1A). We found that PS-5 and, at a lower extent, KIR peptide significantly reduced IFN-γRα phosphorylation. In addition, PS-5 impaired JAK2 phosphorylation, as well as STAT1 phosphorylation at the tyrosine 701 residue. In contrast, STAT1 phosphorylation at serine 727 residue, which is constitutively detected in keratinocyte cultures, was not affected either by PS-5 or KIR. As a direct consequence Selleck X-396 of STAT1 inactivation, the expression of IRF1, which is induced by
IFN-γ in late phase, was reduced in IFN-γ-activated keratinocytes treated with KIR or, more efficiently, with PS-5. We further evaluated the effect of PS-5 peptide on STAT1 transcriptional activity (Fig. 1B). To this end, keratinocyte cultures were transfected with a STAT1-responsive plasmid, pGAS-Luc, pretreated or not with the SOCS1 mimetics and then, stimulated with IFN-γ. In line with data previously described, we found
that PS-5 impaired the luciferase activity of pGAS-Luc as compared with irrelevant peptide. To evaluate the selectivity of PS-5 on JAK2 activity, we also analyzed the activation of ERK1/2, whose phosphorylation and activity are strongly induced by IFN-γ in primary cultures of keratinocytes. Interestingly, we found that PS-5 did not affect significantly ERK1/2 phosphorylation, as well as basal ERK1/2 expression (Fig. 1A). Finally, since the SOCS1 KIR domain can inhibit various molecular cascades, we evaluated the selectivity of PS-5 effects on another signaling pathway, particularly important during pathogenetic skin processes, the IL-22/STAT3 signaling Nivolumab chemical structure [8, 17]. We found that keratinocyte cultures pretreated with PS-5 had a reduced STAT3 activation in response to IL-22. However, this inhibitory effect was less pronounced than that observed Cediranib (AZD2171) for STAT1 phosphorylation, indicating a likely higher affinity of PS-5 peptido-mimetic for JAK2 than for TYK2, the kinase protein mediating IL-22 signal
[17]. As a whole, these data indicate that the SOCS1 mimetic PS-5 greatly reduces the proximal molecular cascades triggered by IFN-γ in human keratinocytes, and, specifically, those leading to STAT1 activation and function. During immune-mediated skin diseases, the exposure to IFN-γ stimulates the epidermal keratinocytes to produce inflammatory mediators, such as membrane molecules, cytokines, and chemokines, which actively participate to the amplification of the local pathogenetic processes [18, 19]. Due to limited existing information on the IFN-γ-dependent transcriptional regulation of these mediators in human keratinocytes, we firstly identified the inflammatory genes whose expression is strictly dependent on STAT1 activity. To this end, we transfected keratinocyte cultures with specific STAT1 siRNA molecules and evaluated the consequence of STAT1 knockdown on the expression of ICAM-1 and HLA-DR membrane molecules in IFN-γ-activated or resting cells.