Long-term treatment with EGFR inhibitor erlotinib attenuates renal inflammatory cytokines but not nephropathy in Alport syndrome mouse model.

BACKGROUND: Alport syndrome (AS) is a hereditary kidney disease caused by mutation of type IV collagen. Loss of collagen network induces collapse of glomerular basement membrane (GBM) structure. The previous studies showed that upregulation of some tyrosine kinase receptors signaling accompanied GBM disorder in AS mouse model. EGFR signaling is one of the well-known receptor kinase signaling that is involved in glomerular diseases. However, whether EGFR signaling is relevant to AS progression is still uninvestigated. Here, we determined the involvement of EGFR in AS and the effect of suppressing EGFR signaling by erlotinib treatment on AS progression. METHODS: Phosphorylated EGFR expression was investigated by Western blotting analysis and immunostaining of kidney tissues of Col4a5 mutant mice (a mouse model of X-linked AS). To check the effect of blocking EGFR signaling in AS, we administered erlotinib to AS mice once a day (10 mg/kg/day) orally for 18 weeks. Renal function parameters (proteinuria, serum creatinine, and BUN) and renal histology were assessed, and the gene expressions of inflammatory cytokines were analyzed in renal tissues. RESULTS: Phosphorylated EGFR expression was upregulated in AS mice kidney tissues. Erlotinib slightly reduced the urinary protein and suppressed the expression of renal injury markers (Lcn2, Lysozyme) and inflammatory cytokines (Il-6, Il-1ß and KC). Erlotinib did not improve renal pathology, such as glomerular sclerosis and fibrosis. CONCLUSION: These findings suggest that EGFR signaling is upregulated in kidney, but although inhibiting this signaling pathway suppressed renal inflammatory cytokines, it did not ameliorate renal dysfunction in AS mouse model.

The transcription factor MEF/Elf4 is dually modulated by p53-MDM2 axis and MEF-MDM2 autoregulatory mechanism.

Myeloid Elf-1-like factor (MEF) or Elf4 is an ETS transcription factor that activates innate immunity-associated genes such as lysozyme (LYZ), human ß-defensin 2 (HßD2), and interleukin-8 (IL-8) in epithelial cells and is also known to influence cell cycle progression. MEF is transcriptionally activated by E2F1, but the E2F1-mediated transcriptional activation is inhibited by p53 through E2F1-p53 protein interaction. Although the transcriptional activation of MEF has been investigated in depth, its post-translational regulation is not well explored. By overexpressing MEF cDNA in human cell lines, here we show that MEF protein expression is suppressed by p53. By screening a number of E3 ligases regulated by p53, we found that MDM2 is involved in the effect of p53 on MEF. MDM2 is transcriptionally activated by p53 and interacts with MEF protein to enhance MEF degradation. MDM2 reduces MEF protein expression, as well as stability and function of MEF as transcriptional activator. Furthermore, MDM2 was able to down-regulate MEF in the absence of p53, indicating a p53-independent effect on MEF. Notably, MEF transcriptionally activates MDM2, which was previously demonstrated to be the mechanism by which MEF suppresses the p53 protein. These results reveal that in addition to the potential of MEF to down-regulate p53 by transcriptionally activating E3 ligase MDM2, MEF participates with MDM2 in a novel autoregulatory feedback loop to regulate itself. Taken together with the findings on the effect of p53 on MEF, these data provide evidence that the p53-MDM2-MEF axis is a feedback mechanism that exquisitely controls the balance of these transcriptional regulators.

Rb/E2F1 regulates the innate immune receptor Toll-like receptor 3 in epithelial cells.

Tumor suppressor genes regulate the antiviral host defense through molecular mechanisms that are not yet well explored. Here, we show that the tumor suppressor retinoblastoma (Rb) protein positively regulates Toll-like receptor 3 (TLR3) expression, the sensing receptor for viral double-stranded RNA and poly(I · C). TLR3 expression was lower in Rb knockout (Rb(-/-)) mouse embryonic fibroblasts (MEF) and in mammalian epithelial cells transfected with Rb small-interfering RNA (siRNA) than in control cells. Consequently, induction of cytokines interleukin-8 and beta interferon after poly(I · C) stimulation was impaired in Rb(-/-) MEF and Rb siRNA-transfected cells compared to controls. TLR3 promoter analysis showed that Rb modulates the transcription factor E2F1, which directly binds to the proximal promoter of TLR3. Exogenous addition of E2F1 decreased TLR3 promoter activity, while Rb dose dependently curbed the effect of E2F1. Interestingly, poly(I · C) increased the Rb expression, and the poly(I · C)-induced TLR3 expression was impaired in Rb-depleted cells, suggesting the importance of Rb in TLR3 induction by poly(I · C). Together, these data indicated that E2F1 suppresses TLR3 transcription, but during immune stimulation, Rb is upregulated to block the inhibitory effect of E2F1 on TLR3, highlighting a role of Rb-E2F1 axis in the innate immune response in epithelial cells.