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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: J Invest Dermatol. 2015 Nov 18;136(2):436–443. doi: 10.1016/j.jid.2015.11.003

Palmoplantar keratoderma in Slurp2-deficient mice

Christopher M Allan 1,*, Shiri Procaccia 1,*, Deanna Tran 1, Yiping Tu 1, Richard H Barnes II 1, Mikael Larsson 1, Bernard B Allan 4, Lorraine C Young 1, Cynthia Hong 3,5, Peter Tontonoz 3,5, Loren G Fong 1,†, Stephen G Young 1,2,†, Anne P Beigneux 1,†
PMCID: PMC4789766  NIHMSID: NIHMS734515  PMID: 26967477

Abstract

SLURP1, a member of the Ly6 protein family, is secreted by suprabasal keratinocytes. Mutations in SLURP1 cause a palmoplantar keratoderma (PPK) known as mal de Meleda. Another secreted Ly6 protein, SLURP2, 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., PPK), 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 very similar to those of Slurp1−/− mice. To solidify a link between Slurp2 deficiency and PPK 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.

Introduction

Every clinical dermatologist knows that SLURP1 mutations cause a palmoplantar keratoderma (PPK) known as mal de Meleda (Eckl et al., 2003; Fischer et al., 2001a; Marrakchi et al., 2003). Mal de Meleda patients have a thickened epidermis on the palms and soles, occasionally with pseudoainhum formation, but the skin elsewhere is normal or minimally affected. Mal de Meleda is a recessive syndrome; every patient carries two mutant SLURP1 alleles. Heterozygous carriers are free of disease.

SLURP1 is an 8.9-kDa protein of the “Lymphocyte Antigen 6” (Ly6) family. The hallmark of this family is an “Ly6 domain” with 8–10 cysteines, all arranged in a characteristic spacing pattern and all disulfide-linked so as to create a three-fingered motif (Galat et al., 2008; Kieffer et al., 1994). The same structure is found in many secreted toxins in viper and cobra venom (Fry et al., 2003; Kini, 2002). Most Ly6 proteins in mammals are tethered to the plasma membrane by a glycosylphosphatidylinositol (GPI)-anchor, but SLURP1 is an exception. SLURP1 is synthesized and secreted by keratinocytes (Favre et al., 2007), enters the plasma, and can be found in the urine (Andermann et al., 1999; Mastrangeli et al., 2003).

The function of SLURP1 in the skin is not well defined, although several studies have reported that it modulates acetylcholine signaling (Arredondo et al., 2005; Chernyavsky et al., 2010; Chimienti et al., 2003). SLURP1 is often presumed to bind to a cell-surface receptor on keratinocytes (Fischer et al., 2001a), but thus far no one has documented binding of SLURP1 to a specific keratinocyte protein.

Inactivating Slurp1 in mice (either by replacing exon 2 with neo and lacZ cassettes or by introducing a premature stop codon into exon 2) causes PPK (Adeyo et al., 2014). The PPK is obvious by ~6–8 weeks of age. Slurp1 knockout mice also exhibit increased energy consumption and reduced body weight (Adeyo et al., 2014). The mechanism for those phenotypes is not clear, but they might be secondary to increased grooming (as a result of the PPK). Slurp1-deficient mice also exhibit hind limb clasping, a nonspecific neuromuscular phenotype (Dequen et al., 2010; Hayward et al., 2008; Lalonde and Strazielle, 2011). Again, the mechanism is unclear.

In mammals, SLURP1 is not the only secreted Ly6 protein. SLURP2, an ~8-kDa Ly6 protein, is synthesized and secreted by keratinocytes. Studies of human skin with a human SLURP2–specific monoclonal antibody revealed that SLURP2, like SLURP1, is made by suprabasal keratinocytes (Arredondo et al., 2006). SLURP2 was initially identified as a cDNA that is upregulated in psoriasis vulgaris (Tsuji et al., 2003). The gene for SLURP2 is immediately upstream from LYNX1 and 21.9 kb downstream from SLURP1; SLURP2 is ~443 kb upstream from the gene for GPIHBP1, a GPI-anchored Ly6 protein that shuttles lipoprotein lipase to the capillary lumen (Beigneux et al., 2007; Davies et al., 2010; Goulbourne et al., 2014). The function of SLURP2 is unclear, but one paper proposed that SLURP2 modulates acetylcholine signaling (Arredondo et al., 2006). Similar to the situation with SLURP1, no one has yet identified specific interactions between SLURP2 and any keratinocyte protein.

