Enhancement of 26S proteasome functionality connects oxidative stress and vascular endothelial inflammatory response in diabetes mellitus

Abstract

Objective: Although the connection of oxidative stress and inflammation has been long recognized in diabetes mellitus, the underlying mechanisms are not fully elucidated. This study defined the role of 26S proteasomes in promoting vascular inflammatory response in early diabetes mellitus.

Methods and results: The 26S proteasome functionality, markers of autophagy, and unfolded protein response were assessed in (1) cultured 26S proteasome reporter cells and endothelial cells challenged with high glucose, (2) transgenic reporter (Ub(G76V)-green fluorescence protein) and wild-type (C57BL/6J) mice rendered diabetic, and (3) genetically diabetic (Akita and OVE26) mice. In glucose-challenged cells, and also in aortic, renal, and retinal tissues from diabetic mice, enhanced 26S proteasome functionality was observed, evidenced by augmentation of proteasome (chymotrypsin-like) activities and reduction in 26S proteasome reporter proteins, accompanied by increased nitrotyrosine-containing proteins. Also, whereas inhibitor of the nuclear factor κ-light-chain-enhancer of activated B cells α proteins were decreased, an increase was found in nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) nucleus translocation, which enhanced the NF-κB-mediated proinflammatory response, without affecting markers of autophagy or unfolded protein response. Importantly, the alterations were abolished by MG132 administration, small interfering RNA knockdown of PA700 (proteasome activator protein complex), or superoxide scavenging in vivo.

Conclusions: Early hyperglycemia enhances 26S proteasome functionality, not autophagy or unfolded protein response, through peroxynitrite/superoxide-mediated PA700-dependent proteasomal activation, which elevates NF- ĸB-mediated endothelial inflammatory response in early diabetes mellitus.

Figure 1. High glucose, not the osmotic control, reduces the poly-Ub-GFP protein levels, accompanied by enhanced 26S proteasome activity in Ub^G76V-GFP cells

The 26S proteasome reporter Ub^G76V-GFP cells were incubated with high concentration of D-glucose (25 mM) for 4h, in the presence or absence of proteasome inhibitor MG132 (5 μM, pre-incubation for 1h), using mannitol (25 mM 4h) or L-Glucose (25 mM 4h) as an osmotic control. Cells were (A) collected for Western blotting with a rabbit-derived GFP antibody or (B) subjected to immunofluorescent staining against GFP protein with a rabbit-derived GFP antibody and a goat-derived rabbit IgG antibody labeled with Alex Fluor 594. The reporter cells having been treated with D-glucose (25 mM) for up to 4h were subjected to (C) 26S proteasome (the Chymotrypsin-like) activity assay, or Western blotting with (D) rabbit-derived GFP antibody, or (E) individual antibody against PA700 (S10B), Grp94, LC3B, HSP90, HSP 70, and β-actin, respectively. All blots shown are representative of three independent experiments. The results (n=3) were analyzed with a one-way ANOVA. * indicates significant difference vs control.

Figure 2. Scavenging of peroxynitrite prevents both 26S proteasome activation and poly-Ub-GFP protein reduction by high glucose

The 26S proteasome reporter Ub^G76V-GFP cells were incubated with a high concentration of D-glucose (25 mM) for 4h, with pre-incubation for 1h with (A) L-NAME (2 mM); (B) mTempol (1 mM)); or (C) uric acid (100 μM), followed by cell lysates preparation for Western blotting with a rabbit-derived GFP antibody and a mouse-derived β-actin antibody. All blots shown are representative of three independent experiments. The results (n=3) were analyzed with a one-way ANOVA. * indicates significant difference vs control.

Figure 3. High glucose induces NF-κB nucleus translocation, which can be blocked by proteasome inhibition or superoxide scavenging in both Ub^G76V-GFP cells and primary endothelial cells

The 26S proteasome reporter Ub^G76V-GFP cells were incubated with a high concentration of D-glucose (25 mM) for 4h, with pre-incubation of mTempol (1 mM for 1h). The reporter cells were then subjected to (A) cell immunofluorescent staining using an NF-κB antibody or (B) isolation of the nucleus faction using a commercial kit followed by Western blotting with an NF-κB antibody and a histone antibody, both of which were rabbit-derived. Human umbilical vein endothelial cells (HUVEC) were incubated with a high concentration of D-glucose (25 mM) for 4h, with pre-incubation of the proteasome inhibitor MG132 (5 μM, pre-incubation for 1h), followed by fractionation (NE-PER^® Nuclear and Cytoplasmic Extraction Reagents Kit, Thermo Scientific) and Western blotting with the same aforementioned NF-κB antibody and histone antibody. All blots shown are representative of three independent experiments. The results (n=3) were analyzed with a one-way ANOVA. *P<0.05.

