Novel role of C terminus of Hsc70-interacting protein (CHIP) ubiquitin ligase on inhibiting cardiac apoptosis and dysfunction via regulating ERK5-mediated degradation of inducible cAMP early repressor

Abstract

Growing evidence indicates a critical role of ubiquitin-proteosome system in apoptosis regulation. A cardioprotective effect of ubiquitin (Ub) ligase of the C terminus of Hsc70-interacting protein (CHIP) on myocytes has been reported. In the current study, we found that the cardioprotective effect of insulin growth factor-1 (IGF-1) was mediated by ERK5-CHIP signal module via inducible cAMP early repressor (ICER) destabilization. In vitro runoff assay and Ub assay showed ICER as a substrate of CHIP Ub ligase. Both disruption of ERK5-CHIP binding with inhibitory helical linker domain fragment (aa 101-200) of CHIP and the depletion of ERK5 by siRNA inhibited CHIP Ub ligase activity, which suggests an obligatory role of ERK5 on CHIP activation. Depletion of CHIP, using siRNA, inhibited IGF-1-mediated reduction of isoproterenol-mediated ICER induction and apoptosis. In diabetic mice subjected to myocardial infarction, the CHIP Ub ligase activity was decreased, with an increase in ICER expression. These changes were attenuated significantly in a cardiac-specific constitutively active form of MEK5α transgenic mice (CA-MEK5α-Tg) previously shown to have greater functional recovery. Furthermore, pressure overload-mediated ICER induction was enhanced in heterozygous CHIP(+/-) mice. We identified ICER as a novel CHIP substrate and that the ERK5-CHIP complex plays an obligatory role in inhibition of ICER expression, cardiomyocyte apoptosis, and cardiac dysfunction.

Figure 1.

ERK5 activation inhibited ICER induction via the ubiquitin-proteosome system. A) Neonatal rat cardiomyocytes were cotransduced with Ad-ICER and Ad-LacZ or Ad-CA-MEK5α for 24 h and incubated with CHX for the indicated times (see Materials and Methods). Expression of ICER, CA-MEK5α, and tubulin was detected by Western blotting with the respective antibodies (middle panel) and the relative expression of ICER was summarized by a line graph from 3 independent experiments (right panel; n=3). B) Cardiomyocytes were cotransduced with Ad-ICER and Ad-CA-MEK5α or Ad-LacZ at 50 MOI for 24 h and additionally incubated with forskolin (Fsk; 10 μM) for 12 h. Cells were treated further with MG132 for 8 h and subjected to a Western blot analysis with anti-ICER, anti-HA, and anti-tubulin antibodies (left panel). C) Myocytes were transduced with Ad-LacZ or Ad-CA-MEK5 for 24 h and then exposed to 10 μM ISO for an additional 24 h. Ad-CA-MEK5α-transduced cells were treated with MG132 for 8 h, as indicated, and subjected to immunoblotting with the stated antibodies (left panel). Bar graph results are means ± sd from 3 independent experiments. *P < 0.05; ** P < 0.01.

Figure 2.

ICER is a novel substrate of CHIP Ub ligase. A) Neonatal rat cardiomyocytes were cotransduced with Ad-ICER and Ad-LacZ, or Ad-CHIP for 24 h and incubated with CHX for the indicated times. Top panel: expression of ICER and CHIP was detected by Western blotting with the respective antibodies. Botttom panel: line graph summary of the relative expression of ICER, determined by densitometry (n=3). B) Myocytes were transfected with pcDNA3, pMyc-CHIP, pMyc-CHIP-H260Q, and pFlag-ICER in the presence or absence of 10 μM MG132. Top panel: protein expression was determined by Western blotting. Bottom panel: bar graph summary from 3 independent experiments. C) Top panel: ICER ubiquitination was determined by immunoprecipitation with anti-Flag antibody followed by immunoblotting with anti-ubiquitin antibody. Bottom panel: bar graph summary from 3 independent experiments. Values are means ± sd. *P < 0.05; **P < 0.01.

Figure 3.

ERK5-CHIP association was critical for the up-regulation of CHIP Ub ligase activity. A) Cardiomyocytes were transduced with Ad-CA-MEK5α or IGF-1 treatment to activate ERK5. Lysates were immunoprecipitated with anti-ERK5 antibody, followed by Western blotting with anti-CHIP antibody to determine ERK5-CHIP association. Ad-lacZ transduction served as a negative control. Expression of CHIP and CA-MEK5α was determined by immunoblotting with anti-CHIP and anti-HA antibodies, respectively. B) To confirm the binding specificity, a mammalian 2-hybrid assay was performed as described in Materials and Methods. CHO cells were cotransfected with pG5-Luc, pBIND-CHIP, and pACT-ERK5 or pACT in the presence or absence of pCA-MEK5α. Protein binding affinity was determined by luciferase activity using dual luciferase assay. C) CHO cells were cotransfected with reporter gene mixture and pBIND-CHIP fragments (CHIP-Fr1, Fr2, Fr3) in the presence or absence of pCA-MEK5α. Binding affinity was determined by luciferase activity. D) Cardiomyocytes were cotransfected with pFlag-ERK5, pMyc-CHIP, and pcDNA3 or pXp/His-CHIP fragments (CHIP-Fr1, Fr2, Fr3). After immunoprecipitation with anti-Flag antibody, ERK5-CHIP interaction was determined by immunoblotting with anti-Myc antibody. Expression of His-tagged CHIP fragments was determined by Western blotting with anti-HisG antibody (Invitrogen). E) Disruption of ERK5-CHIP association with CHIP-Fr2 fragment transfection inhibited CA-MEK5α-mediated CHIP ligase activation. Data are representative of 3 independent experiments. *P < 0.05; **P < 0.01.

