Granular Layer Keratinocytes Research Articles

Cell-Specific and Variant-Linked Alterations in Expression of ERAP1, ERAP2, and LNPEP Aminopeptidases in Psoriasis

ERAP1, ERAP2, and LNPEP are aminopeptidases implicated in autoimmune pathophysiology. In this study, we show that ERAP2 is upregulated and ERAP1 is downregulated in patients with psoriasis who are homozygous for autoimmune-linked variants of ERAP. We also demonstrate that aminopeptidase expression is not uniform in the skin. Specifically, the intracellular antigen-processing aminopeptidases ERAP1 and ERAP2 are strongly expressed in basal and early spinous layer keratinocytes, whereas granular layer keratinocytes expressed predominantly LNPEP, an aminopeptidase specialized in the processing of extracellular antigens for presentation to T cells. In psoriasis, basal keratinocytes also expressed the T-cell- and monocyte-attracting chemokine, CCL2, and the T-cell-supporting cytokine, IL-15. In contrast, TGF-β1 was the major cytokine expressed by healthy control basal keratinocytes. SFRP2-high dermal fibroblasts were also noted to have an ERAP2-high expression phenotype and elevated HLA-C. In psoriasis, the SFRP2-high fibroblast subpopulation also expressed elevated CXCL14. From these results, we postulate that (i) an increased ERAP2/ERAP1 ratio results in altered antigen processing, a potential mechanism by which ERAP risk alleles predispose individuals to autoimmunity; (ii) ERAP2-high expressing cells display a unique major histocompatibility complex-bound peptidome generated from intracellular antigens; and (iii) the granular layer peptidome is skewed toward extracellular antigens.

Proprotein convertase subtilisin/kexin type 9 is a psoriasis-susceptibility locus that is negatively related to IL36G.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a posttranslational regulator of the LDL receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-Seq–based variant discovery, we identified a possible psoriasis-susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C > T). This finding was verified in independently acquired genomic and RNA-Seq data sets. Single-cell RNA-Seq (scRNA-Seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-Seq and IHC demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression, including IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C > T was associated with lower PCSK9 expression and higher IL36G expression, when compared with heterozygous skin or cell lines. Together, these results support PCSK9 as a psoriasis-susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.

427 The autophagy receptor TEX264 initiates endoplasmic reticulum degradation in differentiating epidermal keratinocytes

During late stages of differentiation, epidermal keratinocytes break down organelles and nuclei to become compact corneocytes that form a barrier for the body. Our prior work uncovered a novel pathway driving degradation of mitochondria in granular layer keratinocytes, which engage the selective autophagy machinery to route mitochondrial fragments into lysosomes as they begin to cornify. These findings led us to ask if the epidermis uses distinct autophagy receptors to orchestrate breakdown of other organelles as keratinocytes transition into the stratum corneum.

Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins

BackgroundProfilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 (“A”) domain and a unique “B” domain with a nuclear localization sequence. ObjectiveTo determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal “AB” domain and compare it to other S100 fused-type proteins. MethodsWe used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins. ResultsComparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins. ConclusionThe N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions.

Palmoplantar hyperkeratotic projections

Palmoplantar hyperkeratotic projections

Oleic acid enhances keratinocytes differentiation via the upregulation of miR-203 in human epidermal keratinocytes.

Oleic acid (OA) is an unsaturated free fatty acid (FFA) constituent of sebum. FFAs modulate keratinocyte differentiation. In this study, we determined whether OA affects keratinocyte differentiation in neonatal human epidermal keratinocytes (HEKn). HEKn was grown in EpiLife medium. The cells were treated with various concentrations of OA. The expression levels of keratin 10 and involucrin were determined using Western blotting (for the proteins) and quantitative real time polymerase chain reaction (qRT-PCR) (for the mRNAs). Cytoskeletal changes were investigated by immunofluorescent staining. The levels of microRNA (miR)-203 were determined by stem-loop qRT-PCR. The effect of miR-203 on keratinocyte differentiation was evaluated using anti-miR-203. Treatment with OA promoted the expression of keratin 10 and involucrin, which are markers of spinous and granular layer keratinocytes, respectively. Treatment with OA also induced cell stratification and cytoskeletal changes such as the concentric ring organization of actin, a loss of planar polarity, and increased localization of epithelial cadherin (E-cadherin) at cell-cell contacts. OA increased the expression of miR-203, which is associated with keratinocyte differentiation, and reduced the expression of p63, a target of miR-203, in HEKn. Furthermore, transfection with anti-miR-203 suppressed the OA-induced expression of involucrin. Oleic acid accelerates keratinocyte differentiation via the upregulation of miR-203 in HEKn under sub-confluent conditions.

