Insulin-Like Growth Factor-II Receptor Mediates Angiotensin II-Induced Rab9-Dependent Macroautophagy and Apoptosis in H9c2 Cardiomyoblast Cells

(1)

Ding-Yu Lin (林町餘) 1_{, Wei-Wen Kuo (}_郭薇雯₎2_{, Fu-Jen Tsai (}_蔡輔仁₎3_{, Chang-Hai Tsai (}_蔡長海₎4_{, Chih-Yang Huang (}_黃志揚₎1,5

1Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan; 2 Department of Biological Science and Technology, China Medical University, Taichung, Taiwan 3 Department of Pediatrics, Medical Research and Medical Genetics, China Medical University Hospital, Taichung, Taiwan 4 Department of Healthcare Administration Asia University, Taichung, Taiwan 5Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan.

Background

Macroautophagy (hereafter referred to as autophagy) has emerged as an important process in the pathogenesis of cardiovascular diseases. Angiotensin II (AngII) plays an important role in pathogenesis of heart disease. Our previous studies demonstrated that up-regulation of IGF-IIR (Insulin-like growth factor -II receptor) gene and the subsequent activation of IGF-IIR signaling contributed to AngII-induced myocardial cell hypertrophy, and apoptosis.

Insulin-Like Growth Factor-II Receptor Mediates Angiotensin II-Induced

Rab9-Dependent Macroautophagy and Apoptosis in H9c2 Cardiomyoblast Cells

類胰島素生長因子第二型受體調控血管收縮素

II

在

H9c2

心肌纖維母細胞所誘導的

Rab9

依賴

型自噬作用與細胞凋亡

AV AU

Figure 2. Autophagy was induced by IGF2R activation. (A).Typical autophagic

vacuoles were observed in Leu27-IGFII treated H9c2 cells by transmission electron microscopy. AU: autophagosome; AV: Autolysosome . (B).Increase of autophagy level by IGF2R activation. Measurement by Lyso Tracker Red staining of flow cytometry.

Fig2(A). Fig2 (B.)

Control

Figure 6. Rab9-dependent autophagy contributes to IGF2R-induced apoptosis in H9c2 cells..

Figure 7. Rab9-depedent autophagy induced by AngII is mediated by IGF2R.

*

Fig 6 (A). Leu27IGF-II induced apoptosis were attenuated markedly by autophagy inhibition.

Fig 7 (A). Increase of igf2r gene expression by AngII in a time depenent manner. Transcription levels were analyzed by RT-PCR.

hypertrophy, and apoptosis.

Methods

Objectives

To investigate whether autophagy is involved in AngII-induced IGF2R signaling and further clarify the induced autophagy is associated with IGF2R-induced apoptosis in H9c2 cells..

Figure 1. (A.) Illustration of the IGF-IIR dependent signaling pathway in cardiac remodeling (Endocrinology, 2009). (B).Comparison of canonical and non-canonical autophagy (Nature. 2009).

α -Tubulin LC3 I LC3 II BafA1(10-8M) －－－－－－－－－－－－－－－－－－－－＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋ Hours 0 4 6 8 24 4 4 6 6 8 8 24 24 Leu27IGF-II (10-8 M) －－－－＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋－－－－＋＋＋＋－－－－＋＋＋＋－－－－＋＋＋＋－－－－＋＋＋＋ 16 kDa 14 55

Figure 3. Canonical autophagy was suppressed by IGF2R activation. LC3

turnover assay indicated that significant reduction of canonical autophagy after Leu27IGF-II 24hr incubation compared with no-stimuli cells .

Fig 1 (A). Fig 1. (B.)

Figure 4. Rab9-dependet autophagy is mediated through IGF2R-Gαq signal.

(A).Up-regulation of rab9 by IGF2R activating. (B).Rab9-depednet autophagy was induced by IGF2R signaling. (C).Inhibition of Rab9-depedent autophagy by Type III PI3K inhibition or Gαq knockdown.