There have been no insights into possible consequences of SLURP2 deficiency. One could easily imagine that the consequences of SLURP2 and SLURP1 deficiency might be similar, given that both are members of the Ly6 family and both are secreted by keratinocytes. On the other hand, one could be skeptical about that possibility, given that different Ly6 family members can play very diverse functions in mammalian biology (Galat et al., 2008). Moreover, the level of amino acid sequence identity between SLURP1 and SLURP2 is extremely low—only 17% after the 10 cysteines of the Ly6 domain are excluded from consideration (Fig. S1). The level of sequence identity between the Ly6 domains of SLURP2 and GPIHBP1 (the LPL transporter) is 21%.

To define the in vivo functional relevance of SLURP2 in mammals and to determine whether SLURP2 might be relevant to skin disease, we characterized two independent lines of Slurp2 knockout mice.

Results

We first examined Slurp2 knockout mice (Slurp2−/−) that were created by replacing an exon 2–3 fragment with neo and lacZ cassettes (Fig. S2). As expected, Slurp2 transcripts were half-normal in heterozygotes and absent in homozygotes (see Fig. 3 below). We attempted to visualize mouse SLURP2 in the skin of wild-type mice by western blotting and immunohistochemistry with our rabbit antiserum against a mouse SLURP2 peptide, but we were unable to detect a specific signal.

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

Figure 3

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(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.

Slurp2−/− mice appeared normal at birth and at weaning, but hyperkeratosis on the volar surface of the paws (i.e., PPK) was invariably present by 6–8 weeks of age (Fig. 1a). Grossly, the PPK in Slurp2−/− mice was indistinguishable from that in Slurp1−/− mice (Adeyo et al., 2014). On H&E–stained sections, the epidermis of the paw in Slurp2−/− mice exhibited hyperkeratosis, and the stratum granulosum was poorly demarcated (Fig. 1b). The stratum corneum contained many tiny lipid droplets, as judged by H&E and BODIPY staining (Fig. 1c–d). There was no inflammation in the dermis or epidermis (confirmed by a UCLA dermatopathologist, Dr. Peter G. Sarantopoulos). Also, we did not observe consistently higher cytokine transcripts in the paw skin of Slurp2X−/− mice, whereas the expression of each of the cytokines was increased in the skin of Apoe−/−Lxra−/− mice (where cholesterol accumulation in the skin is accompanied by histologic evidence of inflammation) (Fig. S3) (Bradley et al., 2007).

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

Figure 1

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(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.

BrdU incorporation into basal keratinocytes was increased in the paws of Slurp2−/− mice (Fig. 1e). These findings are similar to those in Slurp1−/− mice (Adeyo et al., 2014). Apart from the paw, the skin in Slurp2−/− mice was normal, both by gross appearance and by routine histology (Fig. S4). Heterozygous knockout mice (Slurp2+/−) were free of disease and indistinguishable from wild-type mice.

Like Slurp1−/− mice (Adeyo et al., 2014), Slurp2−/− mice clasped their hind limbs when picked up by the tail (a phenotype often observed in mice with cerebellar disease, myopathy, or peripheral neuropathy) (Dequen et al., 2010; Hayward et al., 2008; Lalonde and Strazielle, 2011) (Fig. 2a–b). The onset of the hind limb clasping in Slurp2−/− mice coincided with the development of obvious PPK (~6–8 weeks of age). Also, like Slurp1−/− mice (Adeyo et al., 2014), Slurp2−/− mice had lower body weights than littermate wild-type mice (Fig. 2c–d) despite consuming similar amounts of food (Fig. 2e). The lower body weight in Slurp2−/− mice was primarily due to reduced adiposity (Fig. 2f). Metabolic cage studies (n = 3 mice/group) revealed increased oxygen consumption but reduced numbers of laser beam breaks in Slurp2−/− mice (Fig. 2g–h). The plasma cholesterol levels in Slurp2−/− mice were lower than in wild-type mice (p = 0.002); the plasma glucose levels were similar (Fig. S5).

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

Figure 2

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(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.

Slurp2 transcripts were absent in the paw skin of Slurp2−/− mice. We previously showed that replacing exon 2 of Slurp1 with lacZ and neo cassettes resulted in reduced expression of several nearby genes, including Slurp2 (Adeyo et al., 2014). Given those results, we examined the expression of Slurp1 and two “nearby Ly6 genes” (Lypd2, Ly6d) in the paw skin of Slurp2−/− mice. Slurp1 transcripts were reduced by ~60% in Slurp2−/− mice and ~50% in Slurp2+/− mice (Fig. 3a). The expression of Lypd2 (located ~11.8 kb upstream from Slurp2) was reduced by ~35% in the paw skin of Slurp2−/− mice. The expression of Ly6d (~15 kb downstream from Slurp2) was not reduced and actually appeared to be increased in Slurp2−/− mice (Fig. 3a).

