Normal Epidermal Differentiation Research Articles

Loss-of-Function Variants in SERPINA12 Underlie Autosomal Recessive Palmoplantar Keratoderma

Inherited palmoplantar keratodermas refer to a large and heterogeneous group of conditions resulting from abnormal epidermal differentiation and featuring thickening of the skin of the palms and soles. Here, we aimed at delineating the genetic basis of an autosomal recessive form of palmoplantar keratodermas manifesting with erythematous hyperkeratotic plaques over the palms and soles, extending to non-palmoplantar areas. Whole-exome sequencing in affected individuals revealed homozygous nonsense variants in the SERPINA12 gene. SERPINA12 encodes the visceral adipose tissue-derived serpin A12, a serine protease inhibitor. The pathogenic variants were found to result in reduced visceral adipose tissue-derived serpin A12 expression in patients' skin biopsies in comparison to healthy controls. In addition, SERPINA12 downregulation in three-dimensional skin equivalents was associated with marked epidermal acanthosis and hyperkeratosis, replicating the human phenotype. Moreover, decreased SERPINA12 expression resulted in reduced visceral adipose tissue-derived serpin A12-mediated inhibition of kallikrein 7 activity as well as decreased levels of desmoglein-1 and corneodesmosin, two known kallikrein 7 substrates, which are required for normal epidermal differentiation. The present data, taken collectively, demarcate a unique type of autosomal recessive palmoplantar keratodermas, attribute to visceral adipose tissue-derived serpin A12 a role in skin biology, and emphasize the importance of mechanisms regulating proteolytic activity for normal epidermal differentiation.

298 Both sphingosine kinase 1 and 2 coordinately regulate cathelicidin antimicrobial peptide production during keratinocyte differentiation

Cathelicidin antimicrobial peptide (CAMP) is a key antimicrobial peptide in skin. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. We recently identified a novel endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)-dependent mechanism of CAMP synthesis. In this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis; and conversely, another isoform SPHK2 likely has a suppressor or no role in CAMP synthesis. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here show that: increased ER stress occurs in differentiated cultured keratinocytes (KC); 2) increases in both CAMP and S1P production depend upon differentiation level of KC (proliferated<early-<late-stage of differentiation); 3) expression of SPHK1 and SPHK2 is increased and decreased, respectively, during KC differentiation; and 4) dihydroS1P that is preferentially synthesized by SPHK2 does not increase CAMP production. Finally, overexpression of wild type, but not dominant negative SPHK2, suppresses CAMP production. Our study suggests that alterations of both SPHK1 and SPHK2 levels coordinately increase CAMP production during epidermal differentiation.

Both Sphingosine Kinase 1 and 2 Coordinately Regulate Cathelicidin Antimicrobial Peptide Production during Keratinocyte Differentiation

The innate immune element, cathelicidin antimicrobial peptide (CAMP), is vital in the formation of the antimicrobial barrier in skin. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. We recently identified an endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)- dependent mechanism of CAMP synthesis. Interestingly, in this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis; and conversely, another isoform SPHK2 likely has a suppressor role or no role in CAMP production. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here demonstrate that: increased ER stress is evident in differentiated cultured keratinocytes (KC); 2) increases in both CAMP and S1P production depend upon differentiation level of KC (proliferated<early-<late-stage of differentiated KC); 3) expression of SPHK1 and SPHK2 is increased and decreased, respectively, during KC differentiation; and 4) dihydroS1P that is preferentially synthesized by SPHK2 does not increase CAMP production. Finally, overexpression of wild type, but not dominant negative SPHK2, suppresses CAMP production in both proliferated and differentiated KC. Our current study suggests that alterations of both SPHK1 and SPHK2 levels coordinately increase CAMP production during epidermal differentiation.

Hyaluronan metabolism enhanced during epidermal differentiation is suppressed by vitamin C.

