Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis

Abstract

The membrane-anchored serine protease, matriptase, is consistently dysregulated in a range of human carcinomas, and high matriptase activity correlates with poor prognosis. Furthermore, matriptase is unique among tumor-associated proteases in that epithelial stem cell expression of the protease suffices to induce malignant transformation. Here, we use genetic epistasis analysis to identify proteinase-activated receptor (PAR)-2-dependent inflammatory signaling as an essential component of matriptase-mediated oncogenesis. In cell-based assays, matriptase was a potent activator of PAR-2, and PAR-2 activation by matriptase caused robust induction of nuclear factor (NF)κB through Gαi. Importantly, genetic elimination of PAR-2 from mice completely prevented matriptase-induced pre-malignant progression, including inflammatory cytokine production, inflammatory cell recruitment, epidermal hyperplasia and dermal fibrosis. Selective ablation of PAR-2 from bone marrow-derived cells did not prevent matriptase-driven pre-malignant progression, indicating that matriptase activates keratinocyte stem cell PAR-2 to elicit its pro-inflammatory and pro-tumorigenic effects. When combined with previous studies, our data suggest that dual induction of PAR-2-NFκB inflammatory signaling and PI3K-Akt-mTor survival/proliferative signaling underlies the transforming potential of matriptase and may contribute to pro-tumorigenic signaling in human epithelial carcinogenesis.

Figure 1. Matriptase-mediated pre-malignant progression of mouse squamous epithelium is PAR-2-dependent

(A). Representative examples of the outward appearance of littermate K5-Mat^+/0;F2rl1^+/+ (left panel), K5-Mat^+/0;F2rl1^−/− (second panel from left), F2rl1^−/− (second panel from right), and wildtype mice (right panel) at 52 weeks of age. Matriptase-induced alopecia and ichthyosis are prevented by genetic elimination of PAR-2. (B). Enumeration of epidermal thickness in littermate K5-Mat^+/0;F2rl1^+/+ (N=11, purple circles), K5-Mat^+/0;F2rl1^−/− (N=7, green circles), wildtype (N=5, red circles), and F2rl1^−/− mice (N=6, grey circles) at 52 weeks of age. *** P < 0.0006 (Mann-Whitney U test, two-tailed). N.S. = not significant (C). Epidermal keratinocyte proliferation rates in littermate K5-Mat^+/0;F2rl1^+/+ (N=6, purple circles), K5-Mat^+/0;F2rl1^−/− (N=7, green circles), wildtype mice (N=3, red circles), and F2rl1^−/− (N=3, grey circles) at 52 weeks of age. Horizontal bars in C and B indicate median values. * P < 0.02, ** P < 0.004, *** P < 0.0006, N.S. = not significant (Mann-Whitney U test, two-tailed). (D–O). Representative histological appearance of dorsal skin of littermate K5-Mat^+/0;F2rl1^+/+ (D, H, and L), K5-Mat^+/0;F2rl1^−/− (E, I, and M), wildtype mice (F, J, and N) and F2rl1^−/− (G, K, and O) mice at 52 weeks of age stained with H&E (D–G), keratin-6 antibodies (H–K), and Masson’s trichrome (L–O). Multifocal dysplasia (examples with arrowhead in D), interfollicular epidermal keratin-6 expression (examples with arrowhead in H), and dermal fibrosis (example with star in L) are completely prevented by genetic elimination of PAR-2. Size bars = 50 µm.

Figure 2. Dysregulated matriptase does not promote chemical carcinogenesis in the absence of PAR-2

