Analysis of the molecular mechanisms that mediate the therapeutic actions of glucocorticoids in skin

Resumen

Synthetic glucocorticoids (GCs) have been widely used for the treatment of autoimmune and inflammatory diseases and exert their actions through binding to the GC receptor (GR) (Ramamoorthy and Cidlowski, 2016). GR is a transcription factor (TF) belonging to the nuclear receptor (NR) superfamily that mediates a wide range of important physiological processes (Sacta et al., 2016). Upon ligand binding, GR translocates to the nucleus and exert its actions either through binding to DNA or interacting with other TFs. Direct transcriptional regulation by GR involves its binding to specific DNA regulatory sequences called GC responsive elements or GREs. For a long time, it was postulated that GR induced their anti-inflammatory effects exclusively through transrepression by inhibiting known inflammatory pathways, including NF-kB, AP-1 and MAPKs (Weikum et al., 2017). However, it is now accepted that transcriptional induction of several classic GRE-containing targets is also key for the anti-inflammatory actions of the GR (De Bosscher and Haegeman, 2009). Continuous treatment or high doses of GCs may lead to unwanted side effects (skin atrophy), which may have a big impact in population (Sacta et al., 2016). Therefore it is important to understand the mechanisms by which GR regulates transcription in skin and their impact on the therapeutic and adverse effects of GCs. In part I of this dissertation, we studied the regulation of GC-induced leucine zipper (Gilz/Tsc22d3) and Zinc Finger Protein 36/Tristetraprolin (Zfp36/Ttp), two primary transcriptional GR targets containing GRE binding motifs, in epidermal keratinocytes. These and other GC-targets were previously identified in our laboratory by GR ChIP-seq assays in which GRE, Krüppel-like factor (KLF), and AP-1 were the most overrepresented binding motifs. Given the relevance of GR and Krüppel-like factor 4 (KLF4) in skin, we have analysed the functional interactions between these TFs and their impact on Gilz/Tsc22d3 and Zfp36/Ttp gene expression. Loss of function experiments of GR and KLF4 demonstrated total GR but partial KLF4 requirement for full gene induction in response to dexamethasone (Dex). In calcium-induced terminally differentiatied keratinocytes, GR and KLF4 protein expression increased concomitant with Gilz/Tsc22d3 and Zfp36/Ttp up-regulation. However, GR-deficient cells failed to differentiate or fully induce Gilz, Zfp36 and Klf4 correlating with an augmented expression of Trp63, a known transcriptional repressor of KLF4 which is epithelium-specific. The identified transcriptional cooperation between GR and KLF4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases. In part II of this dissertation, we have investigated GILZ, which has been shown to act as anti-inflammatory mediator in several cell types similar to GCs but without producing the GC-associated side effects. In contrast, little is known about the role of GILZ in skin function. Thus, we decided to study consequences of GILZ overexpression in psoriasis, an autoimmune inflammatory skin disease, where keratinocytes and immune cells play an important role. For this purpose we used an Imiquimod (IMQ)-induced psoriasis model in mice with generalized overexpression of GILZ (GILZ-Tg). Unexpectedly, GILZ-Tg showed more susceptibility to the IMQ-induced psoriasis, displaying more severe skin alterations shown by histological (increased epidermal hyperplasia and parakeratosis), and biochemical analysis (increased cytokine levels in serum and skin). Importantly, these alterations correlated with skin-specific overactivation of the TGF-β/SMAD signaling, which was recapitulated in cultured keratinocytes. Overall, these findings show that GILZ acts as a pro-inflammatory protein in skin in an IMQ-induced model of psoriasis and thus caution is advised in its therapeutic use.