The fact that the Slurp2 knockout allele reduced Slurp1 transcripts in paw skin suggested the possibility that the disease phenotypes in Slurp2−/− mice might be caused by reduced Slurp1 expression. To examine this issue, we measured Slurp1 transcript levels in the paw skin of an independent group of Slurp2−/− mice as well as an age-matched group of Slurp1+/− mice (where the paw skin was normal). Again, Slurp1 expression in the paw skin of Slurp2−/− mice was reduced by ~60%. Not surprisingly, Slurp1 expression in Slurp1+/− mice was reduced by ~50% (Fig. 3b).

It seems unlikely that a ~60% reduction in Slurp1 transcripts in Slurp2−/− mice would lead to PPK, reduced body weight, and hind limb clasping, while a 50% reduction in Slurp1 in Slurp1+/− mice would yield no disease phenotypes at all. Nevertheless, to further examine the link between SLURP2 deficiency and PPK, we created a new Slurp2 knockout allele (designated Slurp2X) by inserting a simple nonsense mutation into exon 2 (i.e., no lacZ or neo insertions) (Fig. S6). Heterozygous Slurp2X knockout mice (Slurp2X+/−) were normal, indistinguishable from wild-type littermates. Homozygous knockout mice (Slurp2X−/−) exhibited PPK (Fig. 4a–b) that was similar to that in the original Slurp2−/− mice (Fig. 1). The Slurp2X−/− mice also exhibited increased trans-epidermal water loss from the skin of the paw (Fig. 4c). Like the original line of Slurp2−/− mice, Slurp2X−/− mice exhibited hind limb clasping and reduced body weight (Fig. 5). Given that the nonsense mutation in Slurp2X−/− mice was located ~20 kb downstream from Slurp1, it is difficult to avoid the conclusion that Slurp2 is a “PPK gene” in mice.

Figure 4. Slurp2X−/− mice develop PPK.

Figure 4

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(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.

Figure 5

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(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.

Given the severity of the PPK in Slurp2X−/− mice, we predicted that the expression of many “keratinocyte genes” would be perturbed in paw skin. Indeed, this was the case, as judged by qRT-PCR studies on selected keratinocyte genes (Table S1). For example, keratin 6b and keratin 16 expression levels were markedly upregulated in Slurp2X−/− mice. Similar gene-expression changes were observed in the paw skin of Slurp1X−/− mice (Table S1), although we caution against comparing levels of transcripts in Slurp2X−/− and Slurp1X−/− mice because the two groups of mice were not perfectly matched for age. Several transcripts that we found to be increased in the setting of SLURP2 deficiency, for example transcripts for keratin 6b, keratin 16, late cornified envelope protein 3a, and defensin 4B were also increased in the setting of the PPK associated with pachyonychia congenital (Cao et al., 2015). Slurp1 transcripts in Slurp2X−/− mice were reduced by 50%, regardless of whether the Slurp1 transcript levels were normalized to cyclophilin A (expressed in all cells) or LYPD5 (expressed in suprabasal keratinocytes). Given the striking epidermal pathology and massive changes in the expression of many keratinocyte genes (Table S1), the relatively small change in Slurp1 transcripts is probably not surprising. Again, we would contend that a ~50% reduction in Slurp1 transcripts in the Slurp2X−/− mice is unlikely to be relevant to PPK, given that a 50% reduction in Slurp1 transcripts in heterozygous Slurp1 knockout mice does not elicit PPK or any other disease phenotype.

Heterozygosity for the Slurp1X or Slurp2X alleles lowered transcript levels by one-half but caused no disease (Fig. 3), whereas homozygosity for either allele caused severe PPK. A formal possibility is that SLURP1 and SLURP2 play redundant functions and that a threshold level of “SLURP protein” (i.e., SLURP1 plus SLURP2) is necessary to ward off PPK. If that were the case, we reasoned that mice heterozygous for both Slurp1X and Slurp2X alleles (Slurp1X+/−Slurp2X+/− mice) might exhibit PPK. This was not the case. Slurp1X+/−Slurp2X+/− mice did not develop PPK (Fig. S7), whereas PPK was invariably evident in Slurp1X−/− and Slurp2X−/− mice by 6–8 weeks of age. Slurp1X+/−Slurp2X+/− and wild-type mice had normal digits, whereas Slurp1X−/− and Slurp2X−/− mice exhibited PPK (evident by the bulbous appearance of the distal phalanges).

