The psoriasis drug monomethylfumarate is a potent nicotinic acid receptor agonist

Nicotinic acid has been used for several decades to treat dyslipidemia. In mice, the lipid-lowing effect of nicotinic acid is mediated by the Gi coupled receptor PUMA-G. In humans, high (GPR109A) and low (GPR109B) affinity nicotinic acid receptors have been characterized. Here we identify monomethylfumarate as a GPR109A agonist. Monomethylfumarate is the active metabolite of the psoriasis drug Fumaderm. We show that monomethylfumarate activates GPR109A in a calcium based aequorin assay, cAMP assay and demonstrate competitive binding with nicotinic acid. We show that GPR109A is highly expressed in neutrophils and epidermal keratinocytes, and that its expression is increased in human psoriatic lesions. Our findings provide evidence that GPR109A is a target for the drug Fumaderm and suggest that niacin should be investigated to treat psoriasis in addition to its role in treating lipid disorders.     

Leptin Promotes Wound Healing in the Skin

Introduction

Leptin, a 16 kDa anti-obesity hormone, exhibits various physiological properties. Interestingly, skin wound healing was proven to delay in leptin-deficient ob/ob mice. However, little is known on the mechanisms of this phenomenon. In this study, we attempted to elucidate a role of leptin in wound healing of skin.

Methods

Immunohistochemical analysis was performed to confirm the expression of the leptin receptor (Ob-R) in human and mouse skin. Leptin was topically administered to chemical wounds created in mouse back skin along with sustained-release absorbable hydrogel. The process of wound repair was histologically observed and the area of ulceration was measured over time. The effect of leptin on the proliferation, differentiation and migration of human epidermal keratinocytes was investigated.

Results

Ob-R was expressed in epidermal cells of human and mouse skin. Topical administration of leptin significantly promoted wound healing. Histological analysis showed more blood vessels in the dermal connective tissues in the leptin-treated group. The proliferation, differentiation/function and migration of human epidermal keratinocytes were enhanced by exogenous leptin.

Conclusion

Topically administered leptin was proven to promote wound healing in the skin by accelerating proliferation, differentiation/function and migration of epidermal keratinocytes and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the skin.     

Notch signaling regulates late-stage epidermal differentiation and maintains postnatal hair cycle homeostasis

Background

Notch signaling involves ligand-receptor interactions through direct cell-cell contact. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicles during embryonic development and the adult stage. Although Notch signaling plays an important role in regulating differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in late-stage epidermal differentiation and postnatal hair cycle homeostasis.

Methodology and Principal Findings

We applied Cre/loxP system to generate conditional gene targeted mice that allow inactivation of critical components of Notch signaling pathway in the skin. Rbpj, the core component of all four Notch receptors, and Pofut1, an essential factor for ligand-receptor interactions, were inactivated in hair follicle lineages and suprabasal layer of the epidermis using the Tgfb3-Cre mouse line. Rbpj conditional inactivation resulted in granular parakeratosis and reactive epidermal hyperplasia. Pofut1 conditional inactivation led to ultrastructural abnormalities in the granular layer and altered filaggrin processing in the epidermis, suggesting a perturbation of the granular layer differentiation. Disruption of Pofut1 in hair follicle lineages resulted in aberrant telogen morphology, a decrease of bulge stem cell markers, and a concomitant increase of K14-positive keratinocytes in the isthmus of mutant hair follicles. Pofut1-deficent hair follicles displayed a delay in anagen re-entry and dysregulation of proliferation and apoptosis during the hair cycle transition. Moreover, increased DNA double stand breaks were detected in Pofut1-deficent hair follicles, and real time PCR analyses on bulge keratinocytes isolated by FACS revealed an induction of DNA damage response and a paucity of DNA repair machinery in mutant bulge keratinocytes.

Significance

our data reveal a role for Notch signaling in regulating late-stage epidermal differentiation. Notch signaling is required for postnatal hair cycle homeostasis by maintaining proper proliferation and differentiation of hair follicle stem cells.     

Immunolocalization of the Tumor-Sensitive Calmodulin-Like Protein CALML3 in Normal Human Skin and Hyperproliferative Skin Disorders

Background and Objective

Calmodulin-like protein CALML3 is an epithelial-specific protein regulated during keratinocyte differentiation in vitro. CALML3 expression is downregulated in breast cancers and transformed cell lines making it an attractive marker for tumor formation. The objective of this study was to survey CALML3 localization in normal epidermis and in hyperproliferative skin diseases including actinic keratosis, squamous and basal cell carcinoma as well as verruca and psoriasis and to compare CALML3 immunoreactivity with the proliferation marker Ki-67.

Methods

Paraffin-embedded tissue sections from normal human skin and hyperproliferative skin disorders were examined by immunohistochemistry and analyzed for localization and expression of CALML3 and Ki-67.

