Palmoplantar Keratoderma in Slurp2-Deficient Mice

Abstract

SLURP1, a member of the lymphocyte antigen 6 protein family, is secreted by suprabasal keratinocytes. Mutations in SLURP1 cause a palmoplantar keratoderma (PPK) known as mal de Meleda. SLURP2, another secreted lymphocyte antigen 6 protein, is encoded by a gene located ?20 kb downstream from SLURP1. SLURP2 is produced by suprabasal keratinocytes. To investigate the importance of SLURP2, we first examined Slurp2 knockout mice in which exon 2-3 sequences had been replaced with lacZ and neo cassettes. Slurp2(-/-) mice exhibited hyperkeratosis on the volar surface of the paws (i.e., palmoplantar keratoderma), increased keratinocyte proliferation, and an accumulation of lipid droplets in the stratum corneum. They also exhibited reduced body weight and hind limb clasping. These phenotypes are similar to those of Slurp1(-/-) mice. To solidify a link between Slurp2 deficiency and palmoplantar keratoderma and to be confident that the disease phenotypes in Slurp2(-/-) mice were not secondary to the effects of the lacZ and neo cassettes on Slurp1 expression, we created a new line of Slurp2 knockout mice (Slurp2X(-/-)) in which Slurp2 was inactivated with a simple nonsense mutation. Slurp2X(-/-) mice exhibited the same disease phenotypes. Thus, Slurp2 deficiency and Slurp1 deficiencies cause the same disease phenotypes.

Figure 1. Palmoplantar keratoderma in Slurp2^−/− mice

(a) Paws from wild-type and Slurp2^−/− mice. The epidermis of the entire paw was thick but the PPK was quite evident at 6–8 weeks of age by the bulbous appearance of the tips of the digits. (b) H&E–stained sections showing hyperkeratosis in Slurp2^−/− paw skin. Scale bar, 50 μm. (c) Numerous small lipid droplets in the stratum corneum of Slurp2^−/− mice (arrowheads). Scale bar, 10 μm. (d) BODIPY 493/503 staining showing tiny lipid droplets (green) in the stratum corneum of Slurp2^−/− paw skin. DNA was stained with DAPI (blue). In the left-hand panel (wild-type mouse), the stratum corneum is above the white line. (e) Increased BrdU incorporation (green) into the paw skin of Slurp2^−/− mice. DNA was stained with DAPI (red). Scale bar, 50 μm.

Figure 2. “Non-skin” phenotypes in Slurp2^−/− mice

(a) Hind limb clasping in Slurp2^−/− mice. (b) Quantification of hind limb clasping (0 for none; 1 for unilateral retraction; 2 for bilateral retraction). Shown are means ± SEM; n = 12 for Slurp2^+/+ mice; n = 13 for Slurp2^−/− mice; ***p < 0.0001). (c–d) Weight gain in chow-fed male and female Slurp2^+/+ and Slurp2^−/− mice, beginning at ~4 weeks of age. Males: n = 10 Slurp2^+/+ and n = 9 Slurp2^−/−. Females: n = 8 Slurp2^+/+ and n = 5 Slurp2^−/−. Means ± SEM. Males: differences were significant at weeks 8–15 at p values ranging from 0.046 to 0.002. Females, p values for weeks 9–15 were <0.05 except for week 13 (0.066). (e) Chow consumption in Slurp2^−/− and wild-type mice. (f) Adiposity in 7-month-old chow-fed male Slurp2^−/− mice (n = 7/group; p < 0.001 for fat mass and % body fat). (g–h) Increased O₂ consumption but reduced activity (reduced numbers of laser beam breaks) in Slurp2^−/− mice. The differences in O₂ consumption and activity (two light-dark cycles for each of the 3 mice/group) were consistent and significant during the light cycle at p < 0.001 and p < 0.05, respectively.

Figure 3. Expression of Slurp2 and nearby genes in Slurp2^−/− mice

(a) Expression of Slurp2, Slurp1, and genes encoding two other Ly6 proteins (Lypd2, Ly6d) in the paw skin of Slurp2^−/− mice, as judged by qRT-PCR, means ± SEM. Slurp2^+/+ (n = 6), Slurp2^+/− (n = 5), and Slurp2^−/− mice (n = 4/group). The levels of Slurp1 and Slurp2 transcripts in Slurp2^−/− mice were lower than in Slurp2^+/+ mice (p < 0.01) (b) Expression of Slurp1 in the paw skin of wild-type (n = 9), Slurp1^+/− (n = 5), and Slurp2^−/− mice (n = 7). . *p < 0.05; ***p < 0.001.

Figure 4. Slurp2X^−/− mice develop PPK

(a) Paws of Slurp2X^+/+ and Slurp2X^−/− mice at 12 weeks of age. (b) H&E–stained sections of paw skin in Slurp2X^+/+ and Slurp2X^−/− mice, revealing a thickened epidermis in the paw skin of a Slurp2X^−/− mouse. Scale bar, 100 μm. Insert in the right-hand panel shows tiny lipid droplets in the stratum corneum of Slurp2X^−/− paw skin; scale bar, 10 μm. (c) Increased transepidermal water loss (TEWL) from the paw skin of age-matched Slurp2X^+/+ and Slurp2X^−/− male mice (10–15 weeks old) as measured with an RG1 evaporimeter (n = 5/group). Means ± SEM; *p < 0.05).

Figure 5. Slurp2X^−/− mice exhibit hind limb clasping and reduced body weight

(a) Hind limb clasping in male Slurp2X^−/− mice when picked up by the tail (similar results were observed with female Slurp2X^−/− mice). (b) Quantification of the hind limb clasping phenotype (0 for no hind limb retraction; 1 for unilateral retraction; 2 for bilateral retraction) (n = 26 for Slurp2X^+/+ mice and n = 16 for Slurp2X^−/− mice). Means ± SEM; ***p < 0.001. (c–d) Weight gain in chow-fed male and female Slurp2X^+/+ (wild-type) and Slurp2X^−/− mice (4 to 16 weeks of age; n = 9/group). Males: n = 8/group; differences were significant at weeks 12–16 (p < 0.05). Females: n = 13 Slurp2X^+/+ and n = 14 Slurp2X^−/−; p <0.03 for weeks 7–16. Means ± SEM.