Differentiation Of Normal Keratinocytes Research Articles

Promoter Hypermethylation Downregulates MiR-125b-5p and MiR-199b-5p Targeting of ΔNp63, Resulting in PI3K/AKT/mTOR Pathway Activation and Keratinocyte Differentiation.

Normal keratinocyte differentiation is important for epidermal wound healing. ΔNp63 is a major gene regulating epidermal formation and differentiation. We identified miRNAs targeting ΔNp63 and studied the association between the miRNAs and DNA methylation in keratinocyte differentiation. This study aimed to explore the mechanisms regulating ΔNp63 expression during keratinocyte differentiation. Bioinformatics analysis was performed to screen the miRNAs targeting ΔNp63 and uncover potential pathway mechanisms. The differentiation model of keratinocytes was established by CaCl2 treatment. Furthermore, the effects of the miRNA transgenic technique on Δ Np63 and keratinocyte differentiation were studied. In addition, the RNA FISH experiment was conducted to detect the location of different miRNAs. A double luciferase reporter experiment was carried out to verify the potential bindings between the miRNAs and ΔNp63. A rescue experiment was also performed to assess the effects of different miRNAs targeting ΔNp63 on keratinocyte differentiation. We analyzed the methylation patterns of the promoter regions of miRNAs using keratinocytes treated with 5-Aza-2'-deoxycytidine. Finally, we designed a methylation rescue experiment to verify the effects of miRNA promoter methylation on keratinocyte differentiation. Bioinformatics analysis showed that the miR-125b-5p and miR-199b-5p binding to the ΔNp63 3'UTR region decreased during skin development. Moreover, such binding may downregulate the PI3K/AKT/mTOR pathway. The expression levels of CK10, Inv, TG1, ΔNp63, and PI3K/AKT/mTOR were all significantly increased during keratinocyte differentiation. Both miR- 125b-5p and miR-199b-5p were localized in the cytoplasm. Luciferase assay results showed that both miR-125b-5p and miR-199b-5p can bind to the 3'UTR region of ΔNp63. Overexpression of ΔNp63 can significantly counteract the inhibitory effect of miRNA mimics on keratinocyte differentiation. Moreover, the promoter regions of both miR-125b-5p and miR-199b-5p had methylation sites, and the methylation levels in those promoter regions were significantly increased during keratinocyte differentiation. 5-Aza-2'-Deoxycytidine treatment increased the expression of miR- 125b-5p and miR-199b-5p and inhibited the differentiation of keratinocytes. Finally, miRNA inhibitors reversed the inhibitory effects of 5-Aza-2'-deoxycytidine on keratinocyte differentiation. Promoter hypermethylation in miR-125b-5p and miR-199b-5p seem to promote keratinocyte differentiation via upregulation of ΔNp63 expression and the activation of the PI3K/AKT/mTOR pathway. The findings of this study are helpful for future research on skin development and clinical scar-free healing.

Nuclear receptors for epidermal lipid barrier: Advances in mechanisms and applications.

The skin plays an essential role in preventing the entry of external environmental threats and the loss of internal substances, depending on the epidermal permeability barrier. Nuclear receptors (NRs), present in various tissues and organs including full-thickness skin, have been demonstrated to exert significant effects on the epidermal lipid barrier. Formation of the lipid lamellar membrane and the normal proliferation and differentiation of keratinocytes (KCs) are crucial for the development of the epidermal permeability barrier and is regulated by specific NRs such as PPAR, LXR, VDR, RAR/RXR, AHR, PXR and FXR. These receptors play a key role in regulating KC differentiation and the entire process of epidermal lipid synthesis, processing and secretion. Lipids derived from sebaceous glands are influenced by NRs as well and participate in regulation of the epidermal lipid barrier. Furthermore, intricate interplay exists between these receptors. Disturbance of barrier function leads to a range of diseases, including psoriasis, atopic dermatitis and acne. Targeting these NRs with agonists or antagonists modulate pathways involved in lipid synthesis and cell differentiation, suggesting potential therapeutic approaches for dermatosis associated with barrier damage. This review focuses on the regulatory role of NRs in the maintenance and processing of the epidermal lipid barrier through their effects on skin lipid synthesis and KC differentiation, providing novel insights for drug targets to facilitate precision medicine strategies.

