Biological function of laminin-5 and pathogenic impact of its deficiency

The basement membrane glycoprotein laminin-5 is a key component of the anchoring complex connecting keratinocytes to the underlying dermis. It is secreted by keratinocytes as a cross-shaped heterotrimer of alpha3, beta3 and gamma2 chains and serves as a ligand of various transmembrane receptors, thereby regulating keratinocyte adhesion, motility and proliferation. In intact skin, laminin-5 provides essential links to both the hemidesmosomal alpha6beta4 integrin and the collagen type VII molecules which form the anchoring fibrils inserting into the dermis. If the basement membrane is injured, laminin-5 production increases rapidly. It then serves as a scaffold for cell migration, initiates the formation of hemidesmosomes and accelerates basement membrane restoration at the dermal-epidermal junction. Mutations of the laminin-5 genes or auto-antibodies against one of the subunits of laminin-5 may lead to a significant lack of this molecule in the epidermal basement membrane zone. The major contributions of laminin-5 to the resistance of the epidermis against frictional stress but also for basement membrane regeneration and repair of damaged skin are reflected by the phenotype of Herlitz junctional epidermolysis bullosa, which is caused by an inherited absence of functional laminin-5. This lethal disease becomes manifest in widespread blistering of skin and mucous membranes, impaired wound healing and chronic erosions containing exuberant granulation tissue. Here, we discuss current understanding of the biological functions of laminin-5, the pathogenic impact of its deficiency and implications on molecular approaches towards a therapy of junctional epidermolysis bullosa.     

Changes in localization of human discs large (hDlg) during keratinocyte differentiation are [corrected] associated with expression of alternatively spliced hDlg variants

Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.     

Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.     

Aquaporin 5 increases keratinocyte-derived chemokine expression and NF-κB activity through ERK activation

Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in submucosal glands and alveolar epithelial cells in the lungs. Recent studies have revealed that AQPs regulate not only water metabolism, but also some cellular functions such as cell growth and migration. Here, we report the role of AQP5 in inflammatory responses. In MLE-12 cells, knockdown of AQP5 using siRNA (10–50 nM) attenuated TNF-α-induced expression of keratinocyte chemoattractant (KC) mRNA and protein. Conversely, in NIH-3T3 cells, overexpression of AQP5 increased KC expression, NF-κB activation, and ERK phosphorylation. The AQP5-induced increase of KC expression was diminished by treatment with ERK inhibitors. Taken together, we propose a new function of AQP5 as an inflammatory signal potentiator, which may be mediated by increased activation of ERK and NF-κB.     

Switch from αvβ5 to αvβ6 integrin is required for CD9-regulated keratinocyte migration and MMP-9 activation

Our previous research found that tetraspanin CD9 is downregulated in migrating epidermis during wound healing, and CD9 downregulation contributes to keratinocyte migration via matrix metalloproteinase-9 (MMP-9) activation. However, little is known about the mechanisms involved in CD9-regulated keratinocyte migration and MMP-9 activation. In this study, we revealed that the expressions of integrin subunits β5 and β6 were regulated by CD9. Furthermore, CD9 silencing triggered the switch from αvβ5 to αvβ6 integrin in HaCaT keratinocytes and CD9 overexpression reversed the switch. Importantly, integrin αvβ6 functional blocking antibody 10D5 significantly inhibited CD9 silencing-induced keratinocyte migration and MMP-9 activation, suggesting that the switch from αvβ5 to αvβ6 integrin plays a key role in CD9-regulated cell migration and MMP-9 activation in keratinocytes.     

Disruption of epidermal specific gene expression and delayed skin development in AP-2 gamma mutant mice

Summary Sentence: Conditional ablation of AP-2γ results in a delay in skin development and abnormal expression of p63, K14, K1, filaggrin, repetin and secreted Ly6/Plaur domain containing 1, key genes required for epidermal development and differentiation.The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2γ is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2γ, which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2γ in skin development. Mice deficient for AP-2γ exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2γ in skin development, and reveal the existence of regulatory factors that can compensate for AP-2γ in its absence.     

Epithelial Na+ channel delta subunit mediates acid-induced ATP release in the human skin

The amiloride-sensitive epithelial Na⁺ channel (ENaC) regulates Na⁺ homeostasis in cells and across epithelia. Although we described that ENaCδ is a candidate molecule for a pH sensor in the human brain, the physiological and pathological roles of ENaCδ in non-neuronal tissues are still unknown. Here we show a novel physiological function of ENaCδ in peripheral tissues in humans. Expression analyses at the level of mRNA clearly revealed that ENaCδ was abundantly expressed in human epidermis and keratinocytes. In addition, ENaCδ protein was detected in there. In cultured keratinocytes, acidic stress (pH 5.0) evoked ATP release, which was significantly reduced in the presence of 100 μM amiloride or 10 μM benzamil. In conclusion, ENaCδ may be involved in the mechanism underlying pH sensing followed by the regulation of cell viability in the human skin.     

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.     

Changes in the Proliferative Activity of Epidermal Melanocytes in Serum‐Free Primary Culture During the Development of Ultraviolet Radiation B‐Induced Pigmented Spots in Hairless Mice

Long‐term exposure to ultraviolet radiation B (UVB) induced pigmented spots in the dorsal skin of hairless mice of strain (HR‐1 X HR/De)F₁. To clarify the cellular mechanism for the development of these UVB‐induced pigmented spots, we investigated changes in the proliferative activity of epidermal melanoblasts and melanocytes in the dorsal skin at various weeks after UVB irradiation. Epidermal cell suspensions from the dorsal skin of hairless mice were cultured in a serum‐free medium supplemented with dibutyryl adenosine 3′:5′‐cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). The suspensions were prepared from dorsal skins of mice exposed to UVB for 4 weeks (the stage of hyperpigmentation). Suspensions were also prepared from mice at 3 (the stage of depigmentation), 8 (the stage of appearance of pigmented spots), 20 (the stage of development of small‐sized pigmented spots) and 37 (the stage of development of medium‐sized pigmented spots) weeks after the cessation of 8‐week UVB exposure. At the stage of hyperpigmentation the proliferative activity of melanoblasts and melanocytes was suppressed. With the development of pigmented spots, the proliferative activity of undifferentiated melanoblasts gradually increased, and then followed the increase in the proliferative activity of differentiated melanocytes. These results suggest that the proliferative activity of epidermal melanoblasts and melanocytes in UVB‐irradiated skin increases with the development of pigmented spots.     

In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line

In vitro models to study the process of keratinocyte differentiation have been hindered by the stringent culture requirements and limitations imposed by the inherent properties of the cells. Primary keratinocytes only have a finite life span, while transformed cell lines exhibit many phenotypic features not found in normal cells. The spontaneously immortalized HaCaT cell line has been a widely employed keratinocyte model due to its ease of propagation and near normal phenotype, but protocols for differentiation and gene delivery into HaCaT cells vary widely in the literature. Here we report culture conditions for maintaining HaCaT cells in a basal-like state, for efficient differentiation of these cells, and for delivery of transgenes by transfection or adenoviral infection. This technological report will provide guidance to a large audience of scientists interested in investigating mechanisms of differentiation and skin morphogenesis.