IKK1 aggravates ischemia-reperfusion kidney injury by promoting the differentiation of effector T cells

Ischemia-reperfusion injury (IRI) is one of the major causes of acute kidney injury (AKI), and experimental work has revealed detailed insight into the inflammatory response in the kidney. T cells and NFκB pathway play an important role in IRI. Therefore, we examined the regulatory role and mechanisms of IkappaB kinase 1 (IKK1) in CD4+T lymphocytes in an experimental model of IRI. IRI was induced in CD4cre and CD4IKK1Δ mice. Compared to control mice, conditional deficiency of IKK1 in CD4+T lymphocyte significantly decreased serum creatinine, blood urea nitrogen (BUN) level, and renal tubular injury score. Mechanistically, lack in IKK1 in CD4+T lymphocytes reduced the ability of CD4 lymphocytes to differentiate into Th1/Th17 cells. Similar to IKK1 gene ablation, pharmacological inhibition of IKK also protected mice from IRI. Together, lymphocyte IKK1 plays a pivotal role in IRI by promoting T cells differentiation into Th1/Th17 and targeting lymphocyte IKK1 may be a novel therapeutic strategy for IRI. </p

House dust mite-induced endoplasmic reticulum stress mediates MUC5AC hypersecretion via TBK1 in airway epithelium

Purpose: Endoplasmic reticulum (ER) stress regulates mucus hypersecretion, and may activate downstream factors via TBK1 signaling to induce gene expression. However, it remains unclear whether ER stress promotes airway mucus secretion through the TBK1 pathway. We aimed to investigate the role of the TBK1 pathway in the regulation of MUC5AC expression in a mouse model of house dust mite (HDM)-induced allergic asthma. Materials and Methods: Mice with HDM-induced asthma and human bronchial epithelial BEAS-2B cells were treated with amlexanox, an anti-allergy drug (25 μM), or 4-PBA (10 mM). Tissue and cell samples were collected. Tissue samples were stained with hematoxylin and eosin (H&E) or periodic acid Schiff (PAS) to evaluate pathology. Protein expression was analyzed by western blotting and immunofluorescence. Results: Mice exposed to HDM presented ER stress and hypersecretion of mucus Muc5ac from airway epithelial cells (p in vivo and in vitro studies revealed that HDM-induced ER stress induced MUC5AC overexpression via TBK1 signaling. Amlexanox and 4-PBA markedly reduced mucus production and weakened the TBK1 signal, which mediates MUC5AC hypersecretion. Conclusion: TBK1 plays a pivotal role in HDM-induced ER stress, leading to overproduction of MUC5AC in the asthmatic airway epithelium. The overproduction of MUC5AC can be significantly decreased by inhibiting TBK1 or ER stress using 4-PBA. These findings highlight potential target-specific therapies for patients with chronic allergic asthma.</p

Ubiquitin Carboxyl Terminal Hydrolyase L1 -Suppressed Autophagic Degradation of p21^WAF1/Cip1 as a Novel Feedback Mechanism in the Control of Cardiac Fibroblast Proliferation

<div><p>Aims</p><p>Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation; however, the potential roles of DUBs in the heart remain to be determined. This study was aimed to explore the role of a DUB, ubiquitin carboxyl terminal hydrolyase L1 (UCH-L1) in maladaptive cardiac remodeling and dysfunction.</p><p>Methods and Results</p><p>Maladaptive cardiac remodeling and dysfunction were induced in mice by transverse aortic constriction (TAC). UCH-L1 expression was transiently increased and then declined near to the basal level while impairment of cardiac function proceeded. The upregulation of UCH-L1 was observed in cardiac myocytes and fibroblasts. In primary culture of cardiac fibroblasts, UCH-L1 was upregulated by platelet-derived growth factor (PDGF)-BB and PDGF-DD. Adenoviral overexpession of UCH-L1 inhibited the PDGF-induced cardiac fibroblast proliferation without affecting the activation of mitogen activated protein kinases (MAPKs), Akt, and signal transducers and activators of transcription 3 (STAT3). Further signaling dissection revealed that PDGF-BB posttranscriptional upregulated p21^WAF1/Cip1 protein expression, which was inhibited by rapamycin, an activator of autophagy via suppressing mammalian target of rapamycin (mTOR), rather than MG132, a proteasome inhibitor. Overexpression of UCH-L1 enhanced PDGF-BB-induced mTOR phosphorylation and upregulation of p21^WAF1/Cip1 protein expression while suppressed autophagic flux in cardiac fibroblasts.</p><p>Conclusion</p><p>UCH-L1 facilitates PDGF-BB-induced suppression of autophagic degradation of p21^WAF1/Cip1 proteins in cardiac fibroblasts, which may serve as a novel negative feedback mechanism in the control of cardiac fibroblast proliferation contributing to cardiac fibrosis and dysfunction.</p></div

