTRP9 could be a novel strategy to combat pathological cardiac hypertrophy.

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MicroRNA-1253 Suppresses Cell Proliferation Migration and Invasion of Osteosarcoma by Targeting MMP9

Technology in Cancer Research & Treatment ◽

10.1177/1533033821995278 ◽

2021 ◽

Vol 20 ◽

pp. 153303382199527

Author(s):

Jianwen Mo ◽

Tiansheng Zheng ◽

Li Lei ◽

Peng Dai ◽

Jun Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Target Genes ◽

Migration And Invasion ◽

Quantitative Real Time Pcr ◽

Osteoblast Cells ◽

Downstream Target ◽

Cell Functions

Purpose: MicroRNAs play an important role in osteosarcoma (OS) development and progress. Although miR-1253 was considered as a tumor-inhibitor in some cancers, it’s function in the OS is not clear. Methods: In our study, we examined the expression of miR-1253 in OS cells and osteoblast cells using quantitative real-time PCR. The proliferation of OS cells was measured by BrdU assay, and we performed transwell to detect migration and invasion of OS cells. Meanwhile, EMT proteins were tested by western blot. We used Bioinformatics to predict the target genes of miR-1253 and found out Matrix metalloproteinases9 (MMP9) was one of that. The direct combination between miR-1253 and MMP9 was verified by double luciferase reporting experiment. Quantitative real-time PCR and western blot were used to detect the expression of MMP9. Results: We found that the expression level of miR-1253 in OS cells was significantly lower than that in osteoblast cells. Overexpression of miR-1253 could significantly inhibit OS cell proliferation, migration, invasion and EMT. And then, MMP9 was predicted as a downstream target of miR-1253 by Bioinformatics analysis. Further experiments showed that miR-1253 could reduce the protein level of MMP9 by directly binding to the 3’-UTR of MMP9. Afterward, we performed a rescue experiment, in which both MMP9 and miR-1253 were overexpressed. Compared with the groups overexpressed miR-1253 alone, cell proliferation, migration and invasion in co-overexpression groups were improved. Conclusions: In summary, these results suggested that miR-1253 down-regulated in OS cells, and could suppress the proliferation, migration and invasion of OS cells. Its molecular regulatory mechanism was that inhibits the expression of the downstream target gene MMP9 by directly binding, thus affect OS cell functions. Therefore, miR-1253 has the potential to become a biomarker and therapeutic target for OS therapy.

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Biological evaluation of transdichloridoplatinum( II) complexes with 3- and 4-acetylpyridine in comparison to cisplatin

Radiology and Oncology ◽

10.2478/raon-2013-0050 ◽

2013 ◽

Vol 47 (4) ◽

pp. 346-357 ◽

Cited By ~ 4

Author(s):

Lana Filipovic ◽

Sandra Arandelovic ◽

Nevenka Gligorijevic ◽

Ana Krivokuca ◽

Radmila Jankovic ◽

...

Keyword(s):

Western Blot ◽

Mrna Expression ◽

Real Time ◽

Real Time Pcr ◽

Inductively Coupled Plasma ◽

Gelatin Zymography ◽

Biological Evaluation ◽

Emission Spectrometry ◽

Quantitative Real Time Pcr

Abstract Background. In our previous study we reported the synthesis and cytotoxicity of two trans-platinum(II) complexes: trans-[PtCl2(3-acetylpyridine)2] (1) and trans-[PtCl2(4-acetylpyridine)2] (2), revealing significant cytotoxic potential of 2. In order to evaluate the mechanism underlying biological activity of both trans-Pt(II) isomers, comparative studies versus cisplatin were performed in HeLa, MRC-5 and MS1 cells. Materials and methods. The cytotoxic activity of the investigated complexes was determined using SRB assay. The colagenolytic activity was determined using gelatin zymography, while the effect of platinum complexes on matrix metalloproteinases 2 and 9 mRNA expression was evaluated by quantitative real-time PCR. Apoptotic potential and cell cycle alterations were determined by FACS analyses. Western blot analysis was used to evaluate the effect on expression of DNA-repair enzyme ERCC1, and quantitative real-time PCR was used for the ERCC1 mRNA expression analysis. In vitro antiangiogenic potential was determined by tube formation assay. Platinum content in intracellular DNA and proteins was determined by inductively coupled plasma-optical emission spectrometry. Results. Compound 2 displayed an apparent cytoselective profile, and flow cytometry analysis in HeLa cells indicated that 2 exerted antiproliferative effect through apoptosis induction, while 1 induced both apoptosis and necrosis. Action of 1 and 2, as analyzed by quantitative real-time PCR and Western blot, was associated with down-regulation of ERCC1. Both trans-complexes inhibited MMP-9 mRNA expression in HeLa, while 2 significantly abrogated in vitro tubulogenesis in MS1 cells. Conclusions. The ability of 2 to induce multiple and selective in vitro cytotoxic effects encourages further investigations of trans-platinum(II) complexes with substituted pyridines.

