Regulation of Photosensitivity by the Hippo Pathway in Lupus Skin

Abstract

Objective: Photosensitivity is one of the most common manifestations of systemic lupus erythematosus (SLE), yet its pathogenesis is not well understood. The normal-appearing epidermis of patients with SLE exhibits increased ultraviolet B (UVB)-driven cell death that persists in cell culture. Here, we investigated the role of epigenetic modification and Hippo signaling in enhanced UVB-induced apoptosis seen in SLE keratinocytes.

Methods: We analyzed DNA methylation in cultured keratinocytes from SLE patients compared to keratinocytes from healthy controls (n = 6/group). Protein expression was validated in cultured keratinocytes using immunoblotting and immunofluorescence. An immortalized keratinocyte line overexpressing WWC1 was generated via lentiviral vector. WWC1-driven changes were inhibited using a large tumor suppressor kinase 1/2 (LATS1/2) inhibitor (TRULI) and small interfering RNA (siRNA). The interaction between the Yes-associated protein (YAP) and the transcriptional enhancer associate domain (TEAD) was inhibited by overexpression of an N/TERT cell line expressing a tetracycline-inducible green fluorescent protein-tagged protein that inhibits YAP-TEAD binding (TEADi). Apoptosis was assessed using cleaved caspase 3/7 and TUNEL staining.

Results: Hippo signaling was the top differentially methylated pathway in SLE versus control keratinocytes. SLE keratinocytes (n = 6) showed significant hypomethylation (Δβ = -0.153) and thus overexpression of the Hippo regulator WWC1 (P = 0.002). WWC1 overexpression increased LATS1/2 kinase activation, leading to YAP cytoplasmic retention and altered proapoptotic transcription in SLE keratinocytes. Accordingly, UVB-mediated apoptosis in keratinocytes could be enhanced by WWC1 overexpression or YAP-TEAD inhibition, mimicking SLE keratinocytes. Importantly, inhibition of LATS1/2 with either the chemical inhibitor TRULI or siRNA effectively eliminated enhanced UVB-apoptosis in SLE keratinocytes.

Conclusion: Our work unravels a novel driver of photosensitivity in SLE: overactive Hippo signaling in SLE keratinocytes restricts YAP transcriptional activity, leading to shifts that promote UVB apoptosis.

Figure 1.. Hippo pathway is differentially methylated in SLE keratinocytes (KC)

a. Illumina methylation of non-lesional, SLE vs. HC KCs (age, sex, race matched; n=6 each) b. Correlations of genes induced by IFN and methylation signatureso c. Ingenuity pathway analysis of methylation changes

Figure 2.. scRNA seq of ‘normal appearing’ SLE skin and both CLE subtypes show differential activation of Hippo signaling

a. UMAP plot of 39,086 keratinocytes colored by sub-cluster from all samples: CTL, healthy control skin, n=6. NLE, non-lesional lupus skin. LLE, lesional lupus skin, n=10 each. b. UMAP plot of keratinocytes colored by disease state. CTL, healthy control skin, n=6. NLE, non-lesional lupus skin. LLE, lesional lupus skin, n=10 each. c. Violin plots of Hippo module score from scRNA seq analysis of KC cluster d. Violin plots of Hippo module score from microarray analysis of CLE lesions. DLE = discoid lupus erythematosus; n=47, SCLE =subacute cutaneous lupus erythematosus; n=43 e. Violin plots showing the expression level of representative cell proliferation genes in healthy control (n=6), nonlesional and lesional lupus (n=10) in basal keratinocyte subcluster; *=p<0.05, ***=p<0.001 f. Violin plots showing the expression level of representative cell proliferation genes in the basal keratinocyte sub-cluster in healthy control (n=6) and nonlesional psoriasis (n=14); ***=p<0.001, ****=p<0.0001

Figure 3.. Hippo signaling methylation changes translate to significant difference in protein expression in situ.

a. WWC1, p-YAP, pTAZ, pLATS1/2, total LATS1/2 and beta actin levels by Western blot. Blots were divided and probed with respective primary antibody b. Bar graph of ratio of WWC1 to beta actin in 6 SLE and 6 HC KC primary cell lines c. Bar graph of ratio of YAP/beta actin in 6 CTL and 6 SLE KC primary cell lines d. Bar graph of ratio of p-YAP/YAP in 6 SLE and 6 CTL primary cell lines e. Bar graph of ratio of pLATS/LATS in 6 SLE and 6 CTL primary cell lines f. Immunofluorescence of YAP from frozen skin biopsies in 6 CTL and non-lesional frozen skin biopsies from 6 SLE patients and 3 psoriasis patients. Scale bar= 100 μM g. Bar graph of averaged integrated density per cell of YAP in 6 CTL and non-lesional frozen skin biopsies from 6 SLE patients and 3 psoriasis patients. h. Immunofluorescence of p-YAP from frozen skin biopsies in 6 CTL and non-lesional frozen skin biopsies from 6 SLE patients and 3 psoriasis patients. Scale bar= 100 μM i. Bar graph of average integrated density per cell of p-YAP in 6 CTL and non-lesional frozen skin biopsies from 6 SLE patients and 3 psoriasis patients. f,h. Each dot represents the average of 3 images from each patient. Scale bar 100 μM. ns= not significant, * p<0.05, **p<0.01 unpaired t-test.

Figure 4.. WWC1 overexpression and interruption of YAP-TEAD interactions results in exaggerated apoptosis

a. Western blot of WWC1 overexpression and GFP in N/TERT2G keratinocyte line, probed with WWC1 and GFP primary antibody as indicated by box. Separate gel was run for p-YAP and YAP in Ctr-GFP and WWC1OE cell lines. b. Expression of WWC1 in Ctr-GFP and WWC1OE cell lines by RT qPCR (n=3) c. Expression of apoptosis gene SNAI1 in WWC1OE cell lines vs. Ctr-GFP by RT qPCR (n=8 for Ctr-GFP and n=8 for WWC1OE) d. Representative TUNEL images of WWC1OE vs. Ctr-GFP treated with or without UVB, n=4. Scale bar = 100 μM e. Quantification of TUNEL in WWC1OE vs Ctr-GFP (n=5, each), f. Representative cl-caspase 3/7 images of WWC1OE vs. Ctr-GFP treated with or without UVB, n=4. Scale bar = 100 μM. g. Quantification of cl-caspase 3/7 in WWC1OE vs Ctr-GFP in f (n=4, each) h. Western blot of TEAD, GFP and beta-actin in WT and N/TERT TEADi-GFP cell lines i. Representative images of cl-caspase 3/7 staining in WT N/TERT and TEADi (n=7, each) with and without UVB. Scale bar = 100 μM j. Quantification of %cl-caspase 3/7 in WT vs. TEADi N/TERTs in i, * p<0.05, **p<0.01. ***p<0.001 unpaired t-test.

Figure 5.. Inhibition of LATS1/2 represses exaggerated UVB apoptosis in SLE keratinocytes.

a. Western blot showing siRNA knockdown of LATS1/2 (siLATS1/2) or nontarget control (siCtr) in two different primary healthy control KC lines. b. Expression changes of LATS1 in siRNA treated control primary KCs by qPCR, n=3, each. c. Expression changes of LATS2 in siRNA treated control primary KCs by qPCR, n=3, each. d. Representative images of UVB apoptotic response measured by cl-caspase 3/7 in siRNA treated primary KCs, n=4. Scale bar 50 μM. e. Quantification of % active cl-caspase 3/7 of triplicate samples from SLE keratinocytes (n=4) and healthy control (n=4). Un-paired t test. *=p<0.05 f. Representative images of UVB apoptotic response measured by cl-caspase 3/7 in CTL (n=3) and SLE keratinocytes (n=3) following 24 hr pre-treatment with 10 μM LATS1/2 kinase inhibitor, TRULI. **=p<0.01 g. Quantification of cl-caspase 3/7 in primary CTL and SLE KCs treated with and without UVB and TRULI, n=3. Un-paired t test. Scale bar 100 μM h. Representative Western blot of p-YAP and YAP after 24 hr treatment +/− TRULI with and without UV stimulation. i. Quantification of p-YAP/YAP ratios for Western blot in h for three different healthy control and 3 different SLE primary KC lines. *=p<0.05.

Figure 6.. SLE shows skewed pro-apoptotic gene profile that is reversible with TRULI.

a. Change in gene expression following UVB stimulation in CTL (n=7) and SLE (n=6) cultured KCs. Expression changes between comparisons are significant with p<0.05 unless annotated. b. Mean gene expression (+/− SD) of Hippo-regulated pro-apoptosis gene SNAI1 in CTL (n=3) and SLE (n=4) treated with UVB and LATS1/2 siRNA. c. Gene expression of Hippo regulated pro-apoptosis genes SNAI1 and DDL4 in SLE (n=3) and CTL (n=3) KCs treated with and without UVB and TRULI. Dots represent triplicate biological replicates for 3 unique SLE and 3 unique CTL samples.