High Glucose Induces CCL20 in Proximal Tubular Cells via Activation of the KCa3.1 Channel

Background

Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-β1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-β1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN.

Methods

Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34.

Results

In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB.

Conclusions

Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.     

The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation

Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIP_L) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIP_L regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIP_L, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIP_L but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIP_L specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.     

Hyperuricemia Causes Pancreatic β-Cell Death and Dysfunction through NF-κB Signaling Pathway

Accumulating clinical evidence suggests that hyperuricemia is associated with an increased risk of type 2 diabetes. However, it is still unclear whether elevated levels of uric acid can cause direct injury of pancreatic β-cells. In this study, we examined the effects of uric acid on β-cell viability and function. Uric acid solution or normal saline was administered intraperitoneally to mice daily for 4 weeks. Uric acid-treated mice exhibited significantly impaired glucose tolerance and lower insulin levels in response to glucose challenge than did control mice. However, there were no significant differences in insulin sensitivity between the two groups. In comparison to the islets in control mice, the islets in the uric acid–treated mice were markedly smaller in size and contained less insulin. Treatment of β-cells in vitro with uric acid activated the NF-κB signaling pathway through IκBα phosphorylation, resulting in upregulated inducible nitric oxide synthase (iNOS) expression and excessive nitric oxide (NO) production. Uric acid treatment also increased apoptosis and downregulated Bcl-2 expression in Min6 cells. In addition, a reduction in insulin secretion under glucose challenge was observed in the uric acid–treated mouse islets. These deleterious effects of uric acid on pancreatic β-cells were attenuated by benzbromarone, an inhibitor of uric acid transporters, NOS inhibitor L-NMMA, and Bay 11–7082, an NF-κB inhibitor. Further investigation indicated that uric acid suppressed levels of MafA protein through enhancing its degradation. Collectively, our data suggested that an elevated level of uric acid causes β-cell injury via the NF-κB-iNOS-NO signaling axis.     

Shikonin causes cell-cycle arrest and induces apoptosis by regulating the EGFR–NF-κB signalling pathway in human epidermoid carcinoma A431 cells

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal Zicao, has been shown to exhibit antioxidant and anticancer effects. In the present study, we investigated the antiproliferative and pro-apoptotic effects of shikonin on A431 cells and explored the underlying molecular mechanisms. In the present study, our results showed that shikonin significantly inhibited the growth of A431 cells in a concentration- and time-dependent manner, and caused cell cycle arrest by upregulation of p21 and p27, and downregulation of cyclins and cyclin-dependent kinases. In addition, shikonin evidently induced apoptosis due to decreasing Bcl-2 expression, increasing Bax expression, activating caspase and inactivating NF-κB, while pretreatment with a pan-caspase inhibitor Z-Asp-CH2-DCB abrogated shikonin-induced apoptosis. Moreover, EGF could significantly increase the NF-κB DNA-binding activity and reversed the shikonin-induced inactivation of NF-κB. As anticipated AG1478 (EGFR inhibitor) and Bay11-7082 (NF-κB inhibitor) blocked EGF-reversed the inactivation of NF-κB induced by shikonin. Our data also showed that EGF could evidently reverse the shikonin-induced decreases in cell viability and increases in apoptosis. Then, the NF-κB inhibitors such as Bay11-7082, SN50, Helenalin and the EGFR inhibitor AG1478 and its downstream inhibitor such as PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and STAT3 inhibitor Stattic dramatically blocked EGF-reversed decreases in cell viability and increases in apoptosis induced by shikonin. Collectively, our findings indicated that shikonin inhibited cell growth and caused cell cycle arrest of the A431 cells through the regulation of apoptosis. Moreover, these effects were mediated at least partially by suppressing the activation of the EGFR–NF-κB signaling pathways.     