Epidermal Loss of RORα Enhances Skin Inflammation in a MC903-Induced Mouse Model of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease featuring skin barrier dysfunction and immune dysregulation. Previously, we reported that the retinoid-related orphan nuclear receptor RORα was highly expressed in the epidermis of normal skin. We also found that it positively regulated the expression of differentiation markers and skin barrier-related genes in human keratinocytes. In contrast, epidermal RORα expression was downregulated in the skin lesions of several inflammatory skin diseases, including AD. In this study, we generated mouse strains with epidermis-specific Rora ablation to understand the roles of epidermal RORα in regulating AD pathogenesis. Although Rora deficiency did not cause overt macroscopic skin abnormalities at the steady state, it greatly amplified MC903-elicited AD-like symptoms by intensifying skin scaliness, increasing epidermal hyperproliferation and barrier impairment, and elevating dermal immune infiltrates, proinflammatory cytokines, and chemokines. Despite the normal appearance at the steady state, Rora-deficient skin showed microscopic abnormalities, including mild epidermal hyperplasia, increased TEWL, and elevated mRNA expression of Krt16, Sprr2a, and Tslp genes, indicating subclinical impairment of epidermal barrier functions. Our results substantiate the importance of epidermal RORα in partially suppressing AD development by maintaining normal keratinocyte differentiation and skin barrier function.

Epstein–Barr virus LMP1 protein promotes proliferation and inhibits differentiation of epithelial cells via activation of YAP and TAZ

Latent Epstein-Barr virus (EBV) infection promotes undifferentiated nasopharyngeal carcinomas (NPCs) in humans, but the mechanism(s) for this effect has been difficult to study because EBV cannot transform normal epithelial cells invitro and the EBV genome is often lost when NPC cells are grown in culture. Here we show that the latent EBV protein, LMP1 (Latent membrane protein 1), induces cellular proliferation and inhibits spontaneous differentiation of telomerase-immortalized normal oral keratinocytes (NOKs) in growth factor-deficient conditions by increasing the activity of the Hippo pathway effectors, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif). We demonstrate that LMP1 enhances YAP and TAZ activity in NOKs both by decreasing Hippo pathway-mediated serine phosphorylation of YAP and TAZ and increasing Src kinase-mediated Y357 phosphorylation of YAP. Furthermore, knockdown of YAP and TAZ is sufficient to reduce proliferation and promote differentiation in EBV-infected NOKs. We find that YAP and TAZ are also required for LMP1-induced epithelial-to-mesenchymal transition. Importantly, we demonstrate that ibrutinib (an FDA-approved BTK inhibitor that blocks YAP and TAZ activity through an off-target effect) restores spontaneous differentiation and inhibits proliferation of EBV-infected NOKs at clinically relevant doses. These results suggest that LMP1-induced YAP and TAZ activity contributes to the development of NPC.

Separation, characterization and anti-psoriasis effect of self-assembled nanoparticles from Shaoyao Gancao Decoction