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Gualou Guizhi Granule Protects against OGD/R-Induced Injury by Inhibiting Cell Pyroptosis via the PI3K/Akt Signaling Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/6613572 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Yuqin Zhang ◽

Hongyun Wang ◽

Huang Li ◽

Lihong Nan ◽

Wei Xu ◽

...

Keyword(s):

Ischemic Stroke ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Akt Signaling ◽

Akt Pathway ◽

Pcr Analysis ◽

Akt Signaling Pathway ◽

Quantitative Real Time Pcr ◽

Expression Levels

Pyroptosis is a proinflammatory form of regulated cell death that plays an important role in ischemic stroke. Gualou Guizhi granule (GLGZG) is a classic prescription that has been shown to exert neuroprotective effects against cerebral ischemia reperfusion injury. In the present study, we examined the involvement of pyroptosis and its associated mechanism in protecting nerve function. Methods. Primary neurons were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) conditions in the presence or absence of GLGZG. Cellular viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay. The number of apoptoic cells was detected by NeuN and NSE protein expression. The expression levels of the pyroptosis markers, namely, NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, interleukin-18 (IL-18), and IL-1β were determined by quantitative real-time PCR analysis, western blot, and ELISA analyses as appropriate. Moreover, the expression levels of the PI3K/Akt pathway key proteins were determined by quantitative real-time PCR analysis and western blot assays. To determine the PI3K/Akt pathway involvement in GLGZG-mediated neuroprotection, the PI3K inhibitor LY294002 (LY, 10 μM) was added. The expression levels of NeuN, Akt, and p-Akt were evaluated. Results. It was found that GLGZG could inhibit OGD/R-induced cell apoptosis, increase neuronal cell viability, decrease the production of IL-18 and IL-1β, and downregulate the expression levels of pyroptosis markers (NLRP3, ASC, and caspase-1). Furthermore, GLGZG could modulate the PI3K/Akt signaling pathway. Pharmacological inhibition of the PI3K pathway not only abrogated the effects of GLGZG on Akt but also neutralized its prosurvival and antipyroptotic actions. Conclusions. The findings indicated that GLGZG pretreatment effectively reduced OGD/R-induced injury by inhibiting cell pyroptosis and activating the PI3K/Akt pathway. These data provide important evidence for the therapeutic applications of this regimen in ischemic stroke.

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Έκφραση του σηματοδοτικού μονοπατιού Notch σε πλακούντες εγκύων με προεκλαμψία

10.12681/eadd/35915 ◽

2015 ◽

Author(s):

Περσεφόνη Φραγκιαδάκη

Keyword(s):