Kaplan-Meier analysis of tumor-free survival (A), and time to sacrifice and total tumor burden at sacrifice (B) of littermate K5-Mat^+/0;F2rl1^+/− (N=11, purple squares), K5-Mat^+/0;F2rl1^−/− (N=11, green diamonds), F2rl1^+/− (N=10, red circles), and F2rl1^−/− (N=7, grey triangles) mice. The mice were treated with DMBA every 3 weeks and were followed for up to 63 weeks. *** P < 0.0001, K5-Mat^+/0;F2rl1^+/+ versus other genotypes in A (log-rank test, two-tailed). Vertical and horizontal bars in B indicate standard error of the mean. C–F. Representative outward appearance of littermate K5-Mat^+/0;F2rl1^+/− (left panel), K5-Mat^+/0;F2rl1^−/− (second panel from left), F2rl1^+/− (second panel from right), and F2rl1^−/− (right panel) mice at 26 weeks of age. Example of carcinoma (white star) and papilloma (white triangle) in K5-Mat^+/0;F2rl1^+/− mouse is shown. G–N. Representative of H&E-stained sections of squamous cell carcinoma at low (G–J) and high (K–N) magnification in a 25-week-old K5-Mat^+/0;F2rl1^+/− (left panels) mouse, and in 45 to 55-week-old K5-Mat^+/0;F2rl1^−/− (second panels from left), F2rl1^+/− (second panels from right), and F2rl1^−/− (right panels) mice showing similar histological appearance of tumors irrespective of genotype. Stars in G–J show examples of invasion, while arrowheads and arrows in K–N shows examples of cells invading vessels and atypical mitosis, respectively. Size bars. G–J = 100 µm. K–N = 30 µm.

Figure 3. Hematopoietic PAR-2 is dispensable for matriptase-mediated pre-malignant progression

(A) Enumeration of epidermal thickness in 17-week-old lethally-irradiated littermate K5-Mat^+/0;F2rl1^+/+ mice (orange and blue circles) reconstituted with either F2rl1^+/+ (N=5, orange circles) or F2rl1^−/− bone marrow (N=7, blue circles), and wildtype mice (brown and grey circles) reconstituted with F2rl1^+/+ (N=5, brown circles) or F2rl1^−/− bone marrow (N=5, grey circles). Horizontal bars indicate median values. * P < 0.01 and **P < 0.007, respectively, N.S. = not significant (Mann-Whitney U test, two-tailed). (B) Epidermal keratinocyte proliferation rates in 17-week-old lethally irradiated littermate K5-Mat^+/0;F2rl1^+/+ mice (orange and blue circles) reconstituted with either F2rl1^+/+ (N=5, orange circles) or F2rl1^−/− bone marrow (N=4, blue circles), and wildtype mice (brown and grey circles) reconstituted with either F2rl1^+/+ (N=6, brown circles) or F2rl1^−/− bone marrow (N=5, grey circles). Horizontal bars indicate median values. ** P < 0.003, N.S. = not significant (P < 0.06) (Student’s t-test, two-tailed). (C–J) Representative examples of H&E (C–F) and Masson’s trichrome (G–J) -stained epidermal sections from 13-weeks-old lethally irradiated littermate K5-Mat^+/0;F2rl1^+/+ mice (C, D, G, and H) reconstituted with F2rl1^+/+ (C and G) or F2rl1^−/− bone marrow (D and H), and wildtype mice (E, F, I, and J) reconstituted with F2rl1^+/+ (E and I) or F2rl1^−/− (F and J) bone marrow. Stars in G and H indicate dermal fibrosis. Size bars. C–F = 50 µm. G–J = 100 µm.

Figure 4. Matriptase-induced dermal inflammatory cell accumulation is PAR-2-dependent

A–H. Representative examples of toluidine blue-stained dorsal skin sections from 8 to 10-week-old-littermate (A–D) and 52-week-old littermate (E–H) K5-Mat^+/0;F2rl1^+/+ (left panels), K5-Mat^+/0;F2rl1^−/− (second panels from left), F2rl1^+/+ (second panels from right), and F2rl1^−/− (right panels) mice. Examples of dermal mast cells (blue) are indicated with arrows. Size bars = 50 µm. (I and J) Enumeration of mast cell accumulation in 8 to 10-week-old (left panel) and 52-week-old (right panel) K5-Mat^+/0;F2rl1^+/+ (N=9 to 12, purple circles), K5-Mat^+/0;F2rl1^−/− (N=7 to 9, green circles), F2rl1^+/+ (N=6 to 7, red circles), and F2rl1^−/− mice (N=6 to 7, grey circles). * P < 0.01. ** P < 0.001, N.S. = not significant (Mann-Whitney U test, two-tailed).

