Muscle-Specific F-Box Only Proteins Facilitate BK Channel β₁ Subunit Down-Regulation in Vascular Smooth Muscle Cells of Diabetes Mellitus

Abstract

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 down-regulated in diabetic vessels. The ubiquitin-proteasome-system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-β₁ down-regulation 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 up-regulation of F-box only (FBXO) protein-9 (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 siRNA or by the proteasome inhibitor MG132. Co-transfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-β₁ expression by 2.16-fold, compared to expression of Flag-hSlo-β₁V146A (a mutant without the PDZ-binding motif). After co-transfection with atrogin-1, the ubiquitination of Flag-hSlo-β₁ was increased by 1.91-fold, compared with that of hSlo-β₁V146A, while co-transfection with atrogin-1ΔF (a non-functional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor-3a (FOXO-3a) and enhanced atrogin-1 expression, which in turn suppressed BK-β₁ protein levels in human CASMCs.

Conclusions

Down-regulation of vascular BK-β₁expression 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 investigator have reported that impaired BK channel activation was due 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 three enzyme systems: ubiquitin-activation enzyme E1, ubiquitin conjugating enzyme E2, and ubiquitin ligase E3.⁶ There are one E1, more than 25 E2 and more than 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 T-32, 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-1and 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.

Methods

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.

Results

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 I-V 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.

Figure 1. Impaired BK-β₁ function and increased BK-β₁ ubiquitination in diabetes and in HG culture conditions.

Whole-cell K⁺ currents before and after exposure to 100 nmol/L IBTX and the I-V relationship of IBTX-sensitive currents from freshly isolated CASMCs of control and STZ-induced diabetic rats (A). Inside-out single BK channel currents in CASMCs from control and diabetic rats at baseline and after application of DHS-1 (B). “c” represents the channel closed state. Western blots of BK-α and BK-β₁ in control and diabetic aorta (C) and in human CASMCs in NG and HG (D). Increased BK-β₁ ubiquitination in diabetic aorta and CASMC in HG (E).

Down-regulated 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 to those with normal glucose (NG, 5 mmol/L, p<0.05) (Figure 1D), while BK-α expression remained unchanged. The effects of HG on BK-β₁ down-regulation 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). 72-h after, FBXO-9 siRNA (50 nmol/L) was transfected into human CASMCs, FBXO-9 protein level was suppressed by 80%, while BK-β₁ expression was increased by 1.65±0.16 fold, compared with control siRNA (p<0.05) (Figure 2C).

Figure 2. Enhanced FBXO expression and reduced BK-β₁ expression in diabetic vessels and in HG culture conditions.

Increased expression of FBXO-9 and atrogin-1 in STZ-induced diabetic rat aortas (A) and in human CASMCs cultured with HG (B). FBXO-9 siRNA suppressed FBXO-9 but enhanced BK-β₁ expression in human CASMCs (C). BK-β₁ expression in human CASMCs cultured with NG (D) and HG (E) after treatment with 0.1 μmol/L DXR (24 h), 10 μmol/L MG132 (4 h), or DXR (24 h) + MG132 (4 h). DHS-1 had no effect in rat CASMCs pretreated with DXR, but BK channel activation was preserved in cells treated with DXR + MG-132 (F).

After 24-h 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-h treatment with 10 μmol/L MG132 (a proteasome inhibitor). BK channel openings were less frequent with no response to DHS-1 in non-diabetic rat CASMCs after treatment with DXR (p=N.S. 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 co-transfection with ubiquitin and atrogin-1 or atrogin-1ΔF (a non-functional mutant with deletion of the F-box). 72-h 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 co-transfected with atrogin-1, while 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.

Figure 3. Mutation in the PDZ-binding motif of hSlo-β₁ prevented hSlo-β₁ ubiquitination and degradation.

Sequence alignment of human, rabbit, cattle, rat, and mouse KCNMB1 cDNA shows a conserved PDZ-binding motif (A). ▼: applied mutation site. 72-h after co-transfection with atrogin-1 and ubiquitin, Flag-hSlo-β₁V146A expression was significantly higher than that of Flag-hSlo-β₁ wt (B). Immunoprecipitates of anti-Flag antibody against HEK293 cell lysates with following transfection conditions: no transfection, Flag-hSlo-β₁/ubiquitin, Flag-hSlo-β₁/atrogin-1/ubiquitin, Flag-hSlo-β₁V146A/atrogin-1/ubiquitin and Flag-hSlo-β₁/atrogin-1ΔF/ubiquitin, were resolved and blotted against anti-ubiquitin antibody (C).

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 down-regulated 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, while total FOXO-3a protein remained unchanged (Figure 4A and 4B).

Figure 4. Modulation of vascular BK-β₁ expression by Akt/FOXO-3a/FBXO signaling pathway.

Reduced FOXO-3a(T-32) phosphorylation with unchanged total FOXO-3a expression in STZ-induced diabetic rats (A) and in human CASMCs cultured with HG (B). 24-h treatment with 7 μmol/L LY294002 (LY) attenuated the phosphorylation of Akt and FOXO-3a, enhanced the expression of total FOXO-3a and atrogin-1, and reduced BK-β₁ protein level in human CASMCs cultured with NG (C). Illustration showing the signaling mechanisms that underlie vascular BK-β₁ down-regulation in diabetes (D).

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 h incubation with 7 μmol/L LY294002 (a 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) while 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).

Discussion

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 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 down-regulation of BK-β₁ expression in vasculature was due 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 CASMs with HG were mimicked by PI3K/Akt inhibition. It is well known that PI3K/Akt pathway is activated by insulin (Ins) and suppressed by protein kinase C. Insulin receptor (InR) signaling is diminished and PKC 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.

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 down-regulated in vascular smooth muscle cells (SMCs) in diabetes, leading to loss of the β₁-med_iated channel fu_nction, 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.

Summary.

The BK-β₁ subunit plays a pivotal role in BK channel function by modulating channel voltage- and Ca²⁺-sensitivity. Down-regulation 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.

Non-standard Abbreviations and Acronyms

BK

large conductance Ca²⁺-activated K⁺ channel

BK-α

BK channel α subunit

BK-β₁

BK channel β₁ subunit

CASMC

coronary arterial smooth muscle cell

DXR

doxorubicin

FBXO

f-box only protein

FOXO

forkhead box O family transcription factor

HG

high glucose

IBTX

iberiotoxin

InR

insulin receptor

Ins

insulin

NG

normal glucose

siRNA

small interfering RNA

STZ

streptozotocin

UPS

ubiquitin-proteasome system