Role of UCH-L1 in the control of p21^WAF1/Cip1 (p21) protein expression in the presence of rapamycin in rat neonatal cardiac fibroblasts.

<p>Effect of UCH-L1 overexpression on PDGF-BB-induced mTOR phosphorylation and upregulation of p21^WAF1/Cip1 protein expression in the presence of rapamycin. Quiescent cells infected with Ad-control or Ad-UCH-L1 were pretreated with rapamycin (RAPA, 0.1 µM) or vehicle DMSO for 1 h, and followed with co-treatment of RAPA (0.1 µM) and PDGF-BB (20 ng/ml) for additional 24 h. Upper panel: representatives of immunoblotting. Lower panel: quantitatively densitometric analysis of protein expression. Data is presented as fold change of ratio of target protein to internal control β-actin relative to the Control (-) n = 4, *p<0.05. # p<0.05 vs. DMSO (-); & p<0.05 vs. DMSO (-) and PDGF-BB (+); @ p<0.05 vs. Ad-Controls. All results are representatives of at least 4 separated experiments.</p

PDGF-induced p21^WAF1/Cip1 (p21) expression.

<p><b>A</b>. PDGF-induced p21 mRNA expression in rat neonatal cardiac fibroblasts. n = 4. <b>B</b>. Effect of adenoviral UCH-L1 overexpression on PDGF-induced p21 mRNA expression in rat neonatal cardiac fibroblasts. Quiescent cells were treated with or without PDGF-BB (20 ng/ml) for 6 h. n = 4. ns, no statistical significance. <b>C</b>. Effect of adenoviral UCH-L1 overexpression on PDGF-induced p21 protein expression in rat neonatal cardiac fibroblasts. Quiescent cells infected with Ad-control or Ad-UCH-L1 were treated with or without PDGF-BB (20 ng/ml) for 24 h. n = 4, *p<0.05. The results are representatives of 4 separated experiments.</p

Effect of adenoviral UCH-L1 overexpression on ubiquitin proteasome system (UPS)-mediated degradation of p21^WAF1/Cip1 (p21) proteins in cardiac fibroblasts.

<p><b>A</b>. Effect of adenoviral UCH-L1 overexpression on MG132 and PDGF-induced upregulation of p21 protein expression in rat neonatal cardiac fibroblasts. Quiescent cells infected with Ad-control or Ad-UCH-L1 were treated with or without MG132 (0.5 µM) and PDGF-BB (20 ng/ml) for 24 h. Left panel: representatives of immunoblotting. Right panel: quantitatively densitometric analysis of protein expression. Data is presented as fold change of ratio of target protein to internal control β-actin relative to the Ad-Control (-). n = 4, *p<0.05. <b>B</b>. Effect of PDGF on p21 protein ubiquitination as well as interaction of UCH-L1 and p21 proteins in rat neonatal cardiac fibroblasts. Quiescent cells were treated with or without PDGF-BB (20 ng/ml) for 24 h. W, whole cell lysates; IP, immunoprecitated; IB, immunoblotted. Input, 10 µg of whole cell lysates subjected to IB. All results are representatives of at least 4 separated experiments.</p

Expression of UCH-L1 in the heart after TAC.