Leu27IGF-II + 3MA Leu27IGF-II

Fig 4 (A) Western blotting SYBR Green Real-Time PCR

TUNEL DAPI si-ctrl Leu27IGF- II + si-Beclin I Leu27IGF-II + si-ctrl Leu27IGF- II + si-ATG5 TUNEL DAPI

Figure 8. Rab9-depedent autophagy is a mitophagy which may contribute to IGF2R mediated mitochondria-dependent apoptosis. Arrows indicated the image colorization of Lamp2

and Tom20.

Fig 6B. Leu27IGF-II induced activated-caspase3 were reduced by Rab9-depedent autophagy inhibition.

Fig 6(C). Apoptosis level were detected by TUNEL assay. Selective knockdown of Rab9-dependet autophagy required ATGs (Autophagy related genes) attenuated IGF2R-induced apoptosis markedly.

Fig 7 (B). AngII mediated up-regulation of igf2 gene via AT-1 receptor .Transcription levels were analyzed by RT-PCR. PD 123319: AT-1 receptor antagonist; Losartan: AT-2 receptor antagonist.

Fig 7 (C). IGF2R mediated AngII-induced Rab9-depedent autophagy and cell apoptosis.

Results

Conclusions

Our findings provide the novel evidence that the non-canonical autophagy exists in myocardial cells and implicates that IGF2R may be an important nexus point between autophagy, endosomal trafficking and apoptosis. Further studies are warranted to reveal the detail molecular mechanisms governing IGF2R-mediated Rab9-dependent autophagy. The knowledge may help in the development of future therapeutic autophagy agonists/antagonists to cure cardiomyopathy.

Methods

Study design

Cell line: H9c2 cardiomyoblast cells were obtained from the American Type Culture Collection (ATCC) .

H9c2 cells were incubated with Ang II(10-7_{M) or Leu27IGF-II (10}-8_{M) for} the subsequent analysis. Leu27IGF-II is an analog of IGF2 which interacts selectively with the IGF-IIR.

Autophagy assessments

si-RNAs (small interference RNAs) were transfected into H9c2 cells to specifically knockdown the target genes; Gene silencing efficiency was over 60% in all experiment groups.

3-methyladenine(3-MA;10mM) was used for autophagy inhibitor and Bafilomycin A1(BafA1, 100nM) was used for autolysosome formation inhibition.

Autophagy level was quantified by flow cytometr with LysoTracker Red dye.

Ang II-induced Rab9-depentdent autophagy is mediated by IGF-IIR and this non-canonical autophagy contributes to AngII-induced apoptosis in H9c2 cardiomyoblast cells.

Selective activation of IGF-IIR by Leu27IGF-II confirm that the induction of Rab9-depedent autophagy is mediated by IGF-IIR-Gαq signaling in H9c2 cells, which may in turn contribute to mitochondria-mediated apoptosis.

Control Leu27IGF-II Leu27IGF-II+ 3MA si-ctrl Leu27IGF-II + si-ctrl Leu27IGF-II + si-Rab9

Fig 4 (B)

Fig 4 (C) Determine autophagy by Lysotracker Red stain. The data shown are representative of three independent experiments with similar findings.

Figure 5. Rab9-depedent autophagy contributes to IGF2R-induced H9c2 cell death. Cell viability were assessed by Trypan Blue Exclusion Test.

Leu27IGF-II + si-Rab9 Leu27IGF-II + si-Gαq si-ctrl 1.4% 0.5% Leu27IGF-II + si-ctrl 6.2% 5.5% Leu27IGF-II + si-Rab9 2.9% 3.5% Leu27IGF-II + si-G ααα qαqqq * * *

Fig 6 D. Annexin V/PI staining assay of cell apoptosis induced by Leu27IGF-II. The total apoptotic cells (early and late-stage apoptosis) are represented by the right side of the panel (Annexin V staining alone or together with PI) in which the total cell death number. Results indicated that Rab9-depedent autophagy contributes to IGF2R-induced apoptosis.

References

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