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A novel transgenic mouse model reveals deregulation of the ubiquitin-proteasome system in the heart by doxorubicin

Asangi R. K. Kumarapeli^*,, Kathleen M. Horak^*,, Joseph W. Glasford^*,, Jie Li^*,, Quanhai Chen^*,, Jinbao Liu^*,,, Hanqiao Zheng^*,, and Xuejun Wang^*,,1

^* Cardiovascular Research Institute, South Dakota Health Research Foundation, Sioux Falls, South Dakota, USA;
Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Sioux Falls, South Dakota, USA;
Department of Pathophysiology, Guangzhou Medical College, Guangzhou, China; and
Department of Pathology, Pathophysiology and Forensic Medicine, Wuhan University, Wuhan, China

¹Correspondence: Cardiovascular Research Institute, South Dakota Health Research Foundation, University of South Dakota School of Medicine and Sioux Valley Hospitals and Health System, 1100 E. 21st St., 7th Floor, Sioux Falls, SD 57105, USA. E-mail: xwang{at}usd.edu

SPECIFIC AIMS

Ubiquitin-proteasome system (UPS) -mediated proteolysis is responsible for the degradation of most proteins in the cell. UPS dysfunction was suggested in the pathogenesis of numerous disorders, but monitoring in vivo functional changes of the UPS remains a challenge. This study was aimed to 1) create a new transgenic (TG) mouse model that ubiquitously expresses a surrogate protein substrate for the UPS and validate its suitability to monitor in vivo changes of UPS proteolytic function in all major organs or tissues; 2) develop and test primary culture of cardiomyocytes derived from adult GFPdgn TG mice to probe UPS involvement in pathogenesis; and 3) apply these new approaches to determine effects of doxorubicin (Dox) on the proteolytic function of the UPS in the heart.

PRINCIPAL FINDINGS

1. Stable TG mouse lines expressing a new UPS functional reporter
Degradation of a cellular protein by the UPS requires ubiquitination of the protein molecule and subsequent degradation of the ubiquitinated protein by the 26S proteasome which consists of the 20S and the 19S subcomplexes. The proteolytic activity of the 26S resides in the chamber of the 20S but is largely regulated at the access of target proteins to the chamber of the 20S by the 19S subcomplex. The proteasomal activities are conventionally measured by in vitro cleavage of synthetic fluorogenic peptide substrates. These small artificial substrates can freely diffuse into the lumen of the 20S such that assays using them fail to account for impact from other parts of the UPS. Hence, a reporter substrate of a full length protein is required to assess the proteolytic function of the entire UPS. None of the endogenous proteins would be a good candidate for this reporter because the synthesis and degradation of each are subject to inherent regulations in addition to UPS function. A few surrogate substrates have been described. One is GFP^u, an enhanced green fluorescence protein (GFP) modified via carboxyl fusion of the yeast CL1 consensus ubiquitination signal sequence. We and others have showed that degradation of GFP^u in cultured HEK cells and neonatal rat ventricular myocytes depends exclusively on the UPS. Therefore, we have established three stable TG mouse lines that ubiquitously express a unique UPS surrogate substrate, referred to as GFPdgn. GFPdgn was also engineered by fusion of the CL1 sequence to the carboxyl terminus of GFP. Expression of GFPdgn in the mice is driven by a ubiquitous promoter. Transcript and protein analyses consistently showed that GFPdgn is expressed in all major organs (Fig. 1 B). These mice have been monitored >1 year so far. No gross abnormality was observed. Cardiac gravimetric measurements, echocardiography and fetal gene expression analysis on 1- to 12-month-old mice with the intermediate (line 2) and highest (line 3) GFPdgn expression, did not show morphological or functional abnormalities.

View larger version (31K): [in this window] [in a new window]   Figure 1. GFPdgn protein accumulates upon proteasomal inhibition. A) TG mice from line 3 were injected intravenously with 5 µmol/kg of MG-262 (TG MG-262) or vehicle (TG DMSO) 20 h before the tissue samples were collected. MG-262 inhibited chymotryptic activity in the heart, lungs, skeletal (Sk.) muscle, and liver by 50–75% (P<0.05). B) Representative Western blots for GFPdgn in indicated organs of mice described in panel A. For each organ, an equal amount of total protein from an MG-262-treated (+) and a DMSO-treated (–) mouse was loaded. Total proteins loaded to each lane of skeletal (Sk.) muscle, the heart, lungs, small intestine (intestine), liver, kidney, brain, and spleen was 5, 10, 30, 30, 50, 50, 60, and 60 µg, respectively. For the NTG control lane, 50 µg of skeletal muscle protein was loaded. Note that GFPdgn levels in all the indicated organs clearly displayed significant increases upon proteasomal inhibition by MG-262. C) Western blot analysis shows significant increases in GFPdgn in representative organs (the heart and lungs) in response to proteasome inhibition by a different proteasome inhibitor, clasto-lactacystin ß-lactone (lactacystin).

2. GFPdgn protein is accumulated by proteasomal inhibition
GFPdgn protein abundance in all the major organs showed responsive increases upon systemic proteasomal inhibition induced by MG-262 or lactacystin derivatives (Fig. 1) . The increase is readily measurable by conventional methods, including Western blot analysis (Fig. 1B, C ), fluorescence spectrometry, and direct fluorescence microscopy.