The roles of ABCA12 in keratinocyte differentiation and lipid barrier formation in the epidermis

ABCA12 is a member of the large superfamily of ATP-binding cassette (ABC) transporters, which bind and hydrolyze ATP to transport various molecules across limiting membranes or into vesicles. The ABCA subfamily members are thought to be lipid transporters. ABCA12 is a keratinocyte transmembrane lipid transporter protein associated with the transport of lipids in lamellar granules to the apical surface of granular layer keratinocytes. Extracellular lipids, including ceramide, are thought to be essential for skin barrier function. ABCA12 mutations are known to underlie the three main types of autosomal recessive congenital ichthyoses: harlequin ichthyosis, lamellar ichthyosis and congenital ichthyosiform erythroderma. ABCA12 mutations lead to defective lipid transport via lamellar granules in the keratinocytes, resulting in malformation of the epidermal lipid barrier and ichthyosis phenotypes. Studies of ABCA12-deficient model mice indicate that lipid transport by ABCA12 is also indispensable for intact differentiation of keratinocytes.

Murine mCLCA5 is expressed in granular layer keratinocytes of stratified epithelia

CLCA proteins represent a large family of proteins widely expressed in mammalian tissues with a unique expression pattern for each family member analyzed so far. However, their functions in normal and diseased tissues are poorly understood. Here, we present the cellular expression pattern of mCLCA5 in murine tissues using immunohistochemistry, confocal laser scanning microscopy and immune electron microscopy with specific antibodies and RT-qPCR following laser-capture microdissection. The mCLCA5 protein was localized to granular layer keratinocytes of virtually all stratified squamous epithelia of the body. Biochemical protein characterizations revealed that the amino-terminal cleavage product is fully secreted by the cell, while the carboxy-terminal cleavage product remains associated with the cell. The results imply that mCLCA5 may play a role in maturation and keratinization of squamous epithelial cells.

Cutaneous distribution of plasmacytoid dendritic cells in lupus erythematosus. Selective tropism at the site of epithelial apoptotic damage

Recent evidences suggest a significant role of Plasmacytoid dendritic cells (PDC) role in the pathogenesis of lupus erythematosus (LE) via production of type I IFN. Taking advantage on the availability of multiple reagents (CD123, BDCA2, and CD2ap) specifically recognizing PDC on fixed tissues, we investigated the occurrence of PDC in a cohort of 74 LE patients. The large majority of LE biopsies (67/74; 90.5%) showed cutaneous infiltration of PDC. PDC were more frequently observed (96.4 vs 72.2) and numerous in cutaneous LE compared to systemic LE (SLE) and correlated with the density of the inflammatory infiltrate (r=0.40; p<0.001). PDC reduction in SLE might be related to a broader tissue distribution of this cellular population, as indicated by their occurrence in kidneys in 11 out of 24 (45.8%) cases studied. The distribution of cutaneous PDC showed two distinct patterns. More commonly, PDC were observed within perivascular inflammatory nodules in the dermis, associated with CD208+ mature DC and T-bet+ cells [D-PDC]. A second component was observed along the dermal-epithelial junction [J-PDC], in association with cytotoxic T-cells in areas of severe epithelial damage. Notably, chemerin reactivity was observed in 64% of LE biopsies on endothelial cells and in the granular layer keratinocytes. Cutaneous PDC in LE strongly produced type I IFN, as indicated by the diffuse MxA expression, and the cytotoxic molecule granzyme B.This study confirms cutaneous PDC infiltration as hallmark of LE. The topographical segregation in D-PDC and J-PDC suggests a novel view of the role of these cells in skin autoimmunity.

Localization of ABCA12 from Golgi apparatus to lamellar granules in human upper epidermal keratinocytes

ABCA12 is an ATP-binding cassette transporter and is thought to act as a transmembrane lipid transporter. We reported that deleterious ABCA12 mutations cause a disturbance in lamellar granule (LG) lipid transport in the epidermal granular layer keratinocytes, resulting in harlequin ichthyosis, a severe genodermatosis. Detailed localization of ABCA12 in comparison with glucosylceramide and Golgi apparatus markers were studied in order to obtain clues to clarify the function(s) of ABCA12 in human skin. We performed double-labelling immunofluorescent staining using antibodies against ABCA12, glucosylceramide and two Golgi apparatus markers (TGN46 and GM130) in normal human skin and cultured keratinocytes. Immunogold electron microscopy for ABCA12 and glucosylceramide was studied on postembedding and cryoultrathin sections of normal human skin. Confocal laser scanning microscopy demonstrated that ABCA12 and glucosylceramide co-localized in the granular layer keratinocytes as well as in keratinocytes cultured in high Ca2+ conditions through the Golgi apparatus to the cell periphery. Postembedding immunogold electron microscopy revealed that both ABCA12 and glucosylceramide labellings were associated with the LG of the uppermost granular layer keratinocytes. Using cryoultramicrotomy, lamellar structures in the LG were more clearly observed, and ultrastructural localization of ABCA12 and glucosylceramide was better demonstrated to LG in the uppermost granular layer cells. These results indicate that ABCA12 plays an important role in lipid transport from the Golgi apparatus to LG in human granular layer keratinocytes.

Mild recessive epidermolytic hyperkeratosis associated with a novel keratin 10 donor splice-site mutation in a family of Norfolk terrier dogs

Epidermolytic hyperkeratosis in humans is caused by dominant-negative mutations in suprabasal epidermal keratins 1 and 10. However, spontaneous keratin mutations have not been confirmed in a species other than human. To describe an autosomal recessive, mild, nonpalmar/plantar epidermolytic ichthyosis segregating in an extended pedigree of Norfolk terrier dogs due to a splice-site mutation in the gene encoding keratin 10 (KRT10). Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Genomic DNA samples and cDNA from skin RNA were sequenced and defined a mutation in KRT10. Consequences of the mutation were evaluated by assessing protein expression with immunohistochemistry and Western blotting and gene expression with real-time RT-PCR (reverse transcriptase-polymerase chain reaction). Adult dogs with the disease had generalized, pigmented hyperkeratosis with epidermal fragility. Light microscopic examination defined epidermolysis with hyperkeratosis; ultrastructural changes included a decrease in tonofilaments and abnormal filament aggregation in upper spinous and granular layer keratinocytes. Affected dogs were homozygous for a single base GT-->TT change in the consensus donor splice site of intron 5 in KRT10. Keratin 10 protein was not detected with immunoblotting in affected dogs. Heterozygous dogs were normal based on clinical and histological appearance and keratin 10 protein expression. The mutation caused activation of at least three cryptic or alternative splice sites. Use of the cryptic sites resulted in transcripts containing premature termination codons. One transcript could result in shortening of the proximal portion of the 2B domain before the stutter region. Quantitative real-time PCR indicated a significant decrease in KRT10 mRNA levels in affected dogs compared with wild-type dogs. This disease is the first confirmed spontaneous keratin mutation in a nonhuman species and is the first reported recessive form of epidermolytic hyperkeratosis.

Porokeratosis Causing Change in Melanocytic Nevi

Porokeratosis is an uncommon cause of clinical change in pigmented lesions. We report two cases of so‐called “collision” lesions, in which the cause of clinical change in melanocytic nevi was porokeratosis. One biopsy was submitted concurrently with a biopsy from another anatomic site, which was a superficial pattern basal cell carcinoma. Both the melanocytic nevus and the basal cell carcinoma contained coronoid lamellae, focal diminution of the granular layer and subjacent dyskeratotic keratinocytes, consistent with disseminated superficial actinic porokeratosis (DSAP). The melanocytic nevus, from the lower back, was a broad compound melanocytic nevus, associated with elongated, pigmented rete (lentiginous) and small melanocytes in the papillary and superficial reticular dermis (superficial congenital pattern). There was no evidence of external irritation. The second case showed a similar coronoid lamella, focal diminution of the granular layer and dyskeratotic keratinocytes. There was additional parakeratosis away from the parakeratotic column and a lichenoid host response, suggesting external irritation.