Western Blot ◽

Notch Signaling ◽

Real Time ◽

Signaling Pathway ◽

Real Time Pcr ◽

Target Genes ◽

Notch Signaling Pathway ◽

Quantitative Real Time Pcr ◽

Notch 1 ◽

Qrt Pcr

Η προεκλαμψία αποτελεί μια από τις κυριότερες αιτίες μητρικής και εμβρυικής/νεογνικής νοσηρότητας και θνητότητας και επιπλέκει το 3-5% των κυήσεων. Προηγούμενες μελέτες έδειξαν ότι μέλη του σηματοδοτικού μονοπατιού Notch (Notch signaling pathway) εκφράζονται στον πλακούντα και παίζουν σημαντικό ρόλο στην ομαλή ανάπτυξη του ενεργοποιώντας μεταγραφικούς παράγοντες οι οποίοι αλλάζουν την έκφραση γονιδίων στόχων. Διαταραχές στη λειτουργία του μονοπατίου Notch σχετίζονται με παθολογική πλακουντοποίηση. Σκοπός της μελέτης αυτής είναι να εξετάσει την έκφραση των υποδοχέων (receptors) NOTCH1,-2,-3,-4, των συνδετών (ligands) DLL1,-3,-4, JAG1,-2 και των γονιδίων στόχων (target genes) HEY1,-2 σε πλακούντες κυήσεων με προεκλαμψία. Εξετάστηκαν δείγματα πλακουντιακού ιστού από 20 προεκλαμπτικές και από 20 φυσιολογικές κυήσεις. Τα επίπεδα mRNA των υπό εξέταση μορίων μετρήθηκαν με ποσοτική (quantitative) Real-Time PCR (qRT-PCR) ενώ η πρωτεϊνική έκφραση της ενδοκυττάριας περιοχής (intracellular domain) των NOTCH2 (NICD2) και NOTCH3 (NICD3) εκτιμήθηκαν με Western Blot (WB). Τα αποτελέσματα μας έδειξαν ότι οι υποδοχείς Notch-1 και Notch-4 αλλά και ο συνδέτης Dll-1 δεν εκφράζονται στους πλακούντες γυναικών με προεκλαμψία ή με φυσιολογική εγκυμοσύνη. Ωστόσο, τα επίπεδα έκφρασης mRNA των NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 ήταν μειωμένα στα παθολογικά δείγματα σε σχέση με τα φυσιολογικά (p<0.01). Η ανάλυση με WB επιβεβαίωσε ότι τα επίπεδα πρωτεϊνικής έκφρασης των NICD2 και NICD3 ήταν επίσης ελαττωμένα στα δείγματα της προεκλαμψίας (p=0.014 και p<0.001, αντίστοιχα). Περαιτέρω στατιστική ανάλυση έδειξε α) μη έκφραση σε γονιδιακό επίπεδο του υποδοχέα NOTCH3 στις εγκύους εκείνες που κάπνιζαν και στη διάρκεια της εγκυμοσύνης σε σχέση με εκείνες που το έκοψαν (p=0.029) και β) στις περιπτώσεις με προεκλαμψία και βάρος νεογνού μικρότερο από την 5η εκατοστιαία θέση παρατηρήσαμε υψηλότερα επίπεδα πρωτεΐνης του NICD-3 (p=0.028) και υψηλότερα επίπεδα mRNA του Dll-3 (p=0.041). Τα αποτελέσματα της μελέτης μας δείχνουν ότι το σηματοδοτικό μονοπάτι Notch αποτελεί σημαντικό μέρος της παθογένειας αυτής της επιπλοκής της εγκυμοσύνης. Περαιτέρω μελέτες απαιτούνται με σκοπό να διερευνηθεί βαθύτερα και να καθοριστεί ο ρόλος των εξετασθέντων μορίων στην προεκλαμψία και να μελετηθεί η δυνητική τους χρήση στην πρόβλεψη και τη διαγνωστική και θεραπευτική προσέγγιση της νόσου.

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Analysis of Gene and Protein Expression in Atherosclerotic Mouse Aorta by Western Blot and Quantitative Real-Time PCR

Methods in Molecular Biology - Methods in Mouse Atherosclerosis ◽

10.1007/978-1-4939-2929-0_21 ◽

2015 ◽

pp. 309-322 ◽

Cited By ~ 3

Author(s):

José Rivera-Torres

Keyword(s):

Protein Expression ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Gene And Protein Expression ◽

Mouse Aorta

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Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis

Journal of Molecular Endocrinology ◽

10.1677/jme.1.02138 ◽

2007 ◽

Vol 38 (2) ◽

pp. 245-257 ◽

Cited By ~ 50

Author(s):

Leigh J Ellmers ◽

Nicola J A Scott ◽

Jarkko Piuhola ◽

Nobuyo Maeda ◽

Oliver Smithies ◽

...

Keyword(s):

Gene Expression ◽

Cardiac Hypertrophy ◽

Real Time ◽

Real Time Pcr ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

Heart Weight ◽

Pcr Analysis ◽

Male And Female ◽

Altered Gene

The natriuretic peptides, atrial (ANP) and brain natriuretic peptide (BNP) are known to suppress cardiac hypertrophy and fibrosis. Both ANP and BNP exert their bioactivities through the Npr1 receptor, and Npr1 knockout mice (Npr1−/−) exhibit marked cardiac hypertrophy and fibrosis. In this study, we investigated which genes within the hypertrophic and fibrotic pathways are influenced by the lack of Npr1 signalling. cDNA microarray and quantitative real-time PCR (RT-PCR) analyses were performed on cardiac ventricles from Npr1−/−mice. Gene expression at early and late stages during development of hypertrophy wasinvestigated in male and female Npr1−/−mice at8 weeks and 6 months of age. Heart weight to body weight ratios (HW:BW) were maximally increased in 8-week males (P < 0.01), whilst HW:BW in females continued to increase progressively up to 6 months (P < 0.01). This was despite blood pressure being similarly elevated at both the ages in male and female knockout when compared with wild-type (WT) mice (P < 0.001). Microarray analysis identified altered gene expression at the earliest steps in the hypertrophy-signalling cascade in Npr1−/− mice, particularly calcium–calmodulin signalling and ion channels, with subsequent changes in the expression of intracellular messengers including protein kinases and transcription factors. Real-time PCR analysis confirmed significant differences in gene expression of ANP, BNP, calmodulin 1, histone deacetylase 7a (HDAC7a), protein kinase C (PKC)ι, (GATA) 4, collagen 1, phospholamban and transforming growth factor-β1 in Npr1−/− mice when compared with WT (P < 0.05). The present study implicates the calmodulin–CaMK–Hdac–Mef2 and PKC–MAPK–GATA4 pathways in Npr1 mediation of cardiac hypertrophy.

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