This study aims to separate and characterize self-assembled nanoparticles(SAN) from Shaoyao Gancao Decoction(SGD) and determine the content of active compounds. Further, we aimed to observe the therapeutic effect of SGD-SAN on imiquimod-induced psoriasis in mice. The separation of SGD was performed by dialysis, and the separation process was optimized by single factor experiment. The SGD-SAN isolated under the optimal process was characterized, and the content of gallic acid, albiflorin, paeoniflorin, liquiritin, isoliquiritin apioside, isoliquiritin, and glycyrrhizic acid in each part of SGD was determined by HPLC. In the animal experiment, mice were assigned into a normal group, a model group, a methotrexate group(0.001 g·kg~(-1)), and SGD, SGD sediment, SGD dialysate, and SGD-SAN groups of different doses(1, 2, and 4 g·kg~(-1)) respectively. The psoriasis grade of mice was evaluated based on the pathological changes of skin lesions, the content of inflammatory cytokines, organ index and other indicators. The results showed that SAN obtained by centrifugation at 13 000 r·min~(-1) for 30 min was stable after dialysis for 4 times, which were uniform spherical nanoparticles with the particle size of(164.43±1.34) nm, the polydispersity index of(0.28±0.05), and the Zeta potential of(-12.35±0.80) mV. The active compound content accounted for more than 70% of SGD. Compared with the model group, SAN and SGD decreased the skin lesion score, spleen index, and inflammatory cytokine levels(P<0.05 or P<0.01) and alleviated the skin thickening and infiltration of inflammatory cells. However, the sediment group and the dialysate group had no obvious effect. SGD showed a good therapeutic effect on imiquimod-induced psoriasis in mice, and SAN demonstrated the effect equivalent to SGD in a dose-dependent manner. Therefore, we conclude that the SAN formed during decocting is the main active form of SGD, which can lower the levels of inflammatory cytokines, promote the normal differentiation of keratinocytes, and reduce the infiltration of inflammatory cells in the treatment of psoriasis lesions in mice.

Effect of Hypertrophic Scar Fibroblast-Derived Exosomes on Keratinocytes of Normal Human Skin

Epidermal keratinocytes are highly activated, hyper-proliferated, and abnormally differentiated in the post-burn hypertrophic scar (HTS); however, the effects of scar fibroblasts (SFs) on keratinocytes through cell-cell interaction in HTS remain unknown. Here, we investigated the effects of HTSF-derived exosomes on the proliferation and differentiation of normal human keratinocytes (NHKs) compared with normal fibroblasts (NFs) and their possible mechanism to provide a reference for clinical intervention of HTS. Fibroblasts were isolated and cultured from HTS and normal skin. Both HTSF-exosomes and NF-exosomes were extracted via a column-based method from the cell culture supernatant. NHKs were treated for 24 or 48 h with 100 μg/mL of cell-derived exosomes. The expression of proliferation markers (Ki-67 and keratin 14), activation markers (keratins 6, 16, and 17), differentiation markers (keratins 1 and 10), apoptosis factors (Bax, Bcl2, caspase 14, and ASK1), proliferation/differentiation regulators (p21 and p27), and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and vimentin) was investigated. Compared with NF-exosomes, HTSF-exosomes altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, proliferation/differentiation regulators of NHKs, and EMT markers differently. In conclusion, our findings indicate that HTSF-derived exosomes may play a role in the epidermal pathological development of HTS.

217 Distinctive and shared signatures of Th1 and Th2 cytokines on the lipid pathways involved in the epidermal barrier function

Abnormalities of the permeability barrier and impaired epidermal differentiation characterize both psoriasis and atopic dermatitis, which are driven by immune responses mediated by Th1 and Th2 cytokines. The spectrum of interference of the Th1 and Th2 type cytokines with normal terminal differentiation of keratinocytes is not fully characterized. Our aim was to identify specific and common features of epidermal perturbation associated with each group of cytokines. Immortalized human keratinocytes (Ker-CT) were treated with Th1 and Th2 cytokines in pro-differentiative culture conditions with high calcium concentration.

Role of CD99 in regulating homeostasis and differentiation in normal human epidermal keratinocytes

CD99 is a glycoprotein primarily expressed in immune cells. Physiologically, it is involved in the adhesion, migration, and development of immune cells. The presence of CD99 in the skin was first reported in 2016 and its function is yet to be determined. In this study, we aimed to understand the role of CD99 in the skin using normal human epidermal keratinocytes (NHEK). CD99 expression increased with the confluency of NHEK, while the CD99-high expressing NHEK lost their stem cell properties and played a role in barrier function. We characterized CD99-expressing NHEK as cells committed to early differentiation because they expressed early differentiation markers. However, the deficiency of CD99 in NHEK disrupted homeostasis and caused aberrant differentiation, as evidenced by larger cells with lesser Ki67 staining and higher expression of terminal differentiation markers. Hence, we propose that CD99 is involved in maintaining homeostasis and initiating early differentiation in the skin.

The c.323 G>C mutation in LORICRIN causes new-found late-onset autosomal dominant loricrin keratoderma in a Chinese Han Pedigree

BackgroundLoricrin keratoderma is a rare early-onset autosomal dominant skin disorder. At present, no clinical reports have been published on characteristics of progressive aggravation and late-onset. ObjectivesTo identified a new-found pedigree with c.323 G>C mutation leading to progressive aggravation and late-onset loricrin keratoderma. MethodsTargeted next-generation sequencing of 267 genes associated with all skin abnormalities, sanger sequencing, and bioinformatics tools were used to identify the mutation in this new-found pedigree. Palm skin biopsy was used to observe the clinicopathological features of patient. Further, we constructed pcDNA3.1/V5-His-wild-LORICRIN, pcDNA3.1/V5-His-c.323G>C-LORICRIN, and pcDNA3.1/V5-His-730insG-LORICRIN vectors, nucleofected into HaCaT strain to observe the subcellular localization of loricrin by using the laser scanning confocal microscopy. ResultsThe proband and his affected father carried a heterozygous c.323 G>C missense mutation (p.Gly108Ala) on LORICRIN. Bioinformatics analysis hinted that it had potential pathogenicity; the types of ligands, enzyme commission active sites, and the spatial structure of protein changed enormously. Laser scanning confocal microscopy showed that the signals from cells transfected with the pcDNA3.1/V5-His-730insG-LORICRIN vector were distributed mainly in the nucleus, whereas those from cells transfected with the pcDNA3.1/V5-His-c.323G>C-LORICRIN vector were mainly located in the cytoplasm. Wild type loricrin was distributed in the nucleus and cytoplasm homogeneously ConclusionThe heterozygous c.323G>C missense mutation on LORICRIN caused late-onset and progressive loricrin keratoderma in this large Chinese family. Our study revealed that a large number of loricrin gathered in the cytoplasm may disturb the normal proliferation and terminal differentiation of keratinocytes and lead to the late-onset loricrin keratoderma disease.

Oat β-glucan ameliorates epidermal barrier disruption by upregulating the expression of CaSR through dectin-1-mediated ERK and p38 signaling pathways

The integrity of the epidermal barrier and the maintenance of barrier homeostasis depend on the dynamic balance between the proliferation and differentiation of keratinocytes. Calcium (Ca2+) plays a crucial role in maintaining a balance of these two processes as well as in the formation of an epidermal permeability barrier. In this study, we showed that topical application of oat β-glucan (OG) could ameliorate epidermal hyperplasia and accelerate the recovery of the epidermal barrier by promoting epidermal differentiation. Mechanistic studies revealed a positive interaction between OG and the dectin-1 receptor, and this interaction could lead to an upregulated expression of the calcium-sensing receptor (CaSR) via activation of the downstream ERK and p38 pathways. This consequently increased the sensitivity of keratinocytes to extracellular Ca2+ under the condition of calcium loss following the disruption of the epidermal barrier, resulting in the maintenance of normal keratinocyte differentiation in the epidermis, and ultimately promoting the recovery of the epidermal barrier. These findings clearly demonstrated the healing effect of OG on a physically damaged epidermal barrier. Thus, OG could be considered a valuable component in the development of skin repair agents.

