Epithelial cells in the hair follicle bulge do not contribute to epidermal regeneration after glucocorticoid-induced cutaneous atrophy

One of the major adverse effects of glucocorticoid therapy is cutaneous atrophy, often followed by the development of resistance to steroids. It is accepted that epithelial stem cells (SCs) located in the hair follicle bulge divide during times of epidermal proliferative need. We determined whether follicular epithelial SCs and their transit amplifying progeny were stimulated to proliferate in response to the chronic application of glucocorticoid fluocinolone acetonide (FA). After first two applications of FA, keratinocyte proliferation in the interfollicular epidermis (IFE) and hair follicles was minimal and resulted in significant epidermal hypoplasia. We observed that a 50% depletion of the interfollicular keratinocyte population triggered a proliferative response. Unexpectedly, less than 2% of the proliferating keratinocytes were located in the bulge region of the hair follicle, whereas 82% were in IFE. It is known that cell desensitization to glucocorticoids is mediated via temporary decrease of glucocorticoid receptor (GR) expression. We found that GR expression was significantly decreased in IFE keratinocytes after each FA treatment. In contrast, many bulge keratinocytes retained GR in the nucleus. Our results indicate that bulge keratinocytes, including follicular SCs, are more sensitive to the antiproliferative effect of glucocorticoids than basal keratinocytes, possibly due to the incomplete process of desensitization.     

Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA

Please cite this paper as: Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA. Experimental Dermatology 2010; 19: 633–640. Abstract: Connexin 26 is important in keratinocyte proliferation, differentiation and skin pathologies. Cx26 is barely expressed in normal adult epidermis, but its expression is induced during wound healing, psoriasis, and skin hyperplasia stimulated by tumor promoters. In hyperplastic proliferating epidermis, Cx26 is co‐expressed with Cx43 typical for basal and suprabasal keratinocytes. As Cx26 and Cx43 can not form permeable gap junctions, their co‐expression may alter the gap junctional communication between keratinocytes and induce proliferation. To test the effect of persistent co‐expression of Cx26 and Cx43 in epidermis, we generated transgenic mice using keratin5 promoter to target Cx26 to basal Cx43‐positive keratinocytes. We evaluated the effect of ectopic Cx26 on keratinocyte proliferation and differentiation in normal and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA)‐treated skin. The ectopic Cx26 expression in epidermis did not significantly affect skin development, keratinocyte differentiation and proliferation in newborn and adult skin. Unexpectedly, the proliferative effect of tumor promoter TPA was strongly decreased in epidermis of K5.Cx26 transgenics. This correlated with significant down‐regulation of TPA‐induced activity of protein kinase C (PKC) in K5.Cx26 mice.     

Cell proliferative activity of epidermal keratinocytes in hair discs.

To clarify the differences in epidermal keratinocyte proliferative activity and in postnatal developmental changes of this activity between the hair discs and the interfollicular epidermis, the incorporation of 3H-thymidine into the nuclei of the basal cells was measured by autoradiography, and the percentages of basal cells thus labelled were calculated. This percentage was higher in hair discs than in interfollicular epidermis at all stages examined. The percentage of labelled basal cells in the hair discs was 10% up to 15 days after birth, decreasing to about 6% from 20 days postnatally. These results suggest that the degree of cell proliferation in the hair disc epidermis differs from that in the surrounding epidermis and that changes in proliferative activity during the postnatal developmental stages are well correlated with the morphogenesis of the hair discs.     

Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo

BACKGROUND: The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis. OBJECTIVES: We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. METHODS: (i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively. RESULTS: (i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation. CONCLUSIONS: We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.     

Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein

Basal keratinocytes in the epidermis and hair follicle are biologically heterogeneous but must include a stable subpopulation of epidermal stem cells. In animal models these can be identified by their retention of radioactive label due to their slow cycle (label-retaining cells) but human studies largely depend on in vitro characterization of colony forming efficiency and clonogenicity. Differential integrin expression has been used to detect cells of increased proliferative potential but further stem cell markers are urgently required for in vivo and in vitro characterization. Using LHM2, a monoclonal antibody reacting with a high molecular weight melanoma-associated proteoglycan core protein, a subset of basal keratinocytes in both the interfollicular epidermis and the hair follicle has been identified. Coexpression of melanoma-associated chondroitin sulfate proteoglycan with keratins 15 and 19 as well as beta 1 and alpha 6 integrins has been examined in adult and fetal human skin from hair bearing, nonhair bearing, and palmoplantar regions. Although melanoma-associated chondroitin sulfate proteoglycan coexpression with a subset of beta 1 integrin bright basal keratinocytes within the epidermis suggests that melanoma-associated chondroitin sulfate proteoglycan colocalizes with epidermal stem cells, melanoma-associated chondroitin sulfate proteoglycan expression within the hair follicle was more complex and multiple subpopulations of basal outer root sheath keratinocytes are described. These data suggest that epithelial compartmentalization of the outer root sheath is more complex than interfollicular epidermis and further supports the hypothesis that more than one hair follicle stem cell compartment may exist.     