Hyaluronan is a large, linear glycosaminoglycan present throughout the narrow extracellular space of the vital epidermis. Increased hyaluronan metabolism takes place in epidermal hypertrophy, wound healing and cancer. Hyaluronan is produced by hyaluronan synthases and catabolized by hyaluronidases, reactive oxygen species and KIAA1199. To investigate the changes in hyaluronan metabolism during epidermal stratification and maturation, and the impact of vitamin C on these events. Hyaluronan synthesis and expression of the hyaluronan-related genes were analysed during epidermal maturation from a simple epithelium to a fully differentiated epidermis in organotypic cultures of rat epidermal keratinocytes using quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blotting, in the presence and absence of vitamin C. With epidermal stratification, both the production and the degradation of hyaluronan were enhanced, resulting in an increase of hyaluronan fragments of various sizes. While the mRNA levels of Has3 and KIAA1199 remained stable during the maturation, Has1, Has2 and Hyal2 showed a transient upregulation during stratification, Hyal1 transcription remained permanently increased and transcription of the hyaluronan receptor, Cd44, decreased. At maturation, vitamin C downregulated Has2, Hyal2 and Cd44, whereas it increased high-molecular-mass hyaluronan in the epidermis, and reduced small fragments in the medium, suggesting stabilization of epidermal hyaluronan. Epidermal stratification and maturation is associated with enhanced hyaluronan turnover, and release of large amounts of hyaluronan fragments. The high turnover is suppressed by vitamin C, which is suggested to enhance normal epidermal differentiation in part through its effect on hyaluronan.

Blockade of TNF receptor superfamily 1 (TNFR1)–dependent and TNFR1-independent cell death is crucial for normal epidermal differentiation

A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.

Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2

Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. Although transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2). A family of different eIF2α kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2α phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2α phosphorylation by the general control nonderepressible 2 (GCN2) protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2α and translational control in the formation of an intact human epidermis.

414 Both sphingosine kinase 1 and 2 coordinately regulate cathelicidin antimicrobial peptide production during keratinocyte differentiation

The innate immune element, cathelicidin antimicrobial peptide (CAMP), is a vital antimicrobial peptide needed for the formation of the antimicrobial skin barrier. We recently identified a novel endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)-dependent mechanism of CAMP synthesis. Interestingly, in this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis and conversely, another isoform (SPHK2) likely has a suppressor role in CAMP production. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here investigated how CAMP production is increased during epidermal differentiation. We found that 1) increased ER stress is evident in differentiated cultured keratinocytes; 2) increases in both CAMP and S1P production depend upon differentiation level of keratinocyte (proliferatedwt, but not dominant negative SPHK2 suppresses CAMP production in both proliferated and differentiated KC. Our current study suggests that both an increase in SPHK1 and a decrease in SPHK2 expression coordinately stimulate CAMP production during epidermal differentiation.

Differentiation of human keratinocytes requires translational control by the eIF2 kinase GCN2

In the human epidermis, differentiation of keratinocytes is essential to form a barrier to protect against a variety of environmental insults. This differentiation process can become dysregulated in many skin diseases, including psoriasis, eczema, and non‐melanoma skin cancer. Although changes in transcriptional networks during keratinocyte differentiation are well characterized, little is known about the role of translational control. A major mechanism of translational control involves phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2‐P). In response to environmental stresses, induced eIF2‐P represses translation initiation, allowing cells to conserve resources and trigger cytoprotective gene expression. In addition to repression of global protein synthesis, eIF2‐P induces preferential translation of specific mRNAs that promote stress adaptation, including the transcription factors ATF4 and CHOP. Since keratinocytes undergo a massive change in morphology and gene expression during differentiation, we hypothesized that translational control plays a major role. To investigate this hypothesis we measured both genome‐wide and gene‐specific levels of translation. We observed a global reduction in translation initiation as measured by polysome profiling during differentiation in both monolayer and 3D organotypic keratinocyte cultures. Expression of eIF2‐P, ATF4, and CHOP was limited to suprabasal (differentiated) keratinocytes in vivo as visualized histologically in human skin tissue. Keratinocyte differentiation in vitro induced eIF2‐P to levels similar to that seen following endoplasmic reticulum stress, but eIF2‐P alone was not sufficient to induce differentiation. Of interest, the epidermal differentiation transcript involucrin (IVL) was preferentially bound to large polysomes following differentiation, indicative of preferential translation, which was similar to that seen for ATF4 and CHOP. Inhibition of eIF2‐P blocked both general and gene‐specific translational control during differentiation. Loss of either eIF2‐P or the upstream protein kinase GCN2 abrogated both IVL protein and mRNA expression. Strikingly, loss of GCN2 altered epidermal tissue formation in a 3D organotypic model as characterized by disorganized keratinocyte layers and a decreased number of cornified cells. These results provide novel evidence that a whole‐cell repression of protein synthesis alongside gene‐specific translational control occur during normal epidermal differentiation. These findings suggest that translational control through GCN2/eIF2‐P is important for proper epidermal stratification, likely though translational regulation of differentiation genes such as IVL. The GCN2/eIF2‐P pathway in keratinocyte biology provides for novel targets for intervention in dermatological diseases characterized by abnormal differentiation.Support or Funding InformationRuth L. Kirschstein National Research Service Award (1F31ES026517 to AEC)

Tag SNPs of long non-coding RNA TINCR affect the genetic susceptibility to gastric cancer in a Chinese population.