Results

CALML3 was strongly expressed in differentiating layers of normal skin, staining the periphery in suprabasal cells and exhibiting nuclear localization in the stratum granulosum. CALML3 nuclear localization was inversely correlated to Ki-67 staining in each disease, indicating that CALML3 nuclear presence is related to terminal cell differentiation and postmitotic state.

Conclusions

Increased CALML3 expression in suprabasal layers is characteristic for differentiating keratinocytes in normal epidermis, and nuclear expression of CALML3 inversely correlates with expression of the proliferation marker Ki-67. This suggests that CALML3 is a useful marker for normal and benign hyperplastic epidermal development, whereas the loss of nuclear CALML3 indicates progression to a proliferative and potentially malignant phenotype.     

β1 Integrin-Mediated Adhesion Signalling Is Essential for Epidermal Progenitor Cell Expansion

Background

There is a major discrepancy between the in vitro and in vivo results regarding the role of β1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of β1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation.

Methodology/Principal Findings

To elucidate this discrepancy we generated hypomorphic mice expressing reduced β1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with β1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of β1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the β1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of β1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing.

Conclusions/Significance

These data demonstrate that expression of β1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.     

Differential regulation of adhesion complex turnover by ROCK1 and ROCK2

Background

ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.

Methodology/Principal Findings

In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.

Conclusions

ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.     

Wnt5a Exhibits Layer-Specific Expression in Adult Skin,Is Upregulated in Psoriasis,and Synergizes with Type 1 Interferon

Background

Wnt5a is a member of the wingless-type patterning regulators important in pre-natal development. The expression and distribution of Wnt5a and its receptors frizzled (fzd) 3 and fzd 5 in adult human skin have not been comprehensively studied to date.

Methodology/Principal Findings

We here show that Wnt5a, fzd3, fzd5, as well as fzd6 are restricted to specific layers in normal epidermis, analogous to their zonal distribution in hair follicles, suggesting a role in adult skin differentiation. In line, Wnt5a and fzd5 are both overexpressed and re-distributed in the epidermis of psoriasis which involves disturbed keratinocyte differentiation. Functionally, Wnt5a lowers the concentration of IFN required to induce target genes, and increases the magnitude of IFN target gene induction, suggesting a molecular mechanism underlying IFN hypersensitivity in psoriasis. Finally, we identify nedd8 and the amyloid precursor APP, previously shown to be upregulated in psoriasis, as targets of synergistic IFNα/Wnt5a induction.

Conclusions/Significance

The present data (i) suggest that Wnt5a regulates epidermal differentiation even in adult skin and (ii) identify synergistic induction of type 1 IFN target genes as a novel mode of Wnt5a action. Targeting Wnt5a in the skin may reduce IFN hypersensitivity and be of therapeutical value.     

Distinct Roles for ROCK1 and ROCK2 in the Regulation of Keratinocyte Differentiation

Background

The human epidermis is comprised of several layers of specialized epithelial cells called keratinocytes. Normal homoeostasis of the epidermis requires that the balance between keratinocyte proliferation and terminal differentiation be tightly regulated. The mammalian serine/threonine kinases (ROCK1 and ROCK2) are well-characterised downstream effectors of the small GTPase RhoA. We have previously demonstrated that the RhoA/ROCK signalling pathway plays an important role in regulation of human keratinocyte proliferation and terminal differentiation. In this paper we addressed the question of which ROCK isoform was involved in regulation of keratinocyte differentiation.

Methodology and Principal Findings

We used RNAi to specifically knockdown ROCK1 or ROCK2 expression in cultured human keratinocytes. ROCK1 depletion results in decreased keratinocyte adhesion to fibronectin and an increase in terminal differentiation. Conversely, ROCK2 depletion results in increased keratinocyte adhesion to fibronectin and inhibits terminal differentiation.

Conclusion

These data suggest that ROCK1 and ROCK2 play distinct roles in regulating keratinocyte adhesion and terminal differentiation.     

CD109, a TGF-�� co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

Introduction

Scleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-β) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-β co-receptor that inhibits TGF-β signaling. The aim of the present study was to determine the role of CD109 in regulating extracellular matrix (ECM) production in human SSc skin fibroblasts.

Methods

CD109 expression was determined in skin tissue and cultured skin fibroblasts of SSc patients and normal healthy subjects, using immunofluorescence, western blot and RT-PCR. The effect of CD109 on ECM synthesis was determined by blocking CD109 expression using CD109-specific siRNA or addition of recombinant CD109 protein, and analyzing the expression of ECM components by western blot.

Results

The expression of CD109 proteinis markedly increased in SSc skin tissue in vivo and in SSc skin fibroblasts in vitro as compared to their normal counterparts. Importantly, both SSc and normal skin fibroblasts transfected with CD109-specific siRNA display increased fibronectin, collagen type I and CCN2 protein levels and enhanced Smad2/3 phosphorylation compared with control siRNA transfectants. Furthermore, addition of recombinant CD109 protein decreases TGF-β₁-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts.