Itch and autophagy-mediated NF-κB activation contributes to inhibition of cathepsin D-induced sensitizing effect on anticancer drugs

Inhibition of cathepsin D (Cat D) sensitizes cancer cells to anticancer drugs via RNF183-mediated downregulation of Bcl-xL expression. Although NF-κB activation is involved in the upregulation of RNF183 expression, the molecular mechanism of NF-κB activation by Cat D inhibition is unknown. We conducted this study to investigate the molecular mechanism underlying Cat D-mediated NF-κB activation. Interestingly, Cat D inhibition-induced IκB degradation in an autophagy-dependent manner. Knockdown of autophagy-related genes (ATG7 and Beclin1) and lysosome inhibitors (chloroquine and bafilomycin A1) blocked IκB degradation via Cat D inhibition. Itch induced K63-linked ubiquitination of IκB and then modulated the protein stability of IκB by Cat D inhibition. Inhibition of Cat D-mediated Itch activation was modulated by the JNK signaling pathway, and phosphorylated Itch could bind to IκB, resulting in polyubiquitination of IκB. Additionally, inhibition of Cat D increased autophagy flux via activation of the LKB1-AMPK-ULK1 pathway. Therefore, our results suggested that Cat D inhibition activated NF-κB signaling via degradation of autophagy-dependent IκB, which is associated with the upregulation of RNF183, an E3 ligase of Bcl-xL. Cat D inhibition enhances TRAIL-induced apoptosis through Bcl-xL degradation via upregulation of RNF183.Subject terms: Chemotherapy, Ubiquitylation, Autophagy     

Crotepoxide Chemosensitizes Tumor Cells through Inhibition of Expression of Proliferation,Invasion, and Angiogenic Proteins Linked to Proinflammatory Pathway

Crotepoxide (a substituted cyclohexane diepoxide), isolated from Kaempferia pulchra (peacock ginger), although linked to antitumor and anti-inflammatory activities, the mechanism by which it exhibits these activities, is not yet understood. Because nuclear factor κB (NF-κB) plays a critical role in these signaling pathways, we investigated the effects of crotepoxide on NF-κB-mediated cellular responses in human cancer cells. We found that crotepoxide potentiated tumor necrosis factor (TNF), and chemotherapeutic agents induced apoptosis and inhibited the expression of NF-κB-regulated gene products involved in anti-apoptosis (Bcl-2, Bcl-xL, IAP1,² MCl-1, survivin, and TRAF1), apoptosis (Bax, Bid), inflammation (COX-2), proliferation (cyclin D1 and c-myc), invasion (ICAM-1 and MMP-9), and angiogenesis (VEGF). We also found that crotepoxide inhibited both inducible and constitutive NF-κB activation. Crotepoxide inhibition of NF-κB was not inducer-specific; it inhibited NF-κB activation induced by TNF, phorbol 12-myristate 13-acetate, lipopolysaccharide, and cigarette smoke. Crotepoxide suppression of NF-κB was not cell type-specific because NF-κB activation was inhibited in myeloid, leukemia, and epithelial cells. Furthermore, we found that crotepoxide inhibited TAK1 activation, which led to suppression of IκBα kinase, abrogation of IκBα phosphorylation and degradation, nuclear translocation of p65, and suppression of NF-κB-dependent reporter gene expression. Overall, our results indicate that crotepoxide sensitizes tumor cells to cytokines and chemotherapeutic agents through inhibition of NF-κB and NF-κB-regulated gene products, and this may provide the molecular basis for crotepoxide ability to suppress inflammation and carcinogenesis.     

Infected Cell Killing by HIV-1 Protease Promotes NF-κB Dependent HIV-1 Replication

Acute HIV-1 infection of CD4 T cells often results in apoptotic death of infected cells, yet it is unclear what evolutionary advantage this offers to HIV-1. Given the independent observations that acute T cell HIV-1 infection results in (1) NF-κB activation, (2) caspase 8 dependent apoptosis, and that (3) caspase 8 directly activates NF-κB, we questioned whether these three events might be interrelated. We first show that HIV-1 infected T cell apoptosis, NF-κB activation, and caspase 8 cleavage by HIV-1 protease are coincident. Next we show that HIV-1 protease not only cleaves procaspase 8, producing Casp8p41, but also independently stimulates NF-κB activity. Finally, we demonstrate that the HIV protease cleavage of caspase 8 is necessary for optimal NF-κB activation and that the HIV-1 protease specific cleavage fragment Casp8p41 is sufficient to stimulate HIV-1 replication through NF-κB dependent HIV-LTR activation both in vitro as well as in cells from HIV infected donors. Consequently, the molecular events which promote death of HIV-1 infected T cells function dually to promote HIV-1 replication, thereby favoring the propagation and survival of HIV-1.     

