Regulation of the psoriatic chemokine CCL20 by E3 ligases Trim32 and Piasy in keratinocytes

Abstract

Psoriasis is an inflammatory skin disorder with aberrant regulation of keratinocytes and immunocytes. Although it is well known that uncontrolled keratinocyte proliferation is largely driven by proinflammatory cytokines from the immunocytes, the functional role of keratinocytes in the regulation of immunocytes is poorly understood. Recently, we found that tripartite motif-containing protein 32 (Trim32), an E3-ubiquitin ligase, is elevated in the epidermal lesions of human psoriasis. We previously showed that Trim32 binds to the protein inhibitor of activated STAT-Y (Piasy) and mediates its degradation through ubiquitination. Interestingly, the Piasy gene is localized in the PSORS6 susceptibility locus on chromosome 19p13, and Piasy negatively regulates the activities of several transcription factors, including NF-kappaB, STAT, and SMADs, that are implicated in the pathogenesis of psoriasis. In this study, we show that Trim32 activates, and Piasy inhibits, keratinocyte production of CC chemokine ligand 20 (CCL20), a psoriatic chemokine essential for recruitment of DCs and T helper (Th)17 cells to the skin. Further, Trim32/Piasy regulation of CCL20 is mediated through Piasy interaction with the RelA/p65 subunit of NF-kappaB. As CCL20 is activated by Th17 cytokines, the upregulation of CCL20 production by Trim32 provides a positive feedback loop of CCL20 and Th17 activation in the self-perpetuating cycle of psoriasis.

Figure 1. Elevated Trim32 expression in human psoriasis

a) Immunohistochemical analysis of Trim32 expression in psoriasis specimens. Trim32 was visualized in paraffin sections using 0.2 μg/ml chicken anti-Trim32 antibody and stained using the ABC technique. Trim32 was elevated in the epidermis of psoriatic lesions relative to uninvolved skin (200× magnification). b) Indirect immunofluorescence analysis of Trim32 expression in psoriasis specimens. Trim32 was visualized with chicken anti-Trim32 antibody followed by Texas red secondary antibody (600× magnification). The nucleus was stained with Hoechst 33342. The micrographs are representative of 20 Trim32 positive from 33 psoriatic skin specimens. The bar equals 50 micrometers.

Figure 2. Regulation of CCL20 expression in keratinocytes by Trim32 and Piasy

a) CCL20 mRNA expression in mouse keratinocyte line 291 infected with adenovirus expressing GFP, Trim32, or Piasy. b) Analysis of CCL20 mRNA in primary mouse keratinocytes isolated from Trim32 null mice (Trim32^−/−) and wild type littermates (Wt). CCL20 mRNA was normalized to GAPDH. Data are representative of three independent experiments for 1a and two independent experiments for 1b (**, P< 0.01; ***, P< 0.001).

Figure 3. Trim32 and CCL20 expression in human psoriasis

Paraffin embedded psoriasis lesional tissues from 33 patients were sectioned serially and stained with chicken anti-Trim32 antibody and goat anti-CCL20 antibody (100× magnification). The results are summarized in the table. Correlation of overexpression of Trim32 with CCL20 expression was statistically significant (*Two-tailed Fisher exact test, p-value = 0.00015). The bar equals 50 micrometers.

Figure 4. Regulation of Th17 induced CCL20 by Trim32 and Piasy

a) CCL20 mRNA was measured by qRT-PCR in mouse 291 keratinocytes treated with indicated cytokines and LPS. b) CCL20 mRNA was measured by qRT-PCR in mouse 291 keratinocyte infected overnight with adenovirus expressing GFP, Trim32, or Piasy, then treated with indicated cytokines for 4 hours. CCL20 mRNA was normalized to GAPDH. c) CCL20 protein was measured by ELISA in mouse 291 keratinocyte infected overnight with adenovirus expressing GFP, Trim32, or Piasy, then treated with indicated cytokines for 6 hours. d) Human keratinocyte line HEKnV was infected with adenovirus expressing GFP, Trim32, or Piasy, then treated with indicated cytokines for 6 hours. CCL20 protein was measured from the culture supernatant by ELISA. Data are representative of three independent experiments. The statistical analysis was conducted by one-way ANOVA followed by Bonferroni post-test (**, P< 0.01; ***, P< 0.001).

Figure 5. Piasy regulates CCL20 through p65/RelA subunit of NF-κB

a) CCL20 mRNA was measured in 291 keratinocytes transfected with plasmids expressing GFP, p65, Trim32 plus p65, or Piasy plus p65. Data are representative of two independent experiments. Statistical analysis of Trim32 and Piasy effect was conducted relative to p65 alone control (*, P< 0.05). b) Piasy interacts with endogenous p65 in keratinocytes. Flag-tagged Piasy and Flag-tagged Erk (negative control) were transfected into 291 keratinocytes. The bound p65 was immunoprecipitated with an anti-Flag antibody and visualized by immunoblotting with anti-p65 antibody.