The Role of Bifidobacteria in Predictive and Preventive Medicine: A Focus on Eczema and Hypercholesterolemia

Bifidobacteria colonize the human gastrointestinal tract early on in life, their interaction with the host starting soon after birth. The health benefits are strain specific and could be due to the produced polysaccharides. The consumption of probiotics may prevent obesity, irritable bowel syndrome, eczema or atopic dermatitis, and asthma. Non-replicative strains of Bifidobacterium longum (NCC3001 and NCC2705) promote the differentiation of normal human epidermal keratinocytes (NHEKs), inducing a high expression of differentiation markers (keratin —KRT1—, and transglutaminase —TGM1—) and pro-regeneration markers (cathepsins), including β-defensin-1, which plays an important role in modulating the cutaneous immune response. Strains belonging to the genera Bifidobacterium and Lactobacillus can increase tight-junction proteins in NHEKs and enhance barrier function. Bifidobacteria and lactobacilli may be used as prophylactic or therapeutic agents towards enteric pathogens, antibiotic-associated diarrhea, lactose intolerance, ulcerative colitis, irritable bowel syndrome, colorectal cancer, cholesterol reduction, and control of obesity and metabolic disorders. Bifidobacterium bifidum showed an in vitro capability of lowering cholesterol levels thanks to its absorption into the bacterial membrane. Several strains of the species Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus, L. casei, and L. gasseri led to a reduced amount of serum cholesterol due to their ability to assimilate cholesterol (in vitro). Lactococcus lactis KF147 and Lactobacillus plantarum Lp81 have also been shown to reduce cholesterol levels by 12%. Clarifying the specific health mechanisms of Bifidobacterium and Lactobacillus strains in preventing high-cost pathologies could be useful for delineating effective guidelines for the treatment of infants and adults.

MiR-155 Contributes to Normal Keratinocyte Differentiation and Is Upregulated in the Epidermis of Psoriatic Skin Lesions.

The role of microRNAs (miRNAs) during keratinocyte (KC) differentiation and in skin diseases with epidermal phenotypes has attracted strong interest over the past few years. However, combined mRNA and miRNA expression analyses to elucidate the intricate mRNA–miRNA networks of KCs at different stages of differentiation have not been performed yet. In the present study, we investigated the dynamics of miRNA and mRNA expression during KC differentiation in vitro and in normal and psoriatic epidermis. While we identified comparable numbers of up- and downregulated mRNAs (49% and 51%, respectively), miRNAs were predominantly upregulated (76% vs 24%) during KC differentiation. Further bioinformatics analyses suggested an important inhibitory role for miR-155 in KC differentiation, as it was repressed during KC differentiation in normal skin but strongly upregulated in the epidermis of psoriatic skin lesions. Mimicking the inflammatory milieu of psoriatic skin in vitro, we could show that the pro-inflammatory cytokines IL17, IL1β and INFγ synergistically upregulated miR-155 expression in KCs. Forced over-expression of miR-155 in human in vitro skin models specifically reduced the expression of loricrin (LOR) in KCs, indicating that miR-155 interferes with the establishment of a normal epidermal barrier. Together, our data indicate that downregulation of miR-155 during KC differentiation is a crucial step for epidermal barrier formation. Furthermore, its strong upregulation in psoriatic lesions suggests a contributing role of miR-155 in the altered keratinocyte differentiation observed in psoriasis. Therefore, miR-155 represents as a potential target for treating psoriatic skin lesions.

Markers for Ca++ -induced terminal differentiation of keratinocytes in vitro under defined conditions.

Differentiation of normal human keratinocytes (NHK) grown in vitro as a monolayer to confluency can be triggered with an acute increase in concentration of extracellular Ca++ . Over several days, induced by Ca++ , the cells form pseudostratified sheets that somewhat resemble the basic organization of the intact skin. This experimental system is widely used in studies of keratinocyte biology and skin pathology. However, expression pattern of the genes considered as markers for cells in specific layers of epidermis in vivo does not always match the specific pattern observed in vitro and might lead to misinterpretation of data. Here, we demonstrate that among 18 markers of terminally differentiated keratinocytes of stratum granulosum (SG) and stratum corneum (SC) in vivo, only four (CDSN, KPRP, LCE1C and SPRR4) have reproduced their expression pattern in vitro. Our data suggest that findings based on two-dimensional (2D) Ca++ -induced terminal differentiation of NHK in vitro should be subjected to additional scrutiny before conclusions could be made and, if possible, verified in other experimental system that might more faithfully represent the in vivo microenvironment.