SCUR Abstracts

Tight junctions (TJs) are specialized areas that create an impermeable barrier to solute diiusion between epithelial cells and partition membrane surfaces.We performed indirect immuno£uorescence on samples from diierent gestational ages (6^17 weeks’ gestation) of developing human epidermis using anti-ZO-1, -2, -3, anti-claudin 4 (CL-4) and anti-occludin antibodies. Occludin staining was restricted to the basal and lateral borders of periderm cells in developing epidermis or the uppermost, 2^3 living granular cell layers in adult epidermis. This was in contrast to ZO-1, ZO-2 and CL-4 labeling that extended throughout the upper half of the epidermis at all stages of development. ZO-3 was not expressed in early fetal skin (6^13 weeks gestation) but was weakly expressed between 15^17 weeks and adult epidermis in the granular layers. Ultrastructural analysis of developing skin revealed that classical TJ structures (similar to those in the apicolateral region of enterocytes) were present at the apical borders of adjacent periderm cells and this was con¢rmed by ZO-1 and CL-4 immunoelectron microscopy (IEM) labeling at 6 and 9 weeks gestation. ZO-1 and CL-4 immunogold also labeled the periderm borders at sites of relatively loose membrane apposition (distinct from classical TJ structures). Adult epidermal TJs were di⁄cult to identify using conventional electron microscopy and less frequent than in developing epidermis. TJs were apparent between the uppermost living granular layer keratinocytes. Adult epidermal IEM demonstrated focal areas of ZO-1 and/or CL-4 staining between desmosomes in the upper supra-basal and granular layer keratinocytes that showed a similar localization to an anti-pan (classical) cadherin antiserum at putative adherens junction sites. TJ component expression is therefore carefully regulated during epidermal ontogeny and may play an important role in cell cohesion and epidermal barrier formation.

Characterization of Mouse Profilaggrin: Evidence for Nuclear Engulfment and Translocation of the Profilaggrin B-Domain during Epidermal Differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N- terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal antibody raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Identification of the Fas antigen in human gingiva.

The Fas antigen is a cell-surface glycoprotein that mediates apoptosis from the cell surface into the cytoplasm. Polyclonal antibody (Fas D) was raised against a synthetic polypeptide selected from the extracellular part of the human Fas antigen (amino acid residues 104-114) and was used to detect the Fas antigen in human gingiva. Biopsy specimens of human gingiva were prepared, and the paraffin sections were reacted with the Fas D antibody by an immunohistochemical method. The antibody localized to the prickle-cell layer and to granular layer keratinocytes of human gingiva. Proteins were also prepared from human gingiva and subjected to SDS-PAGE, followed by Western-blotting analysis with the Fas D antibody. The antibody interacted with a band corresponding to an estimated molecular weight of 35 kDa. The incidence of the immunoreactive 35-kDa protein was detected in the gingiva of 90% of the 20 individuals examined. The Fas antigen detected in human gingiva may be related to the physiological turnover of oral mucosa.

Epidermodysplasia verruciformis-associated papillomavirus infection complicating human immunodeficiency virus disease

Three males infected with the human immunodeficiency virus (HIV) were noted to have extensive flat warts of the face and/or body. In two there were also pityriasis versicolor-like lesions. Biopsies showed foamy, basophilic, distended cytoplasm in granular layer keratinocytes, characteristic of the human papillomavirus types seen in epidermodysplasia verruciformis. DNA hybridization techniques demonstrated the presence of HPV-type 8 in one patient and HPV 5 and 8 in another. Patients with immune suppression due to HIV infection may demonstrate the clinical features of epidermodysplasia verruciformis with the same potentially oncogenic HPV types.

Monoclonal Anti-Interleukin 2 (15–2) Antibody Binding to Granular Layer Keratinocytes of Human Skin

Among several monoclonal antibodies (moABs) directed against human interleukin 2 (IL-2), the 15-2 moAB raised in our laboratory against unglycosylated recombinant IL-2 (produced in Escherichia coli) cross-reacted with a human skin epitope. This moAB gave a strong staining on the cell-surface membranes of keratinocytes from the granular layer of the epidermis. In addition, the 15-2 moAB stained 15% of epidermal cell suspensions obtained from suction blisters and reacted with cells from the spinous layer in parakeratosis and psoriasis, as well as with spinous epithelioma cells. Preincubation of the 15-2 moAB with pure human recombinant IL-2 abrogated skin binding, whereas a polyclonal antikeratin antiserum did not block 15-2 skin binding. Two other anti-IL-2 moABs, one directed against unglycosylated recombinant IL-2 (17-2 moAB) and one against glycosylated natural IL-2 (9B11 IE5 moAB), were unreactive on skin. Taken together, the data suggest that the 15-2 moAB binds to an epitope cross-reacting with, but different from, IL-2 which is located in the cell-surface membranes of granular layer cells. This cross-reactive epitope may provide a useful probe for the study of human epidermal cell differentiation.

Ultrastructural Studies of Keratinized Epithelia of the Mouse IV. Quantitative Studies of Lysosomes

Electron microscope cytochemical and simple morphometric studies have permitted an estimate to be made of the absolute numbers of lysosomes present in various cell layers of murine epidermis. Most lysosomes appear to be present in basal layer keratinocytes with few being detected in Langerhans cells or in granular layer keratinocytes. The observation that lysosomes are not numerous in any of the strata is discussed with respect to an alternative explanation for the presence of diffuse acid hydrolase activity in the granular layers.