Figure 4.

ERK5 activation increased CHIP Ub ligase activity. A) To check the role of ERK5 activation in CHIP-mediated autoubiquitination, cells were cotransfected with pHA-Ubiquitin and pMyc-CHIP or pMyc-CHIP-H260Q in the presence of CA-MEK5α as indicated. Left panel: lysates were immunoprecipitated with anti-Myc antibody, followed by Western blotting with anti-ubiquitin antibody. Right panel: relative ubiquitination activity was summarized by a bar graph. B) CHIP Ub ligase activity was determined by an in vitro ubiquitination assay kit (BostonBiochem) with GST-fused ICER protein as a substrate. Cardiomyocytes were transfected with siRNA against rat CHIP or control and then followed by IGF-1 treatment and transduction of Ad-CA-MEK5α for 24 h. Immunoprecipitated CHIP was incubated with recombinant proteins, including ubiquitin and E1/E2 enzyme mixture, for 60 min at 37°C. Left panel: CHIP ligase activity was determined by immunoblotting with anti-ubiquitin antibody. Right panel: bar graph summary of relative ubiquitination activity from 3 independent experiments. Amounts of protein expression were determined by Western blot analysis with specific antibodies. Data are representative of 3 independent experiments. *P < 0.05; **P < 0.01.

Figure 5.

CHIP was responsible for ERK5 activation-mediated inhibition of ICER degradation. Cardiomyocytes were transfected with control or rat CHIP specific si-RNA for 24 h, followed by transduction with Ad-CA-MEK5α or Ad-LacZ at 50 MOI for additional 24 h (A) or 20 ng/ml IGF-1 treatment for 14 h (B). Left panels: cells were further treated with ISO for 24 h, harvested, and subjected to immunoblotting with anti-ICER, anti-CHIP, anti-HA (HA-CA-MEK5α), or anti-tubulin antibodies. Right panels: bar graph summary of relative ICER expression from 3 independent experiments. *P < 0.05; **P < 0.01.

Figure 6.

CHIP was critical for ERK5 activation-mediated inhibition of myocyte apoptosis. A, B) Cardiomyocytes were transfected with control or rat CHIP specific si-RNA for 24 h, followed by transduction with Ad-CA-MEK5α or Ad-LacZ at 50 MOI (A) or IGF-1 treatment (B) for an additional 24 h. After further treatment with ISO for 48 h, cells were assayed by TUNEL staining using the In Situ Cell Death Detection Kit (Roche). Myocytes were counter stained with anti-cardiomyocyte-specific sarcomeric α-actinin antibody. Bar graphs present percentage of TUNEL positive cells from total myocytes counted. **P < 0.01. C) Representative photomicrographs of the above experiment showing TUNEL (green), actin (red) and DAPI (blue) signals and their merged images.

Figure 7.

ERK5 activation increased CHIP Ub ligase activity and decreased ICER induction after coronary artery ligation in diabetic mice. Heart tissues were harvested from NLC and CA-MEK5α-Tg mice without (A) or with coronary artery ligation (B). Tissue extracts were subjected to an ubiquitination assay for 60 min at 37°C with GST-ICER as a substrate. Reaction was stopped with an SDS sample buffer, followed by immunoblotting with anti-ubiquitin antibody. Amounts of protein expression were determined by Western blot analysis with specific antibodies as indicated. Relative ubiquitination of GST-ICER was measured by densitometric quantification and summarized in bar graphs Values are means ± sd; n=3–4. *P < 0.05.

Figure 8.

Pressure overload-mediated ICER induction, cleaved caspase 3 expression, and cardiac dysfunction were accelerated in CHIP^−/+ mice. A) After TAC, echocardiographically determined cardiac function (see Table 1) presented as percentage fractional shortening (% FS) at the indicated times. B) Heart weight 6 wk after TAC was increased in CHIP^−/+ mice compared with NLC mice (NLC, n=7; CHIP^−/+, n=6). C) At 6 wk after TAC or sham operation, heart tissues were collected from wild-type NLC (n=6; open bars in D, E) and CHIP^−/+ mice (n=6; solid bars in D, E). Tissue extracts were immunoblotted with anti-ICER, anti-Bcl-2, anti-cleaved caspase 3, and anti-tubulin antibodies. D, E) Bar graphs summarize expression levels of ICER (D) and Bcl-2 (E). F) Expression level of pERK5, ERK5, pERK1/2, ERK1/2, pAKT, AKT, CHIP, and tubulin in the heart 6 wk after TAC in NLC and CHIP^−/+. Six weeks after TAC or sham operation, heart tissues were collected from NLC and CHIP^−/+ mice. Tissue extracts were applied for Western blot analysis. Expression levels of pERK5, ERK5, pERK1/2, ERK1/2, pAKT, AKT, CHIP, and tubulin were determined by immunoblotting with specific antibodies. *P < 0.05; **P < 0.01.