Discussion

The link between SLURP1 deficiency and PPK is well documented. SLURP1 is produced by keratinocytes, and many different SLURP1 mutations have been uncovered in mal de Meleda patients (Adeyo et al., 2015b; Bakija-Konsuo et al., 2002; Chimienti et al., 2003; Fischer et al., 2001b; Marrakchi et al., 2003; Nellen et al., 2013). Also, two independent lines of Slurp1 knockout mice (Slurp1−/− and Slurp1X−/− mice) developed PPK (Adeyo et al., 2014). In contrast, the functional relevance of SLURP2, another secreted Ly6 protein, has been unclear. In the current studies, we initially investigated Slurp2 knockout mice (Slurp2−/− mice) in which exon 2–3 sequences were replaced with neo and lacZ cassettes. The Slurp2−/− mice developed PPK, similar to that in Slurp1−/− mice (Adeyo et al., 2014). The PPK in Slurp2−/− mice was accompanied by increased keratinocyte proliferation, a poorly demarcated stratum granulosum, and an accumulation of small lipid droplets in the stratum corneum. Like Slurp1-deficient mice (Adeyo et al., 2014), Slurp2−/− mice exhibited hind limb clasping and reduced body weight.

The fact that the phenotypes of Slurp2−/− mice closely resembled Slurp1−/− mice was somewhat surprising because SLURP1 and SLURP2 have negligible levels of sequence identity (apart from the 10 cysteines of the Ly6 domain). We were initially concerned by the fact that Slurp1 transcripts were reduced in Slurp2−/− mice and that the disease phenotypes in Slurp2−/− mice might result from reduced Slurp1 expression (conceivably reflecting the effects of the neo and lacZ insertions on the expression of nearby genes) (Adeyo et al., 2014). That worry was mitigated by the finding that Slurp1 transcripts in the paw skin of Slurp2−/− mice (which manifest severe PPK) were in the same range as those in Slurp1+/− mice (which had no disease). The worry was further mitigated by the discovery that Slurp2X−/− mice (where Slurp2 was inactivated with a nonsense mutation) manifested the same disease phenotypes. Finding disease in mice harboring a simple Slurp2 nonsense mutation strongly supports the idea that Slurp2 is a bona fide “PPK gene” in mice.

We do not fully understand why the PPK in Slurp1- and Slurp2-deficient mice is accompanied by hind limb clasping and metabolic phenotypes. Those phenotypes could be due to the effects of SLURP1 and SLURP2 deficiencies on other tissues, but a more parsimonious explanation would be that these phenotypes are consequences of the PPK. The metabolic phenotypes might relate to increased grooming of paw skin (a form of locomotor activity that often goes undetected in metabolic cages) or to effects of water loss (i.e., increased loss of saliva) during grooming (Gordon, 1990). Hind limb clasping was present at 6–8 weeks of age, coinciding perfectly with visible evidence of PPK. It seems possible that hind limb clasping could relate to impaired nociception/proprioception as a result of the thickened epidermis on the paws. Alternatively, the hind limb clasping could relate to the presence of a passenger gene that segregates with Slurp2 (Ji et al., 2010; Smithies and Maeda, 1995; Westrick et al., 2010).

Thus far, no one has identified SLURP2 mutations in humans. It is possible that SLURP2 is simply dispensable in humans. However, we suspect that, sooner or later, dermatologists will uncover a SLURP2 mutation in a human subject with PPK. It is noteworthy that some patients with PPK resembling mal de Meleda do not have SLURP1 mutations (Charfeddine et al., 2003; Lestringant et al., 2001; van Steensel et al., 2002). In some cases, the linkage data seem inconsistent with SLURP2 but in none of the cases was SLURP2 sequenced.

