Fig. 9

Hyperplasia and transcriptional upregulation of mmp9 in basal keratinocytes of psoriasis mutants is mediated via an aberrant activation of a linear PI3K-Akt-mTorC1-NFκB pathway.

(a-e) Blockade of PI3K, mTorC1, and NFκB signaling rescues epidermal aggregate but not pericardial edema formation in psoriasis mutants. (a-d) Representative live images of phenotypic strength classes of psoriasis -/- embryos at 54 hpf, all with pericardial edema of comparable strengths, but strong (a), intermediate (b), weak (c), or no (d) epidermal aggregates. (e) Quantification of the phenotypes of psoriasis mutants incubated in E3 medium containing 1 µM Wortmannin, 5 µM PIK90, 25 µM LY94002, 1.1 µM Rapamycin, 30 µM AZD8055, or 30 µM Withaferin A compared to the corresponding DMSO controls (n = 16-30). Drugs were added at 34 hpf and embryos scored at 54 hpf. Similar results were obtained in at least two additional independent experiments. For representative live images, see Figure 9- figure supplement 1. f-k. A linear PI3K-Akt-mTORC1-NFκB pathway mediates hyperplasia and upregulation of mmp9 expression in basal keratinocytes. All embryos had been kept in (hypotonic) E3 medium, supplemented with the indicated drugs starting at 34 hpf. (f--f′′′′) IF of cytokeratins (red) at 84 hpf. Distorted keratin localization in the psoriasis mutant (f′, compare to wt (f)) is not restored by Wortmannin (f′′), Rapamycin (f′′′), or Withaferin A (f′′′′). Scale bar: 50 µm. (g-g′′′′) IF of pAkt (red), counterstained with DAPI (blue); transverse sections of 54 hpf psoriasis mutants raised in E3 medium. Elevated pAkt levels in the mutant (g′, compared to the wt (g)) are lowered by Wortmannin (g′′), but not by Rapamycin (g′′′) or Withaferin A (g′′′′). Scale bar: 20 µm. (h-h′′′′) IF of pS6RP and p63 of whole mounts, at 54 hpf. Elevated pS6RP levels in mutant (h′, compared to wt (h)) are alleviated by Wortmannin (h′′), PIK90 (not shown) and Rapamycin (h′′′), but not by Withaferin A (h′′′′). Scale bar: 20 µm. (i- i′′′′) Confocal images of GFP fluorescence in the tail fin of a live 48 hpf wt embryo (i) and an atp1b1a morphant (i′), both carrying the Tg(NFκB-RE:eGFP) transgene. The atp1b1a morphant shows strong upregulation of NFκB activity in keratinocytes, which is restored by treatment with Wortmannin (i′′), Rapamycin (i′′′), and Withaferin A (i′′′′). Scale bar: 100 µm. For quantification, see Figure 9-figure supplement 2. (j-j′′′′) mmp9 WISH at 54 hpf. Elevated mmp9 expression in the mutant epidermis (j′, compare to wt (j)) is downregulated by Wortmannin (j′′), Rapamycin (j′′′), and Withaferin A (j′′′′). Scale bar: 50 µm. (k-k′′′′) IF of incorporated BrdU (red), counterstained with DAPI (blue) at 56 hpf. Elevated cell proliferation in mutant epidermis (k′, compare to wt (k)) is downregulated by Wortmannin (k′′), Rapamycin (k′′′), and Withaferin A (k′′′′). Scale bar: 100 µm.