MECHANISMS OF THE IL-22 ACTIVITY REGULATION IN HUMAN PRIMARY KERATINOCYTES

Interleukin (IL)-22 is a cytokine mainly released by T helper (Th) 17 and Th22 lymphocytes having a pathogenetic role in psoriasis. In this skin disorder, IL-22 is responsible for the altered proliferative and differentiative processes observed in the epidermis, and induces inflammatory molecules in keratinocytes. Signal transducer and activator of transcription (STAT) 3 is the principal mediator of IL-22 signaling and its complete activation requires the phosphorylation in tyrosine (Tyr) 705 and in serine (Ser) 727 residues. Moreover, STAT3 phosphorylation in Tyr705 is proportional to the acetylation in lysine (Lys) 685 residue. STAT3 acetylation is tightly regulated by the acetylation and deacetylation processes, which are controlled by p300 acetylase and histone deacetylase enzymes (HDAC), respectively. Due to the aberrant STAT3 activation in psoriatic epidermis, the aim of this study was to identify the target molecules able to inhibit the IL-22-triggered STAT3 phosphorylation and down-stream effects in human keratinocytes. For this purpose the role of Sirtuin (SIRT) 1, the most characterized Class III HDAC family member, has been studied in the control of IL-22-dependent signaling in keratinocytes. Indeed, it has previously demonstrated that SIRT1 is able to deacetylate STAT3 in Lys685 residue and, consequently, to inhibit STAT3 phosphorylation in Tyr705 in hepatocyte cells. Moreover, it was reported that the sirtuin is able to induce the differentiation and inhibit the proliferation of human keratinocytes. The research activity allowed us to demonstrate that SIRT1 is constitutively expressed by keratinocytes and that it efficiently contributes to STAT3 deacetylation in these cells. During inflammatory skin processes characterized by a concomitant presence of Interferon (IFN)-gamma and IL-22, IFN-gamma strongly down-regulates the keratinocyte expression of SIRT1 and IL-22 activates STAT3. In fact, SIRT1 decrement determines an accumulation of STAT3 acetylated in Lys685, thus favoring STAT3 phosphorylation in Tyr705 and keratinocyte responses to IL-22. SIRT1 negatively modulates the Proliferating cellular nuclear antigen (PCNA), cyclin D1 and phospho-Retinoblastoma (pRB), that is STAT3-dependent and IL-22-induced molecules and play a fundamental role in cellular proliferation. In addition, the sirtuin, by enhancing the level of keratin (KRT) 1, counter-acts the IL-22-triggered de-differentiate effect in keratinocytes. In psoriatic lesions, SIRT1 inhibition could be responsible for the strong activation of STAT3 and, thus, for the exaggerated responses of epidermal keratinocytes to IL-22 and other pro-inflammatory cytokines signaling through STAT3 (i.e., IL-6 and oncostatin M). Suppressor of cytokine signalling (SOCS) 3 represents another possible candidate able to control the IL-22-derived molecular cascades and the biological effects in keratinocytes. Indeed, in hepatocyte cells, the enhanced expression of SOCS3 level reduces the IL-22-dependent STAT3 activation. In this study it has been demonstrated that SOCS3 is the only SOCS family member induced by IL-22 in human keratinocytes. Interestingly, even though SOCS1 was not up-regulated by IL-22, its over-expression in stably trasfected keratinocytes potently inhibited the IL-22-induced Tyr705 and Ser727 phosphorylations of STAT3, likewise to what observed in SOCS3 clones. Consistently, transient transfection of keratinocytes with SOCS1 or SOCS3 plasmids markedly reduced the IL-22-induced STAT3 transcriptional activity. In contrast, stable or transient SOCS2 over-expression in keratinocytes had no effects on STAT3 activation by IL-22. As consequence of STAT3 inactivation, the IL-22-induced proliferation was impaired in SOCS3 and SOCS1 clones compared to mock-trasfected keratinocytes. Vice versa, the mitogenic effect exerted by IL-22 on keratinocytes depleted of SOCS3 was more pronounced. Moreover, SOCS3 reduces the expression of IL-22-dependent pro-inflammatory genes and opposes to the de-differentiative effect of the cytokine in keratinocytes. The SOCS3 inhibitory effect on the IL-22-induced STAT3 activation is executed by SOCS3 KIR (kinase inhibitory region) domain. Therefore, the use of SIRT1 activators or molecules able to mimic SOCS3-KIR region, able to reduce the STAT3 Tyr705 phosphorylation in epidermal keratinocytes, could be therapeutically useful for the treatment of psoriasis as well as other skin diseases characterized by an aberrant STAT3 activation.