Tissue differentiation-inducing non-protein coding RNA (TINCR) is required for normal epidermal differentiation. TINCR is also strongly overexpressed in human gastric cancer (GC) and contributes to carcinogenesis and tumor progression. However, the association between TINCR polymorphisms and the risk of any diseases, such as GC, remains unknown. In the present study, the tag single nucleotide polymorphisms rs8113645, rs2288947, rs8105637, and rs12610531 were analyzed in 602 patients with GC and 602 age- and sex-matched controls. Polymorphisms were genotyped using TaqMan technology. Carriers of variant rs8113645 and rs2288947 alleles indicated reduced risks of GC (p = 0.003 and 0.037, respectively). A allele genotypes of rs8113645 and G allele genotypes of rs2288947 (rs8113645 GA and AA; rs2288947 AG and GG) were also significantly associated with decreased GC risk (p < 0.05). Stratification analysis displayed that the correlations between GC risk and variant genotypes of both rs8113645 and rs2288947were more evident in younger individuals, men, nonsmokers, and individuals from rural areas. We also demonstrated that rs8113645 GA+AA genotype carriers had lower TINCR mRNA expression levels compared with common genotype in both normal and GC tissues (p < 0.05). These results suggest that long non-coding RNA TINCR polymorphisms may be implicated in GC development.

IQGAP1 and IQGAP3 Serve Individually Essential Roles in Normal Epidermal Homeostasis and Tumor Progression

IQGAP scaffolding proteins regulate many essential cellular processes including growth factor receptor signaling, cytoskeletal rearrangement, adhesion and proliferation, and are highly expressed in many cancers. Using genetically engineered human skin tissue in vivo, we demonstrate that diminished, sub-physiologic expression of IQGAP1 or IQGAP3 is sufficient to maintain normal epidermal homeostasis, while significantly higher levels are required to support tumorigenesis. To target this tumor-specific IQGAP requirement in vivo, we engineered epidermal keratinocytes to express individual IQGAP protein domains designed to compete with endogenous IQGAPs for effector protein binding. Expression of the IQGAP1-IQM decoy domain in epidermal tissue in vivo inhibits oncogenic Ras-driven MAPK signaling and antagonizes tumorigenesis, without disrupting normal epidermal proliferation or differentiation. These findings define essential non-redundant roles for IQGAP1 and IQGAP3 in epidermis, and demonstrate the potential of IQGAP antagonism for cancer therapy.

A genetic polymorphism (rs17251221) in the calcium-sensing receptor is associated with ovarian cancer susceptibility.

Calcium-sensing receptor (CaSR) is a G-protein‑ coupled receptor that senses blood calcium. In vivo, CaSR is required for normal epidermal differentiation by mediating calcium signaling. CaSR was confirmed to be a tumor suppressor in colon and breast cancer. The single-nucleotide polymorphism (SNP) rs17251221, located on the intron, is a genetic variation of the CaSR gene. We analyzed rs17251221 in ovarian cancer using an allelic discrimination assay. Cycling probes were used for genotyping 290 ovarian cancer patients and 312 age-matched cancer-free females. rs17251221 and clinicopathological characteristics of ovarian cancer were analyzed statistically. The AG and GG genotypes were confirmed to appear in fewer cancer cases than in controls and the genotype distribution between cases and controls was statistically significant. The AG+GG genotype was correlated with low ovarian cancer risk, while rs17251221 was not associated with clinicopathological variables including age at diagnosis, tumor size, histologic type, pathological subtype, lymph node metastasis, CA-125 expression, clinical stage, or degree of differentiation. The rs17251221 polymorphism genotype was not correlated with survival in ovarian cancer. These results suggest that the G allele of the CaSR rs17251221 polymorphism is protective against ovarian cancer and the homozygous GG genotype may be a protective genotype as well. The rs17251221 may play an important role in the development of ovarian cancer and could be used as a biomarker for predicting ovarian cancer.

AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation.

