The Corticotropin-Releasing Factor (CRF) family of neuropeptides via the CRHR2 receptors induces the expression of Toll Like Receptor-4 (TLR4) expression in macrophages through activation of the transcription factor PU.1

Corticotropin-Releasing Factor (CRF) augments lipopolysaccharide (LPS) -induced production of cytokines by macrophages. Toll like receptor-4 (TLR4) mediates LPS-induced cytokine production by macrophages. Expression of TLR4 is primarily regulated by the transcription factor PU.1. Aim of the present study was to determine the role of CRF and its related peptides Urocortin 1 (UCN1) and Urocortin 2 (UCN2) on TLR4 expression and PU.1 activation in macrophages. Our data are as follows: (a) Exposure of Raw264.7 macrophages to CRF, UCN1 or UCN2 increased TLR4 mRNA by semi-quantitative RT PCR while, as expected, LPS lowered it. Quantitative real-time PCR confirmed these data since CRH increased the levels of TLR4 mRNA by a 3,2±0,22 fold (mean±sem), UCN1 by 2,2±0,08 and UCN2 by 2,5±0,02 while LPS reduced it to 0,75±0,1. (b) To determine whether the effect of the CRH neuropeptides on TLR4 expression occurred at the transcriptional level, Raw264.7 cells were transfected with a construct containing the minimal promoter of mouse TLR4 linked to the luciferase gene. CRH induced the TLR4 promoter at 18,35±2,6 luciferase units compared to 3,4±0,44 of parallel controls while UCN1 induced the TLR4 promoter to 19,18±0,87 and UCN2 to 19,34±1,5. The CRH neuropeptides partly reversed the negative effect of LPS on TLR4 promoter activation since LPS reduced TLR4 promoter activity to 6,2±1,1 luciferase units compared to 16±0,7 of untreated cells, while co-treatment with UCN1 resulted in activation of the TLR4 promoter to 9,8±0,31, co-treatment with UCN2 to 10,1±0,8 and co-treatment with CRF to 11,8±1,8. Real Time RT PCR confirmed the semi-quantitative RT PCR data. (c) The effects of CRH peptides on TLR4 expression were blocked by the CRF2 receptor antagonist anti-sauvagine30. (d) TLR4 expression is primarily controlled by the transcription factor PU.1. We examined the effect of the CRH neuropeptides on PU.1. Western blot analysis showed that the levels of nuclear PU.1 in Raw264.7 cells increased following exposure to CRF, UCN1 or UCN2. Translocation of PU.1 into the nucleus was also assessed by immunofluorescence analysis, which confirmed the Western blot data. Finally, CRF, UCN1 and UCN2 increased PU.1 DNA binding activity as assessed by a functional assay of PU.1 DNA binding activity in nuclear lysates. In conclusion, our data suggest that CRF peptides modify LPS-induced macrophage activation by inducing the expression of TLR4 through activation of the transcription factor PU.1 via CRF2 receptors.