<p><b>A</b>. UCH-L1 mRNA expression in the left ventricles of mice after sham and TAC. n = 6, *p<0.05 vs. sham controls. <b>B</b>. Western blot analysis of UCH-L1 expression in the left ventricles of mice after sham and TAC. n = 3. <b>C</b>. UCH-L1 staining in the left ventricles of mice after sham and TAC. The results are representatives of 4 separated experiments.</p

Role of UCH-L1 in regulating cardiac fibroblast proliferation.

<p><b>A</b>. PDGF-induced UCH-L1 mRNA (<i>Left</i>) and protein (<i>Right</i>) expression in rat neonatal cardiac fibroblasts. Quiescent cells were treated with or without PDGF-AA (50 ng/ml), PGFD-BB (20 ng/ml), PDGF-CC (50 ng/ml), and PDGF-DD (50 ng/ml) for 24 h and subjected to Western blot analysis. n = 4, *p<0.05 vs SF controls. <b>B</b>. CCK-8, cell counting and Ki67 staining detected the role of UCH-L1 in regulating PDGF-induced rat neonatal cardiac fibroblast proliferation. n = 4, *p<0.05 vs. Ad-controls. Scale bar, 50 µm.</p

Role of autophagy in the control of p21^WAF1/Cip1 (p21) protein expression in rat neonatal cardiac fibroblasts.

<p>Left panel: representatives of immunoblotting. Right panel: quantitatively densitometric analysis of protein expression. Data is presented as fold change of ratio of target protein to internal control β-actin relative to the Control (-). <b>A</b>. Effect of rapamycin on PDGF-BB-induced p21^WAF1/Cip1 protein expression and mTOR phosphorylation. Quiescent cells were pretreated with rapamycin (RAPA, 0.1 µM) or vehicle DMSO for 1 h, and followed with co-treatment of RAPA (0.1 µM) and PDGF-BB (20 ng/ml) for additional 24 h. n = 4, *p<0.05. # p<0.05 vs. DMSO (-), & p<0.05 vs. DMSO (-) and PDGF-BB (-). <b>B</b>. Effect of UCH-L1 overexpression on PDGF-BB-induced phosphorylation of mTOR and GSK. Quiescent cells infected with Ad-control or Ad-UCH-L1 were treated with vehicle or PDGF-BB (20 ng/ml) for 2 h. n = 4, *p<0.05. <b>C</b>. Effect of UCH-L1 overexpression on rapamycin-induced suppression of mTOR phosphorylation and downregulation of p21^WAF1/Cip1 protein expression. Quiescent cells infected with Ad-control or Ad-UCH-L1 were treated with vehicle or rapamycin (RAPA, 0.1 µM) for 24 h. n = 4, *p<0.05. All results are representatives of at least 4 separated experiments.</p

Role of UCH-L1 in autophagic clearance of p21^WAF1/Cip1 in rat neonatal cardiac fibroblasts.

<p><b>A</b>. Effect of adenoviral overexpression of UCH-L1 on bafilomycin A1 (BFA)-induced accumulation of LC3. Quiescent cells infected with adenovirus of GFP control or UCH-L1 were treated with or without BFA (5 nM) for 24 h. Left panel: representatives of immunoblotting. Right panel: quantitatively densitometric analysis of protein expression. Data is presented as fold change of ratio of target protein to internal control β-actin relative to the Control (-). n = 4, *p<0.05. <b>B</b>. Effect of BFA on UCH-L1-mediated upregulation of p21^WAF1/Cip1. Quiescent infected cells as indicated were treated with or without PDGF-BB (20 ng/ml) in the presence of BFA (5 nM) for 24 h. Upper panel: representatives of immunoblotting. Lower panel: quantitatively densitometric analysis of protein expression. Data is presented as fold change of ratio of target protein to internal control β-actin relative to the Control (-). n = 4, *p<0.05. All results are representatives of at least 4 separated experiments.</p