Nuclear degradation is a key stage in keratinocyte terminal differentiation and the formation of the cornified envelope that comprises the majority of epidermal barrier function. Parakeratosis, the retention of nuclear material in the cornified layer of the epidermis, is a common histological observation in many skin diseases, notably in atopic dermatitis and psoriasis. Keratinocyte nuclear degradation is not well characterised, and it is unclear whether the retained nuclei contribute to the altered epidermal differentiation seen in eczema and psoriasis. Loss of AKT1 function strongly correlated with parakeratosis both in eczema samples and in organotypic culture models. Although levels of DNAses, including DNase1L2, were unchanged, proteomic analysis revealed an increase in Lamin A/C. AKT phosphorylates Lamin A/C, targeting it for degradation. Consistent with this, Lamin A/C degradation was inhibited and Lamin A/C was observed in the cornified layer of AKT1 knockdown organotypic cultures, surrounding retained nuclear material. Using AKT-phosphorylation-dead Lamin A constructs we show that the retention of nuclear material is sufficient to cause profound changes in epidermal terminal differentiation, specifically a reduction in Loricrin, Keratin 1, Keratin 10, and filaggrin expression. We show that preventing nuclear degradation upregulates BMP2 expression and SMAD1 signalling. Consistent with these data, we observe both parakeratosis and evidence of increased SMAD1 signalling in atopic dermatitis. We therefore present a model that, in the absence of AKT1-mediated Lamin A/C degradation, DNA degradation processes, such as those mediated by DNAse 1L2, are prevented, leading to parakeratosis and changes in epidermal differentiation.

Prodifferentiation, Anti-Inflammatory and Antiproliferative Effects of Delphinidin, a Dietary Anthocyanidin, in a Full-Thickness Three-Dimensional Reconstituted Human Skin Model of Psoriasis

Background: Psoriasis is a chronic inflammatory disorder of skin and joints for which conventional treatments that are effective in clearing the moderate-to-severe disease are limited due to long-term safety issues. This necessitates exploring the usefulness of botanical agents for treating psoriasis. We previously showed that delphinidin, a diet-derived anthocyanidin endowed with antioxidant and anti-inflammatory properties, induces normal epidermal keratinocyte differentiation and suggested its possible usefulness for the treatment of psoriasis [<citeref rid="ref1">1</citeref>]. Objectives: To investigate the effect of delphinidin (0-20 μ<smlcap>M</smlcap>; 2-5 days) on psoriatic epidermal keratinocyte differentiation, proliferation and inflammation using a three-dimensional reconstructed human psoriatic skin equivalent (PSE) model. Methods: PSEs and normal skin equivalents (NSEs) established on fibroblast-contracted collagen gels with respective psoriatic and normal keratinocytes and treated with/without delphinidin were analyzed for histology, expression of markers of differentiation, proliferation and inflammation using histomorphometry, immunoblotting, immunochemistry, qPCR and cultured supernatants for cytokine with a Multi-Analyte ELISArray Kit. Results: Our data show that treatment of PSE with delphinidin induced (1) cornification without affecting apoptosis and (2) the mRNA and protein expression of markers of differentiation (caspase-14, filaggrin, loricrin, involucrin). It also decreased the expression of markers of proliferation (Ki67 and proliferating cell nuclear antigen) and inflammation (inducible nitric oxide synthase and antimicrobial peptides S100A7-psoriasin and S100A15-koebnerisin, which are often induced in psoriatic skin). ELISArray showed increased release of psoriasis-associated keratinocyte-derived proinflammatory cytokines in supernatants of the PSE cultures, and this increase was significantly suppressed by delphinidin. Conclusions: These observations provide a rationale for developing delphinidin for the management of psoriasis.

Secreted HoxA3 Promotes Epidermal Proliferation and Angiogenesis in Genetically Modified Three-Dimensional Composite Skin Constructs.

Objective: Homeobox (HOX) transcription factors coordinate gene expression in wound repair and angiogenesis. Previous studies have shown that gene transfer of HoxA3 to wounds of diabetic mice accelerates wound healing, increasing angiogenesis and keratinocyte migration. In this study, we examined whether HoxA3 can also improve angiogenesis, epidermal integrity, and viability of composite skin grafts. Approach: To determine the effects of HoxA3 on composite skin grafts, we constructed bilayered composite grafts incorporating fibroblasts engineered to constitutively secrete HoxA3. We then transplanted these composite grafts in vivo. Results: The composite grafts produced a stratified epidermal layer after seventeen days in culture and following transplantation in vivo, these grafts exhibit normal epidermal differentiation and reduced contraction compared to controls. In addition, HoxA3 grafts showed increased angiogenesis. Quantitative polymerase chain reaction (PCR) analyses of HoxA3 graft tissue reveal an increase in the downstream HoxA3 target genes MMP-14 and uPAR expression, as well as a reduction in CCL-2 and CxCl-12. Innovation: Expression of secreted HoxA3 in composite grafts represents a comprehensive approach that targets both keratinocytes and endothelial cells to promote epidermal proliferation and angiogenesis. Conclusion: Secreted HoxA3 improves angiogenesis, reduces expression of inflammatory mediators, and prolongs composite skin graft integrity.

Loss of epidermal MMP-14 expression interferes with angiogenesis but not with re-epithelialization

Synthesis and activation of matrix metalloproteinases during wound healing are important for remodeling the extracellular matrix and modulating various cellular functions. The membrane-type 1 matrix metalloproteinase (MMP-14) has been shown to play a key role during these processes. To analyze the function of epidermal-derived MMP-14 during skin repair we generated mice lacking MMP-14 expression in the epidermis (MMP-14ep−/−). These mice displayed overall normal skin morphology and epidermal differentiation patterns. Wound repair in MMP-14ep−/− followed the same kinetics as in wild type mice (MMP-14ep+/+), and infiltration of neutrophils, leukocytes, and macrophages into the wound site was comparable. Microscopic analysis showed no altered re-epithelialization in the absence of epidermal MMP-14. Furthermore, epidermal differentiation at the end of the repair process and scar formation was normal. However, at day 14 post wounding, sustained angiogenesis was observed in MMP-14ep−/− mice in contrast to control mice. Interestingly, decreased levels of endostatin were detected in wound lysates of MMP-14ep−/− mice as well as in cultured keratinocytes. Taken together, these data indicate that MMP-14 expression in keratinocytes is dispensable for skin homeostasis and repair, but plays a crucial role in the epidermal–dermal crosstalk leading to modulation of vessel density.

Imatinib inhibits migration of normal human epidermal melanocytes and suppress the phosphorylation of C-kit

To investigate the inhibitory effect of imatinib on the migration of normal human epidermal melanocytes (NHEM), the growth and differentiation of NHEM were measured by MTT assay. Transwell Cell Migration Assay was performed to determine the migration of NHEM following imatinib treatment. The phosphorylation of c-kit in NHEM after imatinib treatment was measured by western blot assay. Imatinib at 0.1 ?M, 1.0 ?M and 5.0 ?M could inhibit the SCF (50 ng/mL) induced NHEM migration and suppress the phosphorylation of c-kit in NHEM. The result concluded that imatinib can inhibit SCF induced NHEM migration and suppress the c-kit phosphorylation in NHEM. This provide theoretical basis for the prevention of post-treatment recurrence of nevus spilus.

Notch1 and Corneal Cell Fate

I very much enjoyed reading a Developmental Cell paper from Freddy Radtke's laboratory describing exciting findings regarding the requirement of Notch1 signaling for maintenance of corneal integrity during wound repair. The authors showed that Notch1 controls corneal cell fate and prevents keratinization and tissue differentiation into a skin-like epithelium, which is causal to corneal blindness. They further demonstrated that this effect is through negative control of FGF-2 and vascularization and through positive control of vitamin A metabolism. These results provided novel insights into the role of Notch1 as a master regulator of corneal cell fate to prevent skin-like epithelium formation upon injury. I consider these findings particularly interesting because Notch1 signaling in the epidermis is not only required for normal epidermal stratification, differentiation, and skin tumor suppression but is also required for the prevention of cell fate changes upon stress. This discovery serves as a basis for further investigations into the importance of Notch 1 for corneal blindness and also provides a platform to study the differential effects of Notch1 expression in other epithelial cell types and its regulation by upstream or downstream effectors. This PaperPick refers to Corneal Epithelial Cell Fate Is Maintained during Repair by Notch1 Signaling via the Regulation of Vitamin A Metabolism, by S. Vauclair, F. Majo, A.-D. Durham, N. B. Ghyselinck, Y. Barrandon, and F. Radtke, published in August 2007. Video Abstract Freddy Radtke and Craig Nowell discuss what led to the 2007 Vauclair et al. study and the implications of its findings.

BAG-1L promotes keratinocyte differentiation in organotypic culture models and changes in relative BAG-1 isoform abundance may lead to defective stratification

In keratinocytes the human Bag-1 gene produces three different protein isoforms from a single messenger RNA, BAG-1L, BAG-1M and BAG-1S. In this study we questioned whether BAG-1L or the shorter isoforms would promote keratinocyte differentiation in organotypic cultures of HaCaT. HaCaT parental and vector cells showed stratification, but terminal differentiation was not complete. Cultures overexpressing BAG-1L isoform-specifically were of increased thickness, demonstrated pronounced expression of basal cytokeratin 5 and β1-integrin, suprabasal involucrin, cytokeratin 1 and plasma membrane-localised filaggrin, and a marked keratinized layer of cells at the surface. We were unable to overexpress BAG-1S and BAG-1M isoform-specifically. Overexpression of BAG-1M gave rise to organotypic cultures intermediate in differentiation to controls and those overexpressing BAG-1L. Cells overexpressing BAG-1S also exhibited elevated endogenous BAG-1. These produced slow growing cultures with high levels of basal cytokeratin 5, but little involucrin or cytokeratin 1. Suprabasal β1-integrin and Ki67 positive cells indicated defective stratification. The results suggest that BAG-1L potentiates epidermal differentiation, but disruption in the relative balance of isoforms towards overexpression of BAG-1S can lead to defective tissue patterning. Hence, a delicate balance of BAG-1 isoforms may be required to regulate normal epidermal stratification and differentiation, with important implications for aberrant differentiation in cancer.

Asian dust storm particles induce a broad toxicological transcriptional program in human epidermal keratinocytes

Exposure to airborne dust particles originated from seasonal Asian dust storms in Chinese and Mongolian deserts results in increased incidence of a range of diseases including asthma, contact dermatitis and conjunctivitis. The areas affected by Asian dust particles extend from East China to the west coast of North America. In order to study toxicological mechanisms in human skin, we evaluated the effects of dust particles collected during Asian dust storms (Asian dust particles) on gene expression in human epidermal keratinocytes (HEK). In HEK, exposure to Asian dust particles significantly increased gene expressions of cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP1B1, which is an indication of aryl hydrocarbon receptor (AHR) activation. In addition, Asian dust particles increased gene transcription of the cytokines IL-6, IL-8, and GM-CSF, which have broad pro-inflammatory and immunomodulatory properties. Asian dust particles significantly up-regulated expression of caspase 14 in HEK, suggesting that Asian dust particles directly affect keratinocyte differentiation. We also demonstrated that protein extract of pollen, a material frequently adsorbed onto Asian dust particles, potentially contributes to the increased transcription of IL-6, CYP1A1, CYP1A2, and CYP1B1. Taken together, these studies suggest that Asian dust particles can exert toxicological effects on human skin through the activation of the cellular detoxification system, the production of pro-inflammatory and immunomodulatory cytokines, and changes in the expression of proteins essential in normal epidermal differentiation.

Activation of LXRα induces lipogenesis in HaCaT cells

The oxysterol nuclear receptors, LXRα (liver X receptor α; NR1H3) and LXRβ (NR1H2), coordinately regulate the expression of genes involved in lipid metabolism, anti-inflammation, and cholesterol transport. Previous studies have demonstrated that ligands of LXRα are important in the maintenance of the normal epidermal barrier function and keratinocyte differentiation. In this study, we examined whether LXRα and its ligands regulate lipid synthesis in HaCaT cells, a spontaneously transformed human keratinocyte cell line. When HaCaT cells were treated with the LXRα ligand TO901317, lipid droplets accumulated in the majority of cells, which were stained by Oil Red O. A luciferase reporter construct containing the LXR response element was activated about fourfold in HaCaT cells by TO901317 treatment, suggesting that LXR has a role in lipid synthesis in these cells. The expression of LXRα target genes, such as those encoding sterol regulatory binding protein and fatty acid synthase, were induced time dependently by TO901317, as measured by RT-PCR and western blotting. The expression of PPAR-α, -β, and -γ which regulate lipid metabolism, was also increased by TO901317 treatment. In contrast, TO901317 reduced the lipopolysaccharide-induced expression of cyclooxygenase 2 and inducible nitric oxide synthase in HaCaT cells. These results indicate that LXRα activation leads to lipogenesis in keratinocytes, which may enhance the epidermal barrier function of the skin.