Epidermis reconstructed from the outer root sheath of human hair follicle. Effect of retinoic acid

In the present paper, we show that a multilayered and well-differentiated epidermis can easily and rapidly be generated in vitro from the outer root sheath of human hair follicles deposited on de-epidermized demis. Histologically, this epidermis presented characteristic features of normal human epidermis in vivo. Moreover, markers specific for interfollicular keratinocyte terminal differentiation, such as the K10 keratin, involucrin, membrane-bound transglutaminase, filaggrin and loricrin, were expressed in the reconstructed tissue. By in situ hybridization, keratin K5 and K10 mRNAs were detected in the basal and suprabasal cells, respectively, as in normal human epidermis. The differentiation pattern achieved in this reconstructed epidermis confirms the already reported phenotypical shift from outer root sheath cells to interfollicular keratinocytes and shows that this transition takes place in the absence of living fibroblasts. The differentiation of the reconstructed epidermis thus obtained was modulated by retinoic acid in a dose-dependent manner. This culture system on dead dermis is easier to handle than similar cultures on collagen-fibroblast lattices because of the resistance of dermis to mechanical forces and to collagenolysis. It could represent a valuable wound-healing model and a promising tool for pharmacological studies on in vitro reconstructed skin.     

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha5 chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha5 chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (alpha5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha3beta1 and alpha6beta4 integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.     

Ectopic expression of syndecan-1 in basal epidermis affects keratinocyte proliferation and wound re-epithelialization

Epidermal proliferation and differentiation can be regulated by soluble morphogens and growth factors. Heparan sulfate proteoglycans (HSPGs) modulate the action of several of these effector molecules, such as members of the fibroblast growth factor (FGF) and Wnt families. Syndecan-1 is a cell-surface proteoglycan that is expressed in differentiating keratinocytes and transiently upregulated in all layers of the epidermis upon tissue injury. To address the role of syndecan-1 in the regulation of keratinocyte proliferation and differentiation, we generated transgenic mice that overexpress syndecan-1 under K14 keratin promoter in the basal layer of the epidermis. We observed epidermal hyperproliferation in newborn transgenic mice, as evidenced by increased number of suprabasal cell layers, elevated proliferating cell nuclear antigen (PCNA) expression in both basal and suprabasal cell layers and by expression of keratin 6 in the interfollicular epidermis. Compared to both wild-type and syndecan-1-null animals, the transgene expression interfered with skin wound healing in adult mice by decreasing cell proliferation in the re-epithelialized epidermis. Thus, syndecan-1 regulates keratinocyte proliferation differently during skin development and in healing wounds.     

Synergistic effects of epidermal growth factor (EGF) and insulin-like growth factor I/somatomedin C (IGF-I) on keratinocyte proliferation may be mediated by IGF-I transmodulation of the EGF receptor.

The epidermal growth factor (EGF) receptor pathway is an important mediator of keratinocyte growth in vitro and both receptor and ligand components of this pathway are abnormally expressed in hyperproliferative epidermis. The purpose of this study was to examine interactions between the EGF receptor pathway and the insulin-like growth factor I/somatomedin C (IGF-I) receptor pathway in modulating the growth of cultured normal human keratinocytes. Short-term growth of keratinocytes in a chemically defined medium demonstrated that neither EGF nor IGF-I alone could support significant keratinocyte spreading or proliferation, but that a combination of EGF with IGF-I or high-dose insulin could. IGF-I or high-dose insulin transmodulates keratinocyte EGF receptor expression via the IGF-I receptor in a dose- and time-dependent manner, increasing EGF receptor binding an average of 1.8 times up to a maximum of fourfold without altering EGF binding affinity. Staining of normal human epidermis with an IGF-I receptor specific monoclonal antibody demonstrates that IGF-I receptors localize to the basal proliferative cell compartment, suggesting that IGF-I receptor and EGF receptor pathway interactions may play a role in the regulation of epidermal growth and in the pathogenesis of hyperproliferative skin diseases.     

Altered keratin expression in benign and malignant skin diseases revealed with monoclonal antibodies

We studied keratin expression in benign epidermal skin diseases, and in Bowen's disease by using three monoclonal cytokeratin antibodies. In adult normal skin, these antibodies bind only to the follicular epithelium (PKK1), the basal keratinocytes (PKK2), or the suprabasal cells in interfollicular epidermis (KA5). Additionally, in fetal epidermis, the PKK1 antibody reacts with basal keratinocytes. In psoriasis and lichen planus, the PKK2 antibody distinctly revealed all epidermal cell layers by immunostaining. However, a negative basal cell-like layer was revealed in both lesions with the KA5 antibody. In pityriasis rubra pilaris, the basal cell layer was uniformly stained with the PKK2 antibody, but only some keratinocytes in upper cell layers showed fluorescence and, in chronic eczema, the 3-4 lowest epidermal cell layers were reactive. The PKK1 antibody did not stain interfollicular keratinocytes in any of the benign proliferative skin diseases studied. In Bowen's disease, a heterogeneous staining pattern with varying intensity among individual cells was seen with all of the antibodies used. Our results suggest different changes in keratin expression in chronic benign and malignant epidermal diseases that may reflect the mechanisms behind these changes.     

Human umbilical cord blood cells form epidermis in the skin equivalent model

Please cite this paper as: Human umbilical cord blood cells form epidermis in the skin equivalent model. Experimental Dermatology 2010. Abstract: Recently, human embryonic stem cells have been differentiated in vitro into functional epidermal keratinocytes. Here, we demonstrated that these cells can be generated also from non‐embryonic, human umbilical cord blood (hUCB) cells that have the potential to differentiate into cells of non‐hematopoietic lineage. Human UCB mono‐nucleated cells were cultivated in monolayer and in three‐dimensional skin equivalent cultures and assayed for the presence of phenotype‐specific markers. Our results determined that after one month of culturing in serum containing medium, the hUCB cells produced morphologically homogeneous colonies of epithelial cells expressing keratinocyte‐specific markers. They also formed stratified epidermis in organ cultures that contained sporadic CD1a‐positive cells within the accurate strata. We concluded that hUCB cells have the capacity to differentiate into functional epidermal keratinocytes and may serve as a source of high‐quality keratinocytes for clinical applications.     

Induction of senescence pathways in Kindler syndrome primary keratinocytes

Background Individuals with Kindler syndrome (KS) have loss‐of‐function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin‐1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. Objectives To investigate how kindlin‐1 deficiency affects the proliferative potential of primary human keratinocytes. Methods  We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony‐forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin‐1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. Results We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi‐1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle‐arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin‐1 proved that its deficiency was directly responsible for p63, Bmi‐1 and pRb downregulation and p16 induction. Conclusions Our data directly implicate kindlin‐1 in preventing premature senescence of keratinocytes.     

Increased DNA synthesis of uninvolved psoriatic epidermis is maintained in vitro

Clinically uninvolved psoriatic epidermis shows increased DNA synthesis in vivo. We have studied the DNA synthesis of cultured keratinocytes from uninvolved psoriatic skin. Trypsinized epidermal cells were plated on plastic dishes pre-coated with bovine collagen type I. In initial studies, normal human serum was found to be superior to fetal bovine in supporting the growth of human epidermal keratinocytes. Furthermore, keratinocyte cultures established in the presence of normal human serum produced large keratin proteins (68,000 daltons) indicating that the terminal steps in cell differentiation can occur in vitro. In subsequent experiments keratinocyte cultures were grown in medium supplemented with 10% normal human serum. Confluent cultures of keratinocytes from uninvolved psoriatic epidermis had an increased DNA synthesis determined both as the incorporation of [3H]thymidine and as the autoradiographic labelling index. The DNA synthesis of both normal and psoriatic keratinocyte cultures increased in response to incubation in medium with 10% psoriatic serum. The ability of keratinocytes from uninvolved psoriatic epidermis to maintain an increased DNA synthesis suggests the presence of an inherent defect within the population of epidermal keratinocytes in psoriasis. Such a culture system can be used as an in vitro model for the study of psoriasis.     

Cell-matrix interactions of normal and transformed human keratinocytes in vitro are modulated by the synthetic phospholipid analogue hexadecylphosphocholine

Summary We have investigated the effect of the synthetic phospholipid analogue, hexadecylphosphocholine (HePC), a member of a new class of membrane-affecting antiproliferative drugs, on adhesion to extracellular matrix components, reorganization of three-dimensional collagen lattices, and proliferative activity of human keratinocyte populations in vitro. Six transformed keratinocyte lines were compared with their normal counterparts from interfollicular epidermis. Adhesion to collagen types I and IV, laminin, and fibronectin was weakest in normal keratinocytes (binding of 3–10% of the cells), followed by HaCaT and HaCaT-II/3 (25-30% binding), and the squamous carcinoma lines and SV-40 transformed keratinocytes (35-50% binding). Treatment with non-toxic doses of 10–20 μmol/1 HePC led to a clear inhibition of the adhesive interactions by 50–75% in all populations. The impaired adhesion capacity was paralleled by a clear decrease of the ability of all keratinocyte populations to contract three-dimensional collagen lattices, an experimental mental model system for the reorganization of extracellular matrices. Histological examination revealed that these effects were due to a reduced cell number in the collagen lattices, a finding underscored by significant inhibition of proliferative activity of all keratinocyte populations by HePC. Furthermore, HePC led to marked changes of cellular morphology in the contracted gels. FACS analysis revealed that the impaired interaction with components of the extracellular matrix was not due to a specific downregulation of β₁-integrins, the major cell-surface receptors for the respective matrix proteins. In conclusion, our results provide new insights into the potential action of HePC in a variety of skin disorders. Decreased proliferative activity and changes of cellular morphology, combined with impaired interactions with extracellular matrix proteins and a consecutive loss of matrix organization capacity, may cause suppression of different hyperproliferative skin diseases.     

Clear and Dark Basal Keratinocytes in Human Epidermis

Standardized techniques of stereologic cytology were applied to normal human epidermis, in order to comparatively describe the suprabasal and the dark and clear basal keratinocytes. The results, expressed mainly in surface and volume densities of epidermal components, showed that some important stereologic parameters of dark basal keratinocyte constituents exhibited values which could be interpreted as intermediate between those of the clear basal keratinocytes and those of the suprabasal keratinocytes. Although the degenerative nature of dark cells cannot be ruled out, the stereologic characterization of their cell architecture also could indicate that dark basal cells are already differentiating keratinocytes still remaining in the basal layer.     

The expression of differentiation markers in aquaporin-3 deficient epidermis

Aquaporin-3 (AQP3) is a water/glycerol transporting protein expressed strongly at the plasma membrane of keratinocytes. There is evidence for involvement of AQP3-facilitated water and glycerol transport in keratinocyte migration and proliferation, respectively. Here, we investigated the involvement of AQP3 in keratinocyte differentiation. Studies were done using AQP3 knockout mice, primary cultures of mouse keratinocytes (AQP3 knockout), neonatal human keratinocytes (AQP3 knockdown), and human skin. Cells were cultured with high Ca²⁺ or 1α,25-dihydroxyvitamin D₃ (VD₃) to induce differentiation. The expression of differentiation marker proteins and differentiating responses were comparable in control and AQP3-knockout or knockdown keratinocytes. Topical application of all-trans retinoic acid (RA), a known regulator of keratinocyte differentiation and proliferation, induced comparable expression of differentiation marker proteins in wildtype and AQP3 null epidermis, though with impaired RA-induced proliferation in AQP3 null mice. Immunostaining of human and mouse epidermis showed greater AQP3 expression in cells undergoing proliferation than differentiation. Our results showed little influence of AQP3 on keratinocyte differentiation, and provide further support for the proposed involvement of AQP3-facilitated cell proliferation.     

Expression of vascular endothelial growth factor in normal epidermis, epithelial tumors and cultured keratinocytes

Vascular endothelial growth factor (VEGF), an endothelium-specific growth factor and microvessel hypermeability factor, is expressed and secreted by several kinds of cells and is implicated in angiogenesis of tumors. The present study was performed to determine the relationship between the expression of VEGF in normal skin, benign and malignant epithelial lesions and cultured keratinocytes and the proliferative activity and degree of differentiation of keratinocytes. Skin lesions were studied immunohistochemically by staining with two anti-VEGF antibodies and secretion and production of VEGF by keratinocyte cultures were evaluated using an enzyme-linked immunosorbent assay. Low to moderate VEGF expression was observed in normal epidermis. In epithelial tumors, different reactivity patterns were observed and different areas of the same tumor expressed different amounts of VEGF. A more prominent labelling occurred in proliferative layers and/or more differentiated cells of virus-induced lesions, squamous cell carcinomas and Bowen’s disease, whereas basal cell carcinomas always stained weakly for VEGF. In cultured keratinocytes, the amount of cell-associated and secreted VEGF increased with time, and the constitutively produced VEGF was mostly released extracellularly. High calcium concentrations upregulated the intracellular content of VEGF but downregulated its release. Taken together, these results showed a modulated expression and release of VEGