Muscle-Specific F-Box Only Proteins Facilitate BK Channel β1 Subunit Downregulation in Vascular Smooth Muscle Cells of Diabetes MellitusNovelty and Significance
Rationale: Activity of the large conductance Ca2+-activated K+ (BK) channels is profoundly modulated by its β1 subunit (BK-β1). However, BK-β1 expression is downregulated in diabetic vessels. The ubiquitin–proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-β1 downregulation in diabetic vessels is unknown.
Objective: We hypothesize that UPS facilitates vascular BK-β1 degradation in diabetes.
Methods and Results: Using patch clamp and molecular biological approaches, we found that BK-β1–mediated channel activation and BK-β1 protein expression were reduced in aortas of streptozotocin-induced diabetic rats and in human coronary arterial smooth muscle cells (CASMCs) cultured in high glucose. This was accompanied by upregulation of F-box only protein (FBXO)-9 and FBXO-32 (atrogin-1), the key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex. BK-β1 expression was suppressed by the FBXO activator doxorubicin but enhanced by FBXO-9 small interfering RNA or by the proteasome inhibitor MG-132. Cotransfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-β1 expression by 2.16-fold, compared with expression of Flag-hSlo-β1V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-β1 was increased by 1.91-fold, compared with that of hSlo-β1V146A, whereas cotransfection with atrogin-1ΔF (a nonfunctional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor (FOXO)-3a and enhanced atrogin-1 expression, which in turn suppressed BK-β1 protein levels in human CASMCs.
Conclusions: Downregulation of vascular BK-β1 expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-β1 degradation.
The large conductance Ca2+-activated K+ (BK) channels play an important role in the regulation of vascular physiology. Functional BK channels in coronary arterial smooth muscle cells (CASMCs) are composed of the pore-forming α subunits (BK-α, encoded by the Slo gene) and the regulatory β1 subunits (BK-β1) in 4:4 stoichiometry. However, BK channel function is impaired in diabetes,1,2 which is associated with microvessel complications. Recently, we and other investigators have reported that impaired BK channel activation was attributable to reduced BK-β1 expression in diabetic vessels.3,4 However, the underlying molecular mechanisms is unknown.
The ubiquitin–proteasome system (UPS) accounts for 80% to 90% of intracellular protein turnover.5 UPS-mediated protein degradation involves 3 enzyme systems: ubiquitin-activation enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3.6 There are 1 E1, >25 E2, and >1000 E3 enzymes. Each E3 recognizes a specific motif on substrate proteins.
F-box only proteins (FBXOs) are key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex, functioning as sites for enzyme–substrate interaction.7 FBXO expression is controlled by the forkhead box O family transcription factor (FOXO). FOXO activities are negatively regulated by Akt, which phosphorylates FOXO at T32, S253, and S315. Phosphorylated FOXO is extruded from the nucleus with loss of transcriptional function.8 FBOX-9 and FBXO-32 (atrogin-1) are muscle-specific subtypes and are abundantly expressed in myocardium and skeletal muscles.9,10 Atrogin-1 may bind to the PDZ-binding motif (T/S-X-V; X is any amino acid) in substrates.9 Interestingly, the PDZ-binding motif is present in most BK-β1 isoforms in different species including human. However, the role of FBXOs in the regulation of BK-β1 expression is unknown. Here, we hypothesized that enhanced UPS activity facilitates BK-β1 protein degradation in diabetes. We found that expression of atrogin-1 and FBXO-9 was augmented in human CASMCs under high-glucose (HG) culture and in streptozotocin (STZ)-induced diabetic rat vessels, leading to downregulation of BK-β1 expression. Moreover, expression of FBXOs and BK-β1 was regulated by FOXO-3a phosphorylation. Hence, we have delineated a novel fundamental mechanism that underlies vascular BK-β1 dysfunction in diabetes.
Male Sprague–Dawley rats were used. Handling and care of animals were approved by the Institutional Animal Care and Use Committee of Mayo Clinic.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Reduced BK Current Density and Impaired DHS-1–Mediated Channel Activation in CASMCs of Diabetic Rats
Figure 1A shows whole-cell K+ currents from freshly isolated CASMCs of control and STZ-induced diabetic rats before and after application of 100 nmol/L iberiotoxin (IBTX) (a specific BK channel inhibitor). The current–voltage curves of IBTX-sensitive K+ currents (defined as BK currents) were significantly decreased by 4.5-fold in diabetic rats, compared with control. DHS-1 (100 nmol/L, a specific BK-β1 activator) applied to the cytoplasmic membrane surface of CASMCs remarkably increased BK channel open probability from 0.11±0.04 at baseline to 0.33±0.11 with DHS-1 (P<0.05 versus baseline) in control rats but had no effect in diabetic rats (Figure 1B), suggesting that the β1-mediated BK channel activation is lost.
Downregulated BK-β1 Expression and Enhanced BK-β1 Ubiquitination in Diabetic Vessels and in Human CASMCs With HG Culture
BK-β1 protein levels were decreased by 3.06±0.07-fold (P<0.05 versus control) in STZ-induced diabetic rats (Figure 1C) and by 2.10±0.09-fold in human CASMCs with HG (22 mmol/L), compared with those with normal glucose (NG) (5 mmol/L, P<0.05) (Figure 1D), whereas BK-α expression remained unchanged. The effects of HG on BK-β1 downregulation were detectable at day 7 and plateaued at days 10 to 14 of HG culture (Online Figure I). However, BK-β1 mRNA level was not reduced in diabetic rats but actually increased in human CASMCs cultured with HG (Online Figure II).
Accordingly, the ubiquitination of BK-β1 in diabetic rat aortas and in human CASMCs cultured with HG was increased by 3.23±0.36-fold (P<0.05 versus controls) and by 2.40±0.33-fold (P<0.05 versus NG), respectively (Figure 1E).
Increased FBXO Expression in Diabetic Rat Vessels and in Human CASMCs With HG Culture
Atrogin-1 and FBXO-9 were expressed in rat aortas and in human CASMCs (Online Figure III). Protein levels of FBXO-9 and atrogin-1 were increased in aortas of STZ-induced diabetic rats by 1.65±0.16-fold and 4.87±1.17-fold, respectively (P<0.05 versus control in both) (Figure 2A), as well as in human CASMCs cultured with HG by 2.43±0.27-fold and 2.33±0.36-fold, respectively (P<0.05 versus NG in both) (Figure 2B). Seventy-two hours after FBXO-9 small interfering (si)RNA (50 nmol/L) was transfected into human CASMCs, FBXO-9 protein level was suppressed by 80%, whereas BK-β1 expression was increased by 1.65±0.16-fold, compared with control siRNA (P<0.05) (Figure 2C).
After 24 hours of incubation with 0.1 μmol/L doxorubicin (DXR) (a FBXO activator),11 BK-β1 expression was reduced by 1.92±0.11-fold (P<0.05 versus control) in NG (Figure 2D) and by 1.79±0.05-fold (P<0.05 versus control) in HG (Figure 2E). The DXR effects were abolished by 4-hour treatment with 10 μmol/L MG-132 (a proteasome inhibitor). BK channel openings were less frequent, with no response to DHS-1 in nondiabetic rat CASMCs after treatment with DXR (P=NS versus baseline). In CASMCs incubated with DXR+MG-132, however, BK channel activity was robust in the presence of DHS-1 (P<0.05 versus baseline) (Figure 2F), consistent with the immunoblotting results.
Lack of the PDZ-Binding Motif in hSlo-β1 Abolished UPS-Mediated Protein Degradation
The PDZ-binding motif is well conserved in BK-β1 (Figure 3A). We measured protein expression of Flag-hSlo-β1 wt and Flag-hSlo-β1V146A (a mutation in the PDZ-binding motif) after cotransfection with ubiquitin and atrogin-1 or atrogin-1ΔF (a nonfunctional mutant with deletion of the F-box). Seventy-two hours after transfection, expression of hSlo-β1 wt was 2.16±0.16-fold lower than that of hSlo-β1V146A (P<0.05) (Figure 3B). Also, ubiquitination of hSlo-β1 wt was 1.91±0.08-fold higher than that of hSlo-β1V146A (P<0.05) in cells cotransfected with atrogin-1, whereas atrogin-1ΔF transfection had no effect (Figure 3C). Hence, our results suggest that FBXO interacts with BK-β1 through the PDZ-binding motif and facilitates BK-β1 ubiquitination and degradation.
Regulation of Atrogin-1 and BK-β1 Expression by Akt/FOXO-3a Signaling in Human CASMCs
We found that FOXO-3a (T-32) phosphorylation was downregulated by 1.54±0.01-fold (P<0.05 versus control) in STZ-induced diabetic rat aortas and by 4.23±0.02-fold (P<0.05 versus NG) in human CASMCs in HG, whereas total FOXO-3a protein remained unchanged (Figure 4A and 4B).
We further determined the role of Akt/FOXO-3a signaling in the regulation of atrogin-1 and BK-β1 expression in human CASMCs. After 24 hours of incubation with 7 μmol/L LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor), Akt (S473) phosphorylation was attenuated by 2.86±0.03-fold (P<0.05 versus control) with unchanged total Akt. Phosphorylated FOXO-3a protein was reduced by 1.86±0.04-fold (P<0.05 versus control), whereas total FOXO-3a was upregulated by 1.70±0.16-fold (P<0.05 versus control) with LY294002, resulting in 2.44±0.23-fold increase (P<0.05 versus control) in atrogin-1 expression and 3.22±0.06-fold reduction (P<0.05 versus control) in BK-β1 expression in human CASMCs (Figure 4C).