Conclusion

The upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-β signaling in SSc. Our finding that CD109 is able to decrease excessive ECM production in SSc fibroblasts suggest that this molecule has potential therapeutic value for the treatment of SSc.     

Upregulation of GPR109A in Parkinson’s Disease

Background

Anecdotal animal and human studies have implicated the symptomatic and neuroprotective roles of niacin in Parkinson’s disease (PD). Niacin has a high affinity for GPR109A, an anti-inflammatory receptor. Niacin is also thought to be involved in the regulation of circadian rhythm. Here we evaluated the relationships among the receptor, niacin levels and EEG night-sleep in individuals with PD.

Methods and Findings

GPR109A expression (blood and brain), niacin index (NAD-NADP ratio) and cytokine markers (blood) were analyzed. Measures of night-sleep function (EEG) and perceived sleep quality (questionnaire) were assessed. We observed significant up-regulation of GPR109A expression in the blood as well as in the substantia nigra (SN) in the PD group compared to age-matched controls. Confocal microscopy demonstrated co-localization of GPR109A staining with microglia in PD SN. Pro and anti-inflammatory cytokines did not show significant differences between the groups; however IL1-β, IL-4 and IL-7 showed an upward trend in PD. Time to sleep (sleep latency), EEG REM and sleep efficiency were different between PD and age-matched controls. Niacin levels were lower in PD and were associated with increased frequency of experiencing body pain and decreased duration of deep sleep.

Conclusions

The findings of associations among the GPR109A receptor, niacin levels and night-sleep function in individuals with PD are novel. Further studies are needed to understand the pathophysiological mechanisms of action of niacin, GPR109A expression and their associations with night-sleep function. It would be also crucial to study GPR109A expression in neurons, astrocytes, and microglia in PD. A clinical trial to determine the symptomatic and/or neuroprotective effect of niacin supplementation is warranted.     

H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Background

Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Methodology/Principal Findings

Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.

Conclusions/Significance

The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.     

MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R

Background

Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder.

Methodology/Principal Findings

Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca⁺⁺, miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a.

Conclusions/Significance

We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.     

Synergistic effect of hyaluronate fragments in retinaldehyde-induced skin hyperplasia which is a Cd44-dependent phenomenon

Background

CD44 is a polymorphic proteoglycan and functions as the principal cell-surface receptor for hyaluronate (HA). Heparin-binding epidermal growth factor (HB-EGF) activation of keratinocyte erbB receptors has been proposed to mediate retinoid-induced epidermal hyperplasia. We have recently shown that intermediate size HA fragments (HAFi) reverse skin atrophy by a CD44-dependent mechanism.

Methodology and Principal Findings

Treatment of primary mouse keratinocyte cultures with retinaldehyde (RAL) resulted in the most significant increase in keratinocyte proliferation when compared with other retinoids, retinoic acid, retinol or retinoyl palmitate. RAL and HAFi showed a more significant increase in keratinocyte proliferation than RAL or HAFi alone. No proliferation with RAL was observed in CD44^−/− keratinocytes. HA synthesis inhibitor, 4-methylumbelliferone inhibited the proliferative effect of RAL. HB-EGF, erbB1, and tissue inhibitor of MMP-3 blocking antibodies abrogated the RAL- or RAL- and HAFi-induced keratinocyte proliferation. Topical application of RAL or RAL and HAFi for 3 days caused a significant epidermal hyperplasia in the back skin of wild-type mice but not in CD44^−/− mice. Topical RAL and HAFi increased epidermal CD44 expression, and the epidermal and dermal HA. RAL induced the expression of active HB-EGF and erbB1. However, treatment with RAL and HAFi showed a more significant increase in pro-HB-EGF when compared to RAL or HAFi treatments alone. We then topically applied RAL and HAFi twice a day to the forearm skin of elderly dermatoporosis patients. After 1 month of treatment, we observed a significant clinical improvement.

Conclusions and Significance

Our results indicate that (i) RAL-induced in vitro and in vivo keratinocyte proliferation is a CD44-dependent phenomenon and requires the presence of HA, HB-EGF, erbB1 and MMPs, (ii) RAL and HAFi show a synergy in vitro and in vivo in mouse skin, and (iii) the combination of RAL and HAFi seems to have an important therapeutic effect in dermatoporosis.     

Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo

Background

The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras^G12D mice with ablation of Notch1 and/or Notch2.

Methodology/Principal Findings

Surprisingly, mice with activated Kras^G12D and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.

Conclusions/Significance

Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.     

Atopic Dermatitis-Like Disease and Associated Lethal Myeloproliferative Disorder Arise from Loss of Notch Signaling in the Murine Skin

Background

The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth.

Methodology and Principal Findings

To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.

Significance

Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.