251 Skin epidermal keratinocyte differentiation-associated processes regulate homeostatic antiviral protein expression

Epidermal keratinocytes produce antimicrobial peptides and proteins, including antiviral proteins (AVPs) as innate defense against various pathogens. We made the novel observation that distinct genes encoding innate AVPs are transcriptionally activated upon calcium-induced epidermal differentiation of primary normal human epidermal keratinocytes (NHEK). The AVPs include, but are not limited to, members of oligoadenylate synthetase (OAS) family, myxovirus resistance proteins (MX), interferon-induced proteins with tetratricopeptide repeats (IFIT), interferon-induced transmembrane protein 1 (IFITM1), interferon stimulated gene 15 (ISG15), and guanylate-binding proteins (GBP) (p < 0.05).

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Psoriatic Dermal-derived Mesenchymal Stem Cells Reduce Keratinocyte Junctions, and Increase Glycolysis

Although it is known that psoriatic dermal-derived mesenchymal stem cells (DMSCs) dysregulate keratinocyte proliferation, the biological activity profile of keratinocytes influenced by psoriatic DMSCs remain unknown. In the present study, we assessed the impact of psoriatic DMSCs on keratinocyte proliferation, differentiation, and glucose metabolism in normal human epidermal keratinocytes co-cultured with or without psoriatic DMSCs. Co-culture of normal human epidermal keratinocytes with psoriatic DMSCs down-regulated expression levels of proteins associated with cell junction assembly (alpha-actinin-1, catenin beta-1, poliovirus receptor-related protein 4 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2), while upregulating proteins associated with keratinocyte proliferation and differentiation (involucrin, isoform 2 of Histone-binding protein, isoform 3 of Telomeric repeat-binding factor 2 and keratin 13). Moreover, coculture of normal human epidermal keratinocytes with psoriatic DMSCs stimulated keratinocyte proliferation and glycolysis, but reduced keratinocyte junctions. Taken together, these results demonstrate that psoriatic DMSCs increase keratinocyte proliferation and glycolysis, and reduce cell junctions, suggesting a pathogenic role of psoriatic DMSCs in epidermal hyperplasia, aberrant differentiation, and reduction in turnover time of keratinocytes in psoriasis.

Genetics of Inherited Ichthyoses and Related Diseases.

Inherited ichthyoses are classified as Mendelian disorders of cornification (MEDOC), which are defined on the basis of clinical and genetic features and are mainly divided into non-syndromic and syndromic ichthyoses. Numerous genes, which encode for corresponding proteins, are involved in the normal differentiation of keratinocytes (cornification) and participate in the formation of a functional epidermal barrier. To date, mutations in more than 50 genes are known to result in various types of ichthyoses. Thanks to modern genetic diagnostic methods based on targeted next generation sequencing (NGS), approximately 80–90% of cases can be resolved at present. Further sequencing methods covering the whole exome (WES) or whole genome (WGS) will obviously elucidate another portion of the remaining unknown ichthyoses in the future.

Psoriatic Dermal-derived Mesenchymal Stem Cells Reduce Keratinocyte Junctions, and Increase Glycolysis.

Although it is known that psoriatic dermal-derived mesenchymal stem cells (DMSCs) dysregulate keratinocyte proliferation, the biological activity profile of keratinocytes influenced by psoriatic DMSCs remain unknown. In the present study, we assessed the impact of psoriatic DMSCs on keratinocyte proliferation, differentiation, and glucose metabolism in normal human epidermal keratinocytes co-cultured with or without psoriatic DMSCs. Co-culture of normal human epidermal keratinocytes with psoriatic DMSCs downregulated expression levels of proteins associated with cell junction assembly (alpha-actinin-1, catenin beta-1, poliovirus receptor-related protein 4 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2), while upregulating proteins associated with keratinocyte proliferation and differentiation (involucrin, isoform 2 of Histone-binding protein, isoform 3 of Telomeric repeat-binding factor 2 and keratin 13). Moreover, co-culture of normal human epidermal keratinocytes with psoriatic DMSCs stimulated keratinocyte proliferation and glycolysis, but reduced keratinocyte junctions. Taken together, these results demonstrate that psoriatic DMSCs increase keratinocyte proliferation and glycolysis, and reduce cell junctions, suggesting a pathogenic role of psoriatic DMSCs in epidermal hyperplasia, aberrant differentiation, and reduction in turnover time of keratinocytes in psoriasis.

