Activation of a novel calcineurin-mediated insulin-like growth factor-1 receptor pathway, altered metabolism, and tumor cell invasion in cells subjected to mitochondrial respiratory stress

Abstract

We have previously shown that disruption of mitochondrial membrane potential by depletion of mitochondrial DNA (mtDNA) or treatment with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone, initiates a stress signaling, which causes resistance to apoptosis, and induces invasive behavior in C2C12 myocytes and A549 cells. In the present study we show that calcineurin (Cn), activated as part of this stress signaling, plays an important role in increased glucose uptake and glycolysis. Here we report that, although both insulin and insulin-like growth factor-1 receptor levels (IR and IGF1R, respectively) are increased in response to mitochondrial stress, autophosphorylation of IGF1R was selectively increased suggesting a shift in receptor pathways. Using an approach with FK506, an inhibitor of Cn, and mRNA silencing by small interference RNA we show that mitochondrial stress-activated Cn is critical for increased GLUT 4 and IGF1R expression and activation. The importance of the IGF1R pathway in cell survival under mitochondrial stress is demonstrated by increased apoptosis either by IGF1R mRNA silencing or by treatment with IGF1R inhibitors (AG1024 and picropodophyllin). This study describes a novel mechanism of mitochondrial stress-induced metabolic shift involving Cn with implications in resistance to apoptosis and tumor proliferation.

Figure 1. Increased uptake of deoxyglucose by C2C12 cells subjected to partial mtDNA depletion

A, mitochondrial DNA contents of control, mtDNA-depleted, and reverted cells. Total cell DNA (0.5 μg each), digested with BglII, was subjected to Southern blot hybridization using a ³²P-labeled mtDNA probe (cytochrome oxidase subunits I plus II coding region of mtDNA). In the right panel, 10 ng of DNA was used as template for amplifying the nuclear DNA-encoded Cox IVil amplicon. B-D, 2-DOG uptake by control, mtDNA-depleted, and reverted cells. Sub-confluent cells were washed with PBS and incubated for 30 min at 37 °C in transport buffer containing the indicated concentrations of insulin (B), 10 nm FK506 (C), and 25 μm CCCP (D). Cells were incubated at 37 °C for 30 min with 1 μCi of 2-[³H]DOG (1 mCi/0.1 mmol), and the uptake was measured as described under “Experimental Procedures.” Nonspecific background counts with heat-killed cells were subtracted from experimental values. Averages ± S.E. of three independent estimates (each taken in triplicate) are presented. In B the difference in glucose uptake between control and mtDNA-depleted cells is indicated: **, p < 0.001. Statistical differences between mtDNA-depleted cells without added insulin and with added 1 μm insulin are indicated: *, p ≤ 0.05. In control and reverted cells, the values with added insulin (*) compared with or without added insulin show a significance of p ≤ 0.05. In C and D: *, p ≤ 0.05; **, p ≤ 0.001.

Figure 2. Increased GLUT 4 protein and mRNA levels in mtDNA-depleted C2C12 cells

A, C2C12 cells were treated with AG1024 (10 μm, 2 h), CCCP (25 μm, 2 h), or FK506 (10 nm, 2 h) as indicated. Plasma membrane fractions (30 μg each) were subjected to immunoblot analysis, and the blot was co-developed with antibody to GLUT 4 (1:1000 dilution) and Na⁺/K⁺-ATPase (1:1000 dilution). The latter was used as a loading control. B, RT-PCR analysis of total RNA (50 ng each of RT product) from control, mtDNA-depleted, and reverted C2C12 cells. The values represent average ± S.E. of three independent assays. *, p ≤ 0.05; **, p ≤ 0.001. C, real-time PCR analysis of total RNA(50 ng each of RT product) from mtDNA-depleted C2C12 cells. Cells were transfected with 2 μg of IGF1R cDNA for 48 h, and total RNA was isolated. Real-time PCR analysis of GLUT 4 mRNA was done using a TaqMan probe as described under “Experimental Procedures.” *, p ≤ 0.05; **, p ≤ 0.001. D, lawn assay for GLUT 4 contents of plasma membrane fractions from control and mtDNA-depleted cells before or after treatment with insulin (100 nm, 30 min). Details of immunoblot analysis, real-time PCR, and the lawn assay are as described under “Experimental Procedures.”

Figure 3. Activation of IGF1R and inactivation of IR in mtDNA-depleted C2C12 cells