The BK-β1 subunit significantly enhances BK channel voltage sensitivity and Ca2+ cooperativity,4 and its physiological importance is underscored by BK-β1 knockout mice, which showed increases in myogenic tone and arterial blood pressure, with uncoupling of Ca2+ sparks to BK channels in vascular smooth muscle cells (SMCs).12 We and others have demonstrated that BK channel dysfunction in both type I and type II diabetic vessels is associated with reduced BK-β1 expression.3,4 In this study, we provided the first evidence that downregulation of BK-β1 expression in vasculature was attributable to increased FBXO expression in diabetes and in HG culture conditions.
Abnormal expression of atrogin-1 is associated with muscle diseases, and its increased expression leads to skeletal muscle atrophy,9 whereas its reduced expression produces cardiac hypertrophy.10 We have found that atrogin-1 and FBXO-9 expression are upregulated in vascular SMCs in diabetes and HG. This upregulation of FBXOs plays a pivotal role in UPS-mediated BK-β1 degradation and BK channel malfunction. Perturbation in atrogin-1 and hSlo-β1 interaction attenuated hSlo-β1 ubiquitination and preserved its protein level.
Atrogin-1 and FBXO-9 are the target genes of FOXO-3a. We found that in diabetes and in human CASMCs with HG, FOXO-3a phosphorylation level was significantly reduced, accompanied by enhanced FBXO expression. The changes in FOXO-3a, FBXOs, and BK-β1 expression in human CASMCs with HG were mimicked by PI3K/Akt inhibition. It is well known that the PI3K/Akt pathway is activated by insulin and suppressed by protein kinase C. Insulin receptor signaling is diminished and protein kinase C activity is upregulated in type I and type II diabetes, and these may lead to enhanced FBXO transcription and impaired BK channel function (Figure 4D). Hence, our findings provide novel insights into the pathophysiology of diabetic vasculopathy in both type 1 and type 2 diabetes mellitus and render BK-β1 as a potential therapeutic target in treatment of these conditions.
Sources of Funding
This work was supported by American Diabetes Association grant ADA-JFA-07-39 (to T.L.), American Heart Association Grant 09SDG2190046 (to T.H.), and NIH grants HL74180 and HL080118 (to H.L.) and HL91867 (to Z.S.K.).
Atrogin-1 wt and atrogin-1ΔF in pCMC-Tag 3B were provided by Dr Monte Willis (University of North Carolina, Chapel Hill). Human KCNMB1 in pGEM was obtained from Dr Olaf Pongs (University of Hamburg, Germany). DHS-1 was the kind gift of Merck & Co.
In September 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.1 days.
Non-standard Abbreviations and Acronyms
- large conductance Ca2+-activated K+ channel
- BK channel α subunit
- BK channel β1 subunit
- coronary arterial smooth muscle cell
- f-box only protein
- forkhead box O family transcription factor
- high glucose
- normal glucose
- phosphatidylinositol 3-kinase
- small interfering RNA
- smooth muscle cell
- ubiquitin–proteasome system
- wild type
- Received July 15, 2010.
- Revision received October 8, 2010.
- Accepted October 11, 2010.
- © 2010 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Vascular BK channels are key determinants of vascular tone. They are composed of the pore-forming α subunits (BK-α) and the regulatory β1 subunits (BK-β1). The expression of the BK-β1 protein is downregulated in vascular smooth muscle cells (SMCs) in diabetes, leading to loss of the β1-mediated channel function, including channel sensitivity to Ca2+ and voltage.
The ubiquitin-proteasome-system (UPS) is a major mechanism of intracellular protein degradation, accounting for 80% to 90% of intracellular protein turnover.
What New Information Does This Article Contribute?
Impaired β1-mediated BK channel activation is associated with increase in UPS-dependent BK-β1 protein degradation in vascular SMCs in diabetes mellitus and in high glucose culture conditions.
Muscle-specific F-box only proteins (FBXOs), FBXO-9 and FBXO-32 (atrogin-1), which are integral components of E3 ubiquitin ligase complexes, are abundantly expressed in vascular SMCs. The expression of these proteins is upregulated in diabetic rat aortas and in human coronary arterial SMCs cultured with high glucose.
Expression of FBXOs is controlled by Akt and the forkhead box O family transcription factor 3a (FOXO-3a) signaling. Inhibition of Akt reduces FOXO-3a phosphorylation, increases FOXO-3a transcriptional function and facilitates FBXO expression, which in turn accelerates BK-β1 protein degradation in vascular SMCs.
The BK-β1 subunit plays a pivotal role in BK channel function by modulating channel voltage- and Ca2+-sensitivity. Downregulation of BK-β1 expression in vascular SMCs is a common finding in diabetic vessels that produces BK channel dysfunction. However, the molecular mechanisms underlying downregulation of BK-β1 protein expression is unknown. In this study, we report that impaired β1-mediated channel activity in diabetes is associated with FOXO-3a/FBXO-dependent increase in BK-β1 protein degradation. These results indicate that BK-β1 subunits and Akt/FOXO-3a/FBXOs signaling cascade are potential therapeutic targets in the treatment of diabetic vascular complications.