3. Dox enhances cardiac UPS
Cardiotoxicity is a major hurdle for more effective use of Dox to treat cancers. To test whether Dox affects UPS function in the heart, we examined GFPdgn protein and transcript levels in the heart 6 and 24 h after an i.p. injection of Dox (25 mg/kg). Compared with the mock treatment group, GFPdgn protein in the heart was significantly reduced in the Dox-treated groups at both 6 and 24 h (Fig. 2 A) whereas steady-state transcript levels of GFPdgn were not significantly changed at 6 h (Fig. 2B ), indicating that Dox acutely enhances the proteolytic function of the UPS . Activities of all three peptidases (chymotrypsin-like, caspase-like, and trypsin-like) of the 20S proteasomes in the heart were not increased at either 6 or 24 h (Fig. 2C ). This provides an example that peptidase activity assays do not measure the function of the entire UPS and suggests that the action of Dox on the UPS falls likely outside of the ß-rings of 20S proteasomes.

View larger version (27K): [in this window] [in a new window]   Figure 2. Doxorubicin (Dox) depletes GFPdgn protein in the heart. A) A representative Western blot image showing clear reductions of GFPdgn protein levels in the heart at 6 (Dox 6 h) and 24 h (Dox 24 h) after Dox treatment (25 mg/kg, i.p.). B) Total RNA from ventricular myocardium and skeletal muscle (Sk. M.) of the quadriceps femoris at 6 h after Dox or mock treatment was used for dot blot analysis of GFPdgn and GAPDH with P32-labeled transcript-specific probes. GAPDH transcript levels were used as the loading control. Three mice of line 1 and one mouse of line 3 were included for the heart while only mice of line 1 were included for the skeletal muscle. Densitometry analyses showed there is no statistically significant difference between mock and Dox-treated groups in both the heart and skeletal muscle. C) Activities of the three indicated proteasomal peptidases in myocardial crude protein extracts were assayed using synthetic fluorogenic substrates. Note that the activities showed a tendency to decrease rather than increase 6 and 24 h after Dox treatment. Mean + SE; compared with the control, **P < 0.05, *P < 0.01, 1-way ANOVA.

4. Cultured cardiomyocytes from adult GFPdgn mice as a platform to study UPS function
To evaluate the potential use of cultured cardiomyocytes from adult GFPdgn TG mice to study UPS function, we examined the effects of various proteasome inhibitors on GFPdgn protein levels in cultured adult cardiomyocytes. Proteasome inhibition by MG-132, MG-262, and clasto-lactacystin-ß-lactone all caused significant increases in GFPdgn protein in the cells, suggesting that cultured cardiomyocytes from adult GFPdgn mice represent an alternative platform for in vitro studies of UPS function. Using this platform, we further tested the direct effects of Dox treatment at the cellular level. Dox decreased GFPdgn protein levels in cultured cardiomyocytes from GFPdgn mice in a dose-dependent manner. This effect was detected as early as 1 h after treatment (0.5 µM). 3 h after treatment the reduction of GFPdgn was detectable with a Dox dose as low as 0.1 µM. This decrease was abolished by proteasomal inhibition, suggesting that enhancement of UPS-mediated protein degradation by Dox accounts for the depletion of GFPdgn protein. This notion is further supported by induction of additional decreases in GFPdgn protein levels by Dox in cardiomyocytes pretreated with cycloheximide (20 µM) to block protein synthesis. Taken together, in vivo and in vitro findings both indicate that UPS proteolytic function in cardiomyocytes is significantly enhanced by Dox treatment.

CONCLUSIONS AND SIGNIFICANCE

We demonstrate that 1) GFPdgn mice are well suited for monitoring in vivo changes in UPS proteolytic function in all major organs including the heart, brain, and skeletal muscle, organs that could not be monitored by the Ub^G76V-GFP mouse model reported recently by Lindsten et al.; and 2) Dox enhances UPS-mediated proteolysis in the heart, suggesting that UPS hyperfunction may be an important mechanism for acute cardiotoxicity of Dox.

In addition to facilitating physiological, pharmacological, and pathogenic studies of the UPS, the GFPdgn mouse model and associated in vitro methodologies developed and validated here may also benefit screen-ing for new measures to modulate UPS function. Proteasome inhibition has proved very effective in suppressing cell proliferation when treating cancer and preventing restenosis. Our findings also suggest that combining the use of Dox with a proteasome inhibitor that, by itself, has antitumor effects may reduce Dox toxicity. Since UPS malfunction appears to be a nodal pathogenic mechanism, it is reasonable to postulate that controllable UPS activation would help treat protein surplus diseases more effectively.

View larger version (40K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating validation of GFPdgn mice as a reliable reporter for in vivo UPS function as well as deregulations of UPS proteolytic function by doxorubicin (Dox) treatments as revealed by the reporter mice. CLBL: clasto-lactacystin-ß-lactone; UPS () and UPS (): UPS inhibition and activation, respectively.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3973fje;

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