Expression of GITR Enhances Multiple Myeloma Cell Sensitivity to Bortezomib

Recently tumor necrosis factor receptor super family member 18 (TNFRSF18, also called GITR) has been identified as a novel tumor suppressor gene in Multiple Myeloma (MM), undergoing aberrant DNA methylation-mediated gene expression silencing. Furthermore, the expression of GITR blocks canonical NF-κB activation in MM cells in response to TNFα. Bortezomib, a proteasome inhibitor, can induce NF-κB activation, which may significantly influence the drug response in MM patients. In this study, we aim to elucidate if GITR status is associated with response to Bortezomib in MM cells through regulating GITR mediated NF-κB blockade. We found that GITR was significantly downregulated in MM patients and cell lines. Overexpression of GITR inhibited non-canonical NF-κB activation induced by TNFα. Moreover, NF-κB inhibitor induced apoptosis in GITR-deficient MM cells in response to TNFα. In addition, overexpression of GITR could inhibit Bortezomib-induced NF-κB activation and enhance the cytotoxicity of Bortezomib in GITR-deficient MM cell line (MM1.S). In contrast, knockdown of GITR attenuated the cytotoxic effect of Bortezomib on GITR proficient MM (RPMI) cell line and increased NF-κB activation. Finally, overexpression of GITR enhanced the sensitivity to Bortezomib in co-culture with bone marrow stromal cells and significantly reduced the tumor growth in MM1.S xenograft mice. In conclusion, we demonstrated that GITR expression can enhance the sensitivity to Bortezomib by inhibiting Bortezomib-induced NF-κB activation.     

Effects of SGLT2 Inhibition in Human Kidney Proximal Tubular Cells—Renoprotection in Diabetic Nephropathy?

Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3.     

The Role of the Phylogenetically Conserved Cochaperone Protein Droj2/DNAJA3 in NF-κB Signaling

The NF-κB pathway is a phylogenetically conserved signaling pathway with a central role in inflammatory and immune responses. Here we demonstrate that a cochaperone protein, Droj2/DNAJA3, is involved in the activation of canonical NF-κB signaling in flies and in human cultured cells. Overexpression of Droj2 induced the expression of an antimicrobial peptide in Drosophila. Conversely, Droj2 knockdown resulted in reduced expression of antimicrobial peptides and higher susceptibility to Gram-negative bacterial infection in flies. Similarly, Toll-like receptor-stimulated IκB phosphorylation and NF-κB activation were suppressed by DNAJA3 knockdown in HEK293 cells. IκB kinase overexpression-induced NF-κB phosphorylation was also compromised in DNAJA3 knockdown cells. Our study reveals a novel conserved regulator of the NF-κB pathway acting at the level of IκB phosphorylation.     

Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-κB Interaction

Aortic valve calcification causes the progression of calcific aortic valve disease (CAVD). Stimulation of aortic valve interstitial cells (AVICs) with lipopolysaccharide (LPS) up-regulates the expression of osteogenic mediators, and NF-κB plays a central role in mediating AVIC osteogenic responses to Toll-like receptor 4 (TLR4) stimulation. Diseased aortic valves exhibit greater levels of oxidized low-density lipoprotein (oxLDL). This study tested the hypothesis that oxLDL augments the osteogenic responses in human AVICs through modulation of NF-κB and Notch1 activation. AVICs isolated from normal human aortic valves were treated with LPS (0.1 µg/ml), oxLDL (20 µg/ml) or LPS plus oxLDL for 48 h. OxLDL alone increased cellular bone morphogenetic protein-2 (BMP-2) levels while it had no effect on alkaline phosphatase (ALP) levels. Cells exposed to LPS plus oxLDL produced higher levels of BMP-2 and ALP than cells exposed to LPS alone. Further, LPS plus oxLDL induced greater NF-κB activation, and inhibition of NF-κB markedly reduced the expression of BMP-2 and ALP in cells treated with LPS plus oxLDL. OxLDL also induced Notch1 activation and resulted in augmented Notch1 activation when it was combined with LPS. Inhibition of Notch1 cleavage attenuated NF-κB activation induced by LPS plus oxLDL, and inhibition of NF-κB suppressed the expression of BMP-2 and ALP induced by the synergistic effect of Jagged1 and LPS. These findings demonstrate that oxLDL up-regulates BMP-2 expression in human AVICs and synergizes with LPS to elicit augmented AVIC osteogenic responses. OxLDL exerts its effect through modulation of the Notch1-NF-κB signaling cascade. Thus, oxLDL may play a role in the mechanism underlying CAVD progression.