DPP-4 Inhibitor Attenuates Toxic Effects of Indoxyl Sulfate on Kidney Tubular Cells

Diabetic nephropathy is a common causative factor of chronic kidney disease (CKD). DPP-4 inhibitor has the ability to improve kidney function and renal microvasculature. In the present study, we investigate the deleterious effects of IS on proximal tubular cells and the protective role of DPP-4 inhibitor. Human kidney 2 (HK-2) cells were exposed to IS in the presence or absence of DPP-4 inhibitor. Effects of DPP-4 inhibitor on viability of HK-2 cells were determined by MTT assay. Reactive oxygen species (ROS) production was examined using fluorescent microscopy. Levels of cleaved caspase-3, transforming growth factor-beta (TGF-β), α-smooth muscle actin (α-SMA) and NF-kappaB p65 and phosphorylation of AKT and extracellular signal-regulated kinase (ERK) were detected by immunoblotting. Production of ROS and level of cleaved caspase-3 were increased by IS in a dose-dependent manner. The phosphorylation of AKT and ERK p65 were decreased alongside activation of NF-κB. Expression of TGF-β and α-SMA, were upregulated in IS-treated HK-2 cells. Treatment with DPP-4 inhibitor resulted in a significant increase in cell viability and a decrease of ROS production in IS-treated HK-2 cells. DPP-4 inhibitor restored IS-induced deactivations of AKT and ERK and inhibited activation of NF-κB in IS-treated HK-2 cells. Moreover, DPP-4 inhibitor could also attenuate IS-induced up-regulation of TGF-β and α-SMA expression. These findings suggest that DPP-4 inhibitor possesses anti-apoptotic activity to ameliorate the IS-induced renal damage, which may be partly attributed to regulating ROS/p38MAPK/ERK and PI3K-AKT pathways as well as downstream NF-κB signaling pathway.     

Indoxyl sulfate promotes vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A through oxidative stress

We previously demonstrated that indoxyl sulfate (IS), a uremic toxin, induces aortic calcification in hypertensive rats and induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells. This study aimed to clarify whether IS stimulates senescence of cultured human aortic smooth muscle cells (HASMCs) and aorta in Dahl salt-sensitive hypertensive rats and whether AST-120, an oral sorbent, prevents senescence of aorta in subtotally nephrectomized uremic rats. IS increased the mRNA expression of p53 and p21 in HASMCs, whereas it did not change that of p16 and retinoblastoma protein (pRb). The IS-induced expression of p53 and p21 was suppressed by N-acetylcysteine, an antioxidant. IS promoted protein expression of p53, p21, and senescence-associated β-galactosidase (SA-β-gal) activity in HASMCs, and N-acetylcysteine and pifithrin-α,p-nitro, a p53 inhibitor, blocked these effects. IS upregulated prelamin A, a hallmark of vascular smooth muscle cell senescence, and downregulated FACE1/Zempste24 protein expression in HASMCs, and N-acetylcysteine suppressed these effects. Administration of IS to hypertensive rats increased expression of SA-β-gal, p53, p21, prelamin A, and oxidative stress markers such as 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in the cells embedded in the calcification area of arcuate aorta. Further, the uremic rat model showed positive staining for SA-β-gal, p53, p21, prelamin A, 8-OHdG, and MDA in the cells embedded in the calcification area of arcuate aorta, whereas AST-120 reduced the expression of these biomarkers. Taken together, IS accelerates vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A and downregulation of FACE1 through oxidative stress.     

Rosuvastatin Attenuates CD40L-Induced Downregulation of Extracellular Matrix Production in Human Aortic Smooth Muscle Cells via TRAF6-JNK-NF-κB Pathway

CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration in an atherosclerotic plaque. Statins can decrease both the CD40 expression and the resulting inflammation. However, the effects of CD40L and stains on atherosclerotic plaque ECM production and the underlying mechanisms are not well established. Moreover, prolyl-4-hydroxylase α1 (P4Hα1) is involved in collagen synthesis but its correlations with CD40L and statins are unknown. In the present study, CD40L suppressed P4Hα1 expression in human aortic smooth muscle cells (HASMCs) in a dose- and time-dependent manner, with insignificant changes in MMP2 expression and negative enzymatic activity of MMP9. CD40L increased TRAF6 expression, JNK phosphorylation, NF-κB nuclear translocation as well as DNA binding. Furthermore, silencing TRAF6, JNK or NF-κB genes abolished CD40L-induced suppression of P4Hα1. Lower NF-κB nuclear import rates were observed when JNK or TRAF6 silenced HASMCs were stimulated with CD40L compared to HASMCs with active JNK or TRAF6. Together, these results indicate that CD40L suppresses P4Hα1 expression in HASMCs by activating the TRAF6-JNK- NF-κB pathway. We also found that rosuvastatin inhibits CD40L-induced activation of the TRAF6-JNK- NF-κB pathway, thereby significantly rescuing the CD40L stimulated P4Hα1 inhibition. The results from this study will help find potential targets for stabilizing vulnerable atherosclerotic plaques.     

Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase,GCN5

NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.     