Figure 5. Dysregulated matriptase induces persistent skin inflammatory cytokine production through non-hematopoietic PAR-2

(A–G). Skin mRNA levels of Cxcl1 (A), Il1b (B), Tslp (C), Csf2 (D), Tnf (E), Ifng (F), and Icam1 (G), in 8 to 10-week-old littermate K5-Mat^+/0;F2rl1^+/+ (N=5 to 7, purple circles), K5-Mat^+/0;F2rl1^−/− (N=4 to 6), green circles), wildtype (N=3 to 4, red circles), and F2rl1^−/− mice (N=3 to 4, grey circles). * P < 0.01, **, P < 0.001, *** P < 0.01, N.S. = not significant (Mann-Whitney U test, two-tailed). (H–N). Skin mRNA levels of Cxcl1 (H), Il1b (I), Tslp (J), Csf2 (K), Tnf (L), Ifng (M), and Icam1 (N) in 17-week-old lethally-irradiated littermate K5-Mat^+/0;F2rl1^+/+ mice (orange and blue circles) reconstituted with F2rl1^+/+ (N=5, orange circles) or F2rl1^−/− bone marrow (N=5 to 7, blue circles), and control (F2rl1^+/+ and F2rl1^−/−) mice (N=12 to 13, brown circles) reconstituted with either F2rl1^+/+ or F2rl1^−/− bone marrow. Horizontal bars indicate median values. * P < 0.01, **, P < 0.001, N.S. = not significant (Mann-Whitney U test, two-tailed). (O–U). Skin mRNA levels of Cxcl1 (O), Il1b (P), Tslp (Q), Csf2 (R), Tnf (S), Ifng (T), and Icam1 (U) in 52-week-old littermate K5-Mat^+/0;F2rl1^+/+ (N=4 to 12, purple circles), K5-Mat^+/0;F2rl1^−/− (N=4 to 5, green circles), wildtype (N=5, red circles), and F2rl1^−/− mice (N=3 to 5, grey circles). * P < 0.01, ** P < 0.001, N.S. = not significant (Mann-Whitney U test, two-tailed).

Figure 6. Matriptase can activate NFκB through PAR-2 and Gαi

(A–H) HEK293 cells were co-transfected with PAR-2 expression vector (PAR-2, A–D) or PAR-1 expression vector (PAR-1, E–H) or with empty expression vector and either serum response element-luciferase (SRE) (A, B, E, and F) or NFκB -luciferase reporter plasmids (NFκB) (C, D, G, and H), as indicated. The transfected cells in A and C were either treated for 6 h with the PAR-2 agonist, 2-furoyl-LIGRLO-amide (PAR-2 ag., 0.25, 1, 2.5 and 10 µM in bars 2–5 respectively) or with 10 µM dual PAR-1 and -2 agonist, TRAP-6 (lane 6). The transfected cells in E and G were either treated with TRAP-6 (0.25, 1, 2.5 and 10 µM in bars 2–5, respectively) or with 10 µM 2-furoyl-LIGRLO-amide (lane 6) for 6 h. Cells in B, F, D, and H were treated with recombinant human matriptase serine protease domain for 6 h (1 or 15 nM in bars 2–4) or with vehicle (bar 4). I and J. HEK293 cells were co-transfected with empty vector (bars 1 and 2) or PAR-2 expression vector (bars 3–8) in combination with either serum response element-luciferase (I) or NFκB -luciferase reporter (J) plasmids. The transfected cells were treated overnight with vehicle (lanes 1, 3, 5, and 7) or pertussis toxin (PTX) (lanes 2, 4, 6, and 8), and then were treated with vehicle (lanes 3 and 4) or were treated with either 20 µM PAR-2 agonist (bars 1 and 2, 5 and 6) or 15 nM recombinant human matriptase (lanes 7 and 8). All cells were transfected with a pRL-Renilla luciferase reporter plasmid as an internal control for transfection efficiency. Data are shown as the mean ± standard deviation of the mean of triplicate transfections of firefly luciferase light units/Renilla luciferase light units. The data are representative of three similar experiments.