A, effects of receptor inhibitors wortmannin (0.1 μm), LY294002 (50 μm), genistein (0.1 mg/ml), AG1024 (10 μm), and PPP (2.5 μm) on 2-DOG uptake in control, mtDNA-depleted, and reverted cells. Treatments were carried out for 2 h. Details of 2-DOG uptake are as described in Fig. 1. Shown are the differences between control (basal) and mtDNA-depleted cells (**, p ≤ 0.001) and between mtDNA-depleted cells with and without added genistein (*, p < 0.05). Shown also are the levels of inhibition in mtDNA-depleted cells with added wortmannin, LY294002, AG1024, and PPP compared with no inhibitor control (**, p ≤ 0.001). B, basal plasma membrane levels of IRβ in control, mtDNA-depleted, and reverted cells treated with or without of FK506 (10 nm, 2 h). Cell fractions (30 μg each) were subjected to immunoblot analysis with antibody to IRβ as described under “Experimental Procedures.” The blot was co-developed with Na⁺/K⁺-ATPase antibody to assess equal protein loading levels. C, levels of basal IRβ autophosphorylation. Autophosphorylation was carried out in plasma membranes of cells treated with or without FK506 as described under “Experimental Procedures.” An autoradiogram of gels showing ³²P-labeled IRβ is presented. D, immunoblot analysis of plasma membranes with IGF1R antibody following treatment with or without FK506. The blots were co-developed with antibody to Na⁺/K⁺-ATPase to assess loading levels. E, autoradiograms of gels showing ³²P-labeled IGF1R protein are presented. Levels of IGF1R autophosphorylation in cells treated with or without FK506. Details of treatment are as in C. F, IGF1R mRNA levels were quantified by real-time PCR in control, mtDNA-depleted, and reverted C2C12 cells as described under “Experimental Procedures.” In some cases cells were treated with FK506 (10 nm) for 30 min before isolating total RNA. *, p ≤ 0.05; **, p ≤ 0.001. G, autophosphorylation of IGF1R (upper panel) and IR (lower panel) in cells grown in charcoal-treated serum as described under “Experimental Procedures.” In B–E and G, values of relative band intensities presented in parentheses represent averages of two to three independent blots.

Figure 4. Effect of AICAR on calcineurin activity and deoxyglucose uptake in mtDNA-depleted cells

MtDNA-depleted C2C12 cells were treated with FK506 (10 nm, 30 min) and AICAR (2 mm, 2 h). A, total cell lysates (1 μg of protein) were assayed for calcineurin activity as described under “Experimental Procedures.” B, 2-DOG uptake in mtDNA-depleted C2C12 cells was performed as described under “Experimental Procedures.” *, p ≤ 0.05; **, p ≤ 0.001.

Figure 5. Increased glucose uptake, IGF1R, and GLUT 4 levels in A549, H9C2, and 3T3 cells subjected to mitochondrial stress

mtDNA-depleted A549 cells were generated by ethidium bromide treatment as described before (1). Mitochondrial respiratory stress was induced in H9C2 cells and 3T3 cells by treatment with CCCP(25 mm) for 30 min. A, 2-DOG uptake by control, mtDNA-depleted A549 cells, control, and CCCP-treated H9C2 and 3T3 cells was carried out as described under “Experimental Procedures.” **, p ≤ 0.001. B and C, plasma membrane fractions (30 μg each) were subjected to immunoblot analysis, and the blot was developed with antibody to GLUT 4, IGF1Rβ, and IRβ, respectively. Antibody to Na⁺/K⁺-ATPase (1:1000 dilution) was used as a loading control. D, GLUT 4 and IGF1R mRNA levels were quantified by real-time PCR in control and CCCP-treated H9C2 cells as described under “Experimental Procedures.” In some cases cells were treated with FK506 (10 nm) for 30 min before isolating total RNA. *, p ≤ 0.05;**, p ≤ 0.001. In B and C, the relative band intensities represent averages of two to three independent blots.

Figure 6. Effects of calcineurin Aα mRNA silencing in mtDNA-depleted cells

Control and mtDNA-depleted cells were transfected either with specific siRNA or scrambled siRNA (control) constructs as described under “Experimental Procedures.” A, CnAα mRNA levels; B, IGF1R mRNA levels in transfected cells quantitated by real-time PCR using TaqMan probe. C, GLUT 4 mRNA levels in the transfected cells quantitated by TaqMan assay. D, IR mRNA levels in transfected cells quantitated by real-time PCR using TaqMan assay. **, p ≤ 0.001.

Figure 7. Effects of IR and IGF1R mRNA silencing in mtDNA-depleted cells

Control and mtDNA-depleted C2C12 cells were transfected either with scrambled siRNA (control) or IR- and IGF1R-specific siRNA constructs as described under “Experimental Procedures.”A, IRand IGF1R mRNA levels determined by real-time PCR. B, 2-DOG uptake in control, mtDNA-depleted, and mRNA-silenced cells. C, GLUT 4 mRNA levels in control, mtDNA-depleted, and mRNA-silenced cells. D, Matrigel invasion assay with mtDNA-depleted cells transfected with mock (negative control) siRNA, IR siRNA, and IGF1R siRNA. E, Nexin assay for measuring apoptosis in mtDNA-depleted cells. Panels on the left show the cell flow cytometry pattern, and the graph on the right shows the percent apoptotic cells quantified from three independent cell sorting measurements. **, p ≤ 0.001.

Figure 8. Increased sensitivity of mtDNA-depleted C2C12 cells to AG1024- and PPP-mediated apoptosis

Treatment of cells with various inhibitors was as described under “Experimental Procedures” and in Fig. 3A. A, cells undergoing early apoptosis were detected by using a Guava Nexin assay kit for cell labeling. B, results from four independent assays as in A were used for calculating average ± S.D. *, increased cell death (p ≤ 0.05); **, p ≤ 0.001. C, Martrigel invasion assay with control and mtDNA-depleted C2C12 cells treated with or without PPP (2.5 μm, 2 h).