Norisoboldine,an Anti-Arthritis Alkaloid Isolated from Radix Linderae,Attenuates Osteoclast Differentiation and Inflammatory Bone Erosion in an Aryl Hydrocarbon Receptor-Dependent Manner

Norisoboldine (NOR), the primary isoquinoline alkaloid constituent of the root of Lindera aggregata, has previously been demonstrated to attenuate osteoclast (OC) differentiation. Accumulative evidence has shown that aryl hydrocarbon receptor (AhR) plays an important role in regulating the differentiation of various cells, and multiple isoquinoline alkaloids can modulate AhR. In the present study, we explored the role of NOR in the AhR signaling pathway. These data showed that the combination of AhR antagonist resveratrol (Res) or α-naphthoflavone (α-NF) nearly reversed the inhibition of OC differentiation through NOR. NOR could stably bind to AhR, up-regulate the nuclear translocation of AhR, and enhance the accumulation of the AhR-ARNT complex, AhR-mediated reporter gene activity and CYP1A1 expression in RAW 264.7 cells, suggesting that NOR might be an agonist of AhR. Moreover, NOR inhibited the nuclear translocation of NF-κB-p65, resulting in the evident accumulation of the AhR-NF-κB-p65 complex, which could be markedly inhibited through either Res or α-NF. Although NOR only slightly affected the expression of HIF-1α, NOR markedly reduced VEGF mRNA expression and ARNT-HIF-1α complex accumulation. In vivo studies indicated that NOR decreased the number of OCs and ameliorated the bone erosion in the joints of rats with collagen-induced arthritis, accompanied by the up-regulation of CYP1A1 and the down-regulation of VEGF mRNA expression in the synovium of rats. A combination of α-NF nearly completely reversed the effects of NOR. In conclusion, NOR attenuated OC differentiation and bone erosion through the activation of AhR and the subsequent inhibition of both NF-κB and HIF pathways.     

Involvement of Rab28 in NF-κB Nuclear Transport in Endothelial Cells

Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP) were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB). Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs) through a paracrine control of vascular smooth muscles cells (VSMCs), which at least partly via angiotensin II (Ang II). Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.     

Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) and Nuclear Factor-κB (NF-κB): A FEED-FORWARD LOOP FOR SYSTEMIC AND VASCULAR INFLAMMATION*

The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation. However, the mechanisms underlying these effects are not fully understood. Because of the fundamental role that inflammation plays in vascular diseases, we hypothesized that EBP50 mediates macrophage activation and the response of vessels to inflammation. Indeed, EBP50 expression increased in primary macrophages and VSMC, and in the aorta of mice, upon treatment with LPS or TNFα. This increase was nuclear factor-κB (NF-κB)-dependent. Conversely, activation of NF-κB was impaired in EBP50-null VSMC and macrophages. We found that inflammatory stimuli promote the formation of an EBP50-PKCζ complex at the cell membrane that induces NF-κB signaling. Macrophage activation and vascular inflammation after acute LPS treatment were reduced in EBP50-null cells and mice as compared with WT. Furthermore, macrophage recruitment to vascular lesions was significantly reduced in EBP50 knock-out mice. Thus, EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation.     

Lipopolysaccharide Exposure during Pregnancy Leads to Aortic Dysfunction in Offspring Rats

Background

Prenatal exposure to Lipopolysaccharide (LPS) produces hypertension in adult offspring rats. The present study was to explore the effects of prenatal inflammation on morphological and functional changes in the aorta from offspring rats and to further assess its susceptibility to cardiovascular diseases.

Methods and Results

Pregnant rats were treated intraperitoneally on gestation Days 8, 10 and 12 with saline, LPS (0.79 mg/kg), or pyrrolidine dithiocarbamate (PDTC, 100 mg/kg)+LPS, respectively. Aortic ring reactivity and histopathological alteration were analyzed in offspring at the age of 12 weeks. The detections of connexin (Cx) 37, Cx40, Cx43, and Cx45, including immunofluorescent patterns, protein levels and mRNA expression in the aorta, were performed as well. Furthermore, the expressions of Nuclear factor (NF)-κB (p65), IκBα, phospho-IκBα and IκBβ were determined. The results showed that prenatal LPS exposure leads to morphological abnormalities and impaired aortic reactivity in offspring. Prenatal LPS exposure also decreased the protein and mRNA expression of Cx37 in the aorta from offspring rats. NF-κB and phospho-IκBα levels were both increased, IκBα level, however, was decreased in the aorta of offspring from the maternal LPS exposure compared to the controls. Simultaneously, PDTC treatment markedly reversed the action of LPS.

Conclusions

Decreased expression of Cx37 contributed to the aortic dysfunction of prenatal LPS exposure offspring, which should be associated with NF-κB activation.     

Dual Regulation of Myocardin Expression by Tumor Necrosis Factor-α in Vascular Smooth Muscle Cells

De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.     

Cyclophilin A (CypA) Interacts with NF-κB Subunit,p65/RelA,and Contributes to NF-κB Activation Signaling

Background

Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects.

Methodology

Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes.

Results

GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-κB subunit p65 at the N-terminal 170–176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity.

Conclusions

Our findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant activation of NF-κB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases.