Novel insights into the pathways regulating the canine hair cycle and their deregulation in alopecia X

<div><p>Alopecia X is a hair cycle arrest disorder in Pomeranians. Histologically, kenogen and telogen hair follicles predominate, whereas anagen follicles are sparse. The induction of anagen relies on the activation of hair follicle stem cells and their subsequent proliferation and differentiation. Stem cell function depends on finely tuned interactions of signaling molecules and transcription factors, which are not well defined in dogs. We performed transcriptome profiling on skin biopsies to analyze altered molecular pathways in alopecia X. Biopsies from five affected and four non-affected Pomeranians were investigated. Differential gene expression revealed a downregulation of key regulator genes of the Wnt (<i>CTNNB1</i>, <i>LEF1</i>, <i>TCF3</i>, <i>WNT10B</i>) and Shh (<i>SHH</i>, <i>GLI1</i>, <i>SMO</i>, <i>PTCH2</i>) pathways. In mice it has been shown that Wnt and Shh signaling results in stem cell activation and differentiation Thus our findings are in line with the lack of anagen hair follicles in dogs with Alopecia X. We also observed a significant downregulation of the stem cell markers <i>SOX9</i>, <i>LHX2</i>, <i>LGR5</i>, <i>TCF7L1</i> and <i>GLI1</i> whereas <i>NFATc1</i>, a quiescence marker, was upregulated in alopecia X. Moreover, genes coding for enzymes directly involved in the sex hormone metabolism (<i>CYP1A1</i>, <i>CYP1B1</i>, <i>HSD17B14</i>) were differentially regulated in alopecia X. These findings are in agreement with the so far proposed but not yet proven deregulation of the sex hormone metabolism in this disease.</p></div

Differentially expressed genes in dogs with AX and their physiological function during specific hair cycle phases.

<p>Black indicates downregulation and red indicates upregulation of the respective genes.</p

Histogram and pie chart of the GO classification of differentially expressed genes using the PANTHER Classification System.

<p>(A) The functional classification on the basis of the biological process shows an overrepresentation of differentially regulated genes involved in cellular and metabolic processes. (B) The majority of genes that are differentially regulated within the category of cellular process are involved in cell communication, cell cycle and cellular component movement. The percentages in Figs 2A and 2B correspond to the genes assigned to a specific GO term over the total number of differentially expressed genes.</p

Principal component analysis of the samples in the first two component space.

<p>Samples are plotted across the two most variable components (PC1 and PC2); sample clustering is rather based on condition and not sex.</p

Differentially expressed genes playing a role in sex hormone biosynthesis and metabolism.

<p>Sex hormone biosynthesis and metabolism is accomplished by a complex cascade involving hormones and enzymes of the hypothalamic-pituitary-gonadal axis, the vitamin D synthesis and the pineal gland hormone melatonin. Since it has been shown that the skin has its own neuroendocrine system these complex interactions may also occur in the skin. They are depicted in Fig 4. The differentially regulated genes involved in this process are indicated in blue if they were downregulated and red if they were upregulated. Abbreviations: DHEA, dehydroepiandrosterone; DHT, dehydrotestosterone; CYP, cytochrome P 450 family enzymes; 5α-Red, 5α- Reductase; ESR, estrogen receptor; GnRH, gonadotropin- releasing hormone; FSH, follicle- stimulation hormone; LH, luteinizing hormone; <i>KISS1</i>, kisspeptin 1 gene. In bold: endogenous female sex hormones; their affinity for estrogen receptors is marked with * for higher affinity to ESR-α encoded by <i>ESR1</i> and ** for higher affinity to ESR-β encoded by <i>ESR2</i>. ¹ indicates that melatonin decreases the levels of these. ^# Calcitriol increases levels of estradiol.</p

Mapping of HNPK in Labrador Retrievers.

<p>(A) A genome-wide association study using 13 cases and 23 controls indicates a strong signal with multiple associated SNPs on CFA 2. (B) The detailed view of CFA 2 delineates an associated interval of ∼4 Mb. (C) Homozygosity mapping. Each horizontal bar corresponds to one of the 13 analyzed cases. Homozygous regions with shared alleles are shown in black. A shared homozygous interval of ∼1.6 Mb delineates the exact boundaries of the critical interval from 20,818,258–22,414,948 bp (CanFam 3.1 assembly). (D) Gene content of the corresponding human interval on HSA 10 (NCBI annotation, genome build 37.5).</p

Variants detected by whole genome re-sequencing of an affected Labrador Retriever.

a<p>The sequences were compared to the reference genome (CanFam 3.1) from a Boxer. Only variants that were homozygous in the affected Labrador Retriever are reported.</p

Four non-synonymous variants in the critical interval of an HNPK affected Labrador Retriever with respect to the Boxer reference genome (CanFam 3.1).

a<p>Subsequent analyses of the original Sanger reads from the dog genome project revealed that this is not a true variant, but rather an error in the reference genome assembly (data not shown).</p

Evolutionary conservation of the asparagine residue at position 324 in the SUV39H2 protein.

<p>Position 324 within the catalytically active SET domain is perfectly conserved across vertebrates in all known SUV39H2 orthologs. In the lower part of the alignment the sequences of closely related paralogous histone methyltransferases with similar substrate specificity also demonstrate conservation of the asparagine at this position. The sequences were derived from the following database accessions: <i>C. lupus</i> SUV39H2 XP_535179.2, <i>H. sapiens</i> SUV39H2 NP_001180353.1, <i>B. taurus</i> SUV39H2 NP_001032556.1, <i>M. musculus</i> Suv39h2 NP_073561.2, <i>G. gallus</i> SUV39H2 NP_001026541.1, <i>X. laevis</i> suv39h2 NP_001091337.1, <i>H. sapiens</i> SUV39H1 NP_003164.1, <i>H. sapiens</i> EHMT1 NP_079033.4, <i>H. sapiens</i> EHMT2 NP_006700.3, <i>H. sapiens</i> SETDB1 NP_001138887.1, <i>H. sapiens</i> SETDB2 NP_114121.2.</p

Nasal planum tissue of an HNPK affected and a non-affected Labrador Retriever.

<p>(A) Hematoxylin and eosin (HE) staining of an HNPK affected dog shows diffuse parakeratotic hyperkeratosis, hydropic keratinocytes in upper epidermal layers (black arrows), epidermal lymphocytic exocytosis, and a perivascular dermal lymphocytic infiltrate. The formation of elongated and sometimes fused slim rete pegs in the affected dog contributes to the thickening of the epidermis (the boundary of one rete peg is indicated by a white dashed line, one fusion is indicated by the white arrow). (B) HE staining of a control dog. (C–F) Immunodetection of desmoglein 1 (DSG1, green) in two different magnifications. Nuclei are counterstained with Hoechst 33258 (blue). No visible differences in the DSG1 staining patterns are present between HNPK affected and control dog. Scale bar = 100 µm for HE/DSG1 100× and 25 µm for DSG1 400×.</p