The functions of SLURP1 and SLURP2 proteins require more study. Most of the earlier research focused on the possibility that these proteins (like secreted snake toxins) affect acetylcholine signaling (Chimienti et al., 2003) (Arredondo et al., 2006; Arredondo et al., 2005; Chernyavsky et al., 2010). Several studies suggested that SLURP1 affects signaling through the α7 subtype of nicotinic acetylcholine receptors (Chimienti et al., 2003) (Arredondo et al., 2005; Chernyavsky et al., 2010), and one study found that SLURP2 competed with radiolabeled nicotinic ligands for binding to keratinocytes (Arredondo et al., 2006). However, no study has yet demonstrated a direct protein–protein interaction between SLURP1 or SLURP2 and another keratinocyte protein. Adeyo and coworkers were not able to detect SLURP1 binding to CHO cells that overexpressed the α7-nicotinic acetylcholine receptor (Adeyo et al., 2014; Adeyo et al., 2015a), but the possibility that SLURP1 binds to other acetylcholine receptors was not tested.

We considered the possibility that Slurp1 and Slurp2 play purely redundant functions and that heterozygosity for both knockout alleles would elicit the same disease phenotypes found in Slurp1−/− and Slurp2−/− mice. This was not the case; double heterozygous mice (Slurp1+/−Slurp2+/−) were free of disease. This finding points against functional redundancy of Slurp1 and Slurp2, but finding precise roles for SLURP1 and SLURP2 will require more studies. For example, it would be desirable to determine if a complete deficiency of both SLURP1 and SLURP2 (Slurp1−/−Slurp2−/−) would result in particularly severe disease—or whether the double-knockout mice would be indistinguishable from single-knockout mice. If the latter were the case, it would suggest that SLURP1 and SLURP2 are involved in the same pathway or that they were components in the same multi-protein complex. Still another possibility would be that SLURP2 is required for the expression of “the SLURP1 receptor.” Such a scenario could explain why the phenotypes of SLURP1 and SLURP2 deficiencies are so similar.

Inflammation and infection are prominent features of the PPK in mal de Meleda patients, but we found no histologic evidence of inflammation in the paw skin of Slurp1 and Slurp2 knockout mice. The absence of inflammatory infiltrates in the epidermis or dermis of the mouse PPK models suggests that the inflammation in humans with mal de Meleda may be secondary to infections in the markedly thickened palms and soles (as opposed to being a direct consequence of the absence of a functional SLURP protein).

The knockout mice described in the current study represent an important step forward in defining the function of SLURP2. However, more work is needed. In the case of SLURP1 and SLURP2, digging deeper into their precise function and their link to PPK will require improved reagents, for example monospecific antibodies for mouse SLURP1 and SLURP2 and recombinant proteins. Once improved reagents are in hand, it ought to be possible to identify proteins that interact with SLURP1 and SLURP2 and pursue mechanisms for disease. The availability of recombinant proteins could also open the door to testing a therapy for mal de Meleda.

Materials and Methods

Slurp2-deficient mice. Slurp2−/− mice were obtained from the UC Davis Mutant Mouse Regional Resource Center (MMRRC). The Slurp2 knockout allele was originally created by Lexicon Genetics for Genentech (Tang et al., 2010). Genotyping was performed by PCR. The mutant allele was detected with oligonucleotides 5′-GCAGCGCATCGCCTTCTATC-3′ and 5′-CATTGGACAACTATGTGACCCAGGTA-3′ (amplifying a 396-bp fragment from the neo to sequences downstream from Slurp2). The wild-type allele was detected with oligonucleotides 5′-TGGCTCCAATGATTACTG-3′ and 5′-CTAGACGGGTGAGA-3′ (amplifying a 287-bp fragment from intron 1 to exon 2). We created a second line of Slurp2 knockout mice (“Slurp2X mice”) by gene targeting with a sequence-replacement vector designed to change Leu-27 in exon 2 of Slurp2 to a premature stop codon. A description of the Slurp2X knock-in vector is included in Fig. S6. Targeted clones were identified by long-range PCR (Fig. S6). Two targeted clones were injected into C57BL/6 blastocysts to produce chimeric mice, which were then bred to create “Slurp2X knockout mice.” The neo was removed by breeding Slurp2X+/− mice with a deleter Cre transgenic line (Ella-Cre transgenic mice from The Jackson Laboratory). Genotyping was performed by PCR with oligonucleotides 5′-CTGGGCTGGATGCAAGACCT-3′ and 5′-ACACTCACGGGTGGCAATGA-3′ (amplifying a 663-bp fragment spanning the targeted point mutation). The 663-bp product from the mutant alleles could be cleaved by SpeI into 575- and 88-bp fragments.

Slurp1 knockout mice (one in which exon 2 coding sequences were replaced with neo and lacZ cassettes, and a second in which a nonsense mutation was inserted into exon 2) have been described previously (Adeyo et al., 2014). All mice had a mixed genetic background (129/OlaHsd and C57BL/6). Mice were fed a chow diet (LabDiet No. 5001, Purina) and housed in a barrier facility with a 12-h light-dark cycle. All studies were approved by UCLA’s Animal Research Committee.