158 Triggering immortalized keratinocyte growth arrest by protein kinase C delta activation

Normal epidermal keratinocyte (KC) differentiation is marked by irreversible cell cycle exit and a step-wise transition through the suprabasal layers of the epidermis. In contrast, KCs in both actinic keratosis and squamous cell carcinomas (SCCs) continue to proliferate in the suprabasal compartment, indicating a defect in their growth arrest signaling. Activation of multiple protein kinase C (PKC) isoforms have been associated with KC cell cycle withdrawal. To study the involvement of PKC in KC growth arrest, we evaluated the effects of the PKC agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on normal human epidermal keratinocytes (NHEKs) and HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line. Treating NHEKs with 10 nM TPA for as little as 15 minutes induced complete growth arrest over a 7 day period while untreated KCs underwent a 93-fold increase in cell number (p<0.005). 10 nM TPA treatment of HaCaT cells did not induce any growth arrest, indicating that immortalized KCs have defective PKC-dependent growth arrest signaling. To test if PKCδ activation was able to enforce growth arrest in HaCaT cells, we transduced HaCaT cells with retroviruses encoding either EGFP or a PKCδ-EGFP fusion and sorted EGFP positive cells by flow cytometry. HaCaT-EGFP cells behaved similar to HaCaTs and were not growth arrested by TPA treatment. In contrast, HaCaT-PKCδ-EGFP treated with TPA increased in cell number only 3-fold compared to a 15-fold increase in untreated HaCaT-PKCδ-EGFP over a 4 day growth assay. Activation of PKCδ in HaCaT-PKCδ-EGFP cells was confirmed by observing TPA-induced translocation of PKCδ-EGFP to the plasma membrane and increased S299 autophosphorylation. TPA-induced growth inhibition in HaCaT-PKCδ-EGFP cells was due to arrest in G1 and G2/M, not apoptosis. Global phosphoproteomics are being employed to identify PKCδ signaling pathways that can overcome the defective growth arrest in immortalized keratinocytes and may have therapeutic benefit for the treatment of actinic keratosis and SCCs.

Lesional upregulation of SLURP1 immunostaining parallels disease severity in psoriasis vulgaris patients.

The exact pathogenesis of psoriasis is still unclear. SLURP1 is vital for the normal differentiation of keratinocytes, and could therefore be involved in psoriasis. In this study we investigated the immunohistochemical staining reaction of SLURP1 in psoriasis vulgaris patients and its possible relation to disease pathogenesis. 20 patients with psoriasis vulgaris, 20 patients with psoriasiform dermatitis and 20 normal skin samples were studied. Psoriasis severity was measured with a combination of PASI and DLQI scores. Lesional and non-lesional sites were biopsied for each psoriasis patient. A single biopsy sample was taken for cases with psoriasiform dermatitis and for controls. All sections were immunostained for SLURP1 according to the manufacturer's protocol. Significant differences were noted in SLURP1 immunostaining between lesional and non-lesional biopsies of psoriasis patients and between lesional biopsies of psoriasis patients and lesional sites of psoriasiform dermatitis. However, the differences between non-lesional biopsies of psoriasis patients and normal controls were not significant. Furthermore, the grading of SLURP1 immunostaining paralleled the degree of psoriasis severity. SLURP1 immunostaining is significantly increased in lesional skin of psoriasis vulgaris and not in psoriasiform dermatitis, which demonstrates the role of SLURP1 in the pathogenesis of psoriasis. SLURP1 could be used as a biological marker for psoriasis severity, and this hypothesis warrants further investigation.