Senescence is a cellular program involved in physiological and homeostatic processes, as well as in pathological conditions, and characterized by a permanent cell growth arrest in G1- or in G2-cell cycle phase, pointed to prevent the proliferation of damaged cells [50,51]. Senescence is evoked by cellular stress and by tumor suppressive mechanisms that must be overcome to promote malignant transformation.
Psoriatic keratinocytes are characterized by a senescent-like phenotype, manifesting a peculiar resistance to apoptosis and a high expression of P21 and P16 senescence markers, which is supported by several intracellular pathways [4, 15, 16].
In this study, we explored the role of PI3K/AKT pathway in epidermal senescence of psoriatic skin. We found that keratinocyte cultures from psoriatic patients and undergoing cellular senescence exhibit higher levels of p-AKT during in vitro culture passages, compared to keratinocytes from healthy skin. Hyperactivated AKT correlates with high levels of P53, P21 and P16 senescence markers and SA-β-gal activity and with a reduced expression of p-Rb protein.
Together with RAF/ERK axis, PI3K and AKT are effectors of RAS molecule. RAS-RAF-MAPK pathway is upregulated in psoriatic skin [32,33], and induces thickening and inflammation in the epidermis of murine models of psoriasis [52,53]. However, to date, the role of RAS/PI3K/AKT signaling in the mechanisms underlying senescence of psoriatic keratinocytes are not being proven. To this matter, we showed that psoriatic keratinocytes show a lower replicative rate compared to healthy cells and are characterized by a constitutive RAS hyperactivation that correlates with AKT/P53/P21 upregulation. This observation suggests a link between RAS and PI3K/AKT axis in senescence programs occurring in psoriatic keratinocytes and could depend on the continue exposition to cytokine milieu of psoriatic skin during disease evolution. The genetic and epigenetic status also predisposes psoriatic keratinocytes to a pre-senescent status and to respond aberrantly to cytokines.
PI3K/AKT are not the only effectors of cellular senescence. Additional intracellular mediators of senescence have been identified, including the negative regulator of RAS/ERK signaling, neurofibromin 1 (NF1), in human fibroblasts undergoing AKT-induced senescence [54]. Interestingly, NF1 is downregulated in psoriatic skin, thus resulting in the hyper-activation of RAS [55].
In addition, psoriatic keratinocytes exhibit a strong upregulation of P16, which drives senescence programs in parallel to P21, most likely in response to Th1/Th17 cytokines present in the microenvironment of psoriatic plaques. In particular, pro-inflammatory cytokines can activate various intracellular pathways, including PI3K/AKT axis, that result in the upregulation of the downstream P53 and P16 effectors [56]. Activated P53 induces in turn P21, which impedes cell-cycle progression by inhibiting cyclin E/CDK2 complex. P16 also induces cell-cycle arrest by targeting cyclin D/CDK4 and cyclin D CDK6 complexes. Both P21 and P16 inhibit Rb phosphorylation, thus resulting in S-phase arrest [57,58]. Consistently, Th1/Th17-released cytokines enhance RAS activation, and induce AKT phosphorylation, as well as P53, P21, and P16 expression levels. A strong effect was exerted by TNF-α and IL-17A alone and by their combination. In contrast, IL-22 induced a significant induction of P16, not dependent on RAS and AKT up-regulation. This finding suggests a novel pro-senescent function for IL-22, a pathogenic cytokine known to impair the terminal differentiation of human keratinocytes. We hypothesize that IL-22 could sustain the oncogene-induced senescence in psoriatic keratinocytes, differently to what observed in hepatic cells, where IL-22 induces a senescence mediated by P53/P21 axis [59].
In line with these observations, PI3K/AKT inhibition significantly reverted senescent phenotype of psoriatic keratinocytes. In particular, Ly294002, a pan-PI3K inhibitor, significantly inhibited SASP expression, with the exception of CCL20 chemokine, whereas MK2206, a selective AKT1/2/3 inhibitor, weakly reduced SASP profile. This result could depend on the involvement of PI3K in several intracellular pathways upstream of AKT/mTOR axis. Indeed, Hart demonstrated that pharmacological PI3K inhibition prevented STAT3 phosphorylation induced by a Tec kinase in human transformed cells, suggesting an interplay between PI3K and STAT3 (60). Similarly, PI3Kδ isoform sustains IL-22-induced STAT3 activation in psoriatic keratinocytes [9]. However, PI3K/AKT also activates NF-κB pathway, and its inhibition reduces the NF-κB-dependent inflammatory responses in human keratinocytes [61].
In this study, we showed for the first time that AKT hyper-activation mediated by RAS overexpression in the suprabasal epidermal layers is crucial for the establishment of psoriasis phenotype. Mice overexpressing the H-RAS-V12 transgene in the upper epidermis layers show increased levels of p-AKT, together with P21 and P53 senescence markers. In line with in vitro and in vivo findings, the employment of the AKT1/2/3 inhibitor MK2206 in murine psoriasiform model significantly attenuates the disease symptoms by reducing the epidermal thickness and dermal accumulation of immune cell infiltrate. Of note, RAS overexpression in mice did not influence P16 expression, in line with what observed in vitro in healthy keratinocyte cultures transduced with H-RAS and characterized by a senescence status [37] associated to the upregulation of the only AKT/P53/P21 axis. These data contrast with the high P16 expression observed in human psoriasis lesions and in psoriatic keratinocyte cultures undergone senescence, where genetic background could play a critical role. In addition, the low expression levels of P16 could depend also on the absence of IL-22 in RAS-induced disease model. In line with the human disease condition, P21 and P53 levels were upregulated in RAS-induced disease model, and were strongly reduced by MK2206.
Of note, MK2206 drastically inhibited the recruitment and infiltration into the skin of different leukocyte subpopulations responsible for induction of psoriasiform phenotype in RAS-induced model. Among them, skin-infiltrating neutrophils and CD3+/CD8+ T cells were reduced by AKT inhibition, together with molecules involved in neutrophilic chemoattraction and activation (i. e. CXCL1, GM-CSF, G-CSF, CXCL16), and with cytokines released by Th17, γδT and Tc1/Tc17 cells (i. e. IL-17, IL-23 and IFN-γ) and by dendritic cells (i. e. IL-1β, IL-23 and TNF-α).
In conclusion, we propose a novel model of RAS/AKT-induced senescence (Fig. 8), where cytokine-induced RAS upregulation in keratinocytes determines the basal AKT/P53/P21 and P16 activation. In turn, these pathways determine a strong decrease of cell growth rate, and induction of hypersecretory phenotype in keratinocytes, all processes responsible for aberrant epidermal thickening and skin chronic inflammation observed in psoriatic plaque. SASP exhibited by epidermal keratinocytes is implicated in the amplification of inflammatory circuits through paracrine effects on skin infiltrating immune cells and dermal vasculature, and by autocrine action on keratinocytes themselves (Fig. 8). Senescence could also contribute to the onset of various comorbidities, that frequently affect psoriatic patients [62]. Indeed, the secretion of SASP components can cause chronic inflammation not only locally in the skin, but also systemically [14,50,63,64]. However, in psoriatic skin, senescence represents a response to chronic stress stimuli and aberrant cell division, thus it is thought to protect psoriatic lesions from tumorigenesis [65].
Targeting AKT pathway could be a promising novel strategy to counteract multiple psoriatic symptoms, taking account of senescence-associated pathological signs.