Histology and immunofluorescence microscopy

Skin biopsies were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Images were obtained with a Nikon Eclipse E600 microscope (Plan Fluor 20×/0.50 NA or 40×/0.75 NA objectives, air) with a DS-Fi2 camera (Nikon). To detect lipid droplets, skin biopsies were placed in Optimal Cutting Temperature (OCT) compound, frozen, and cryosectioned (10 μm). Sections were fixed in 4% formalin, washed three times in phosphate-buffered saline, and stained with BODIPY 493/503 (Adeyo et al., 2014). To assess BrdU uptake, 4-week-old mice were given an intraperitoneal injection of BrdU (40 mg/kg of body weight) and euthanized 1 h later. Cryosections were fixed with acetone for 10 min, treated with 1 N HCl for 10 min on ice, 2 N HCl for 10 min at room temperature and 10 min at 37°C, and neutralized with 0.1 M sodium borate pH 8.5 for 12 min (Adeyo et al., 2014). Sections were then blocked with 10% donkey serum, and incubated overnight at 4°C with a rat monoclonal antibody against BrdU (Abcam, 1:200), followed by a 1-h incubation with an Alexa Fluor 488–conjugated donkey anti-rat IgG (Life Technologies, 1:200) (Adeyo et al., 2014). DNA was stained with DAPI. Images were obtained with a Zeiss LSM700 laser-scanning microscope [Plan Apochromat 20×/0.80 NA (air) or 63×/1.4 NA (oil) objectives]. We attempted to detect SLURP2 in skin sections from wild-type mice with a rabbit antiserum against a mouse SLURP2 peptide (CVIIATRSPISFTDLPLVTKM), but no signal was detected.

Transepidermal Water Loss

Transepidermal water loss (TEWL) measurements on the skin of the back, rear paws, and ear were recorded at room temperature on age- and sex-matched wild-type and Slurp2X−/− mice (n = 5/group) with an RG1 evaporimeter (cyberDERM) (Adeyo et al., 2014).

Body Weight/Metabolic Phenotypes

Measurements of oxygen consumption were performed using sealed metabolic cages (Oxymax, Columbus Instruments), and physical activity was assessed by numbers of laser beam breaks (Weinstein et al., 2012). Data was collected and analyzed with Oxymax/CLAMS software. Body weights of male and female mice were recorded weekly. Adiposity was assessed by NMR (Weinstein et al., 2010).

Gene Expression

RNA was isolated with TRI reagent (Molecular Research), and qRT-PCR measurements were performed in triplicate on a 7900HT Fast Real-Time PCR System (Applied Biosystems) (Jung et al., 2013; Weinstein et al., 2012; Yang et al., 2011). Gene-expression was calculated with the comparative CT method and normalized to cyclophilin A. Primers for Slurp1 were 5′-CACGGCCATTAACTCATGC-3′ and 5′-CCATGGGACTGTGGTTGAA-3′ (exons 2 and 3, respectively). Primers for Slurp2 were 5′-TGGTCTTGAGCATGGAGCTA-3′ and 5′-TCCATGGGCAGCTAGACG-3′ (exons 1 and 2, respectively). Primers for Lypd2 and Ly6d were described previously (Adeyo et al., 2014). We also used qRT-PCR to assess levels of Krt6b, Krt16, Krt24, Lce1m, Lce31, Lce3f, Areg, and Defb4 expression the paw skin of Slurp2X−/−, Slurp1X−/−, and littermate wild-type mice (primers listed in Table S2).

Supplementary Material

Acknowledgments

This work was supported by P01 HL090553 (SGY). We thank Dr. Oludotun Adeyo for work on this project and Peter G. Sarantopoulos for reviewing skin pathology findings in the Slurp2-deficient mice.

Abbreviations

PPK

palmoplantar keratoderma

Ly6

lymphocyte antigen 6

GPIHBP1

glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1

Footnotes

Conflict of interest

The authors have declared that no conflict of interest exists.

References

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  77. Weinstein MM, Goulbourne CN, Davies BS, et al. Reciprocal metabolic perturbations in the adipose tissue and liver of GPIHBP1-deficient mice. Arterioscler Thromb Vasc Biol. 2012;32:230–5. doi: 10.1161/ATVBAHA.111.241406. [DOI] [PMC free article] [PubMed] [Google Scholar]
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