Figure 4. Ub^G76V-GFP mice present significant reduction in poly-Ub-GFP protein levels as well as elevated 26S proteasome activity in response to acute hyperglycemia, like the diabetic wild type mice or the genetic diabetic Akita and OVE26 mice

Male and age matched (10 wks) wild type (C57BL/6J) and transgenic (Ub^G76V-GFP) mice received STZ-regimen (STZ: 50mg/kg/d; vehicle: sodium citrate, pH 4.5; for 5d; n=5/group). Preparations of tissues obtained 7d post STZ regimen were subjected to (A) Western blotting with a rabbit-derived GFP antibody and a mouse-derived β-actin antibody, followed by (B) quantification of protein band densitometry for protein expression levels. The 26S proteasome (chymotrypsin-like) activity assay (in the presence of ATP) was performed in tissue preparations of the (C) wild type (C57BL/6J) and transgenic (Ub^G76V-GFP) mice and (D) Akita mice and OVE26 mice. Additional Akita mice were implanted with insulin pellets (4 days) which decreased the fasting blood glucose from 450 ± 18 to 135 ± 16 mg/dL (n=5) and aortic tissues were collected for (E) 26S proteasome (the Chymotrypsin-like) activity assay. The results (n=5/group) were analyzed with a one-way ANOVA. *P< 0.05.

Figure 5. Diabetic mice show increase in nitrotyrosine-containing proteins which is associated with increased 26S proteasome activity, likely mediated by increased tyrosine nitration of PA700

Male and age matched (10 wks) wild type (C57BL/6J) and transgenic (Ub^G76V-GFP) mice received STZ-regimen (STZ: 50mg/kg/d; vehicle: sodium citrate, pH 4.5; for 5d; n=5/group). Preparations of the indicated tissues obtained 7d post STZ regimen were subjected to Western blotting with a rabbit-derived anti-nitrotyrosine antibody and a mouse-derived β-actin antibody for (A) C57BL/6J mice and Ub^G76V-GFP mice, followed by quantification of protein band densitometry for levels of nitrotyrosine-containing protein. Male and age-matched (8 and 12 weeks, respectively) OVE26 mice and control FVB mice were used to detect the time course of (B) nitrotyrosine-containing protein levels and (C) 26S proteasome (chymotrypsin-like) activity in renal tissue homogenates. Some STZ-diabetic Ub^G76V-GFP mouse groups were further fed with mTempol (1 mM in drinking water for 4 weeks) and aortic tissues were stained with Western blot and quantified for (D) protein levels of total PA700 and nitrated PA700 after immunoprecipitation. The results (n=5/group) were analyzed with a one-way ANOVA. *P< 0.05 vs the vehicle-treated. mT: mTempol.

Figure 6. Diabetic mice demonstrate IκBα protein reduction and NF-κB activation, which can be reversed either by 26S proteasome inactivation or superoxide scavenging, without affecting markers of autophagy and unfolded protein response

Male and age matched (10 wks) wild type (C57BL/6J) mice received STZ-regimen (STZ: 50mg/kg/d; vehicle: sodium citrate, pH 4.5; for 5d; n=5/group). Preparations of the indicated tissues obtained 7d post STZ regimen were subjected to Western blotting with (A) a rabbit-derived IκBα antibody or individual antibodies against Grp78 or LC3B. Additional groups were further administrated with control siRNA or PA700 (S10B) (Vehicle/STZ mice, 25 μg/100μl of the control/PA700 siRNA in in vivo jetPEI^™ solution, single injection, retro-orbital; n=5/group) on next day of the last STZ dose. Aortic tissue preparations collected 7d after siRNA injections were subjected to (B) Western blotting with rabbit-derived antibodies respectively against PA700 (S10B) and IκBα, and a mouse-derived β-actin antibody, followed by protein band densitometry or to (C) 26S proteasome (chymotrypsin-like) activity assay. Additional groups were either treated with (D) MG132 injections (MG132: 5 mM/kg, vehicle: DMSO, i.p., 2d) or (E) mTempol (mTempol: 1 mM in drinking water, vehicle: normal drinking water, 4 weeks); 30 mg of lung tissues were subjected to ChIP assay (NF-κBp65), with an IgG antibody as the negative control and total DNA before immunoprecipitation as an input. PCR products of promoter specific primers were separated on a 1.2% agarose gel with respectively sizes of 202 bp (IkBα), 164bp (Ccl5) and 216 bp (ICAM-1). The results (n=3–5/group) were analyzed with a one-way ANOVA. *P< 0.05 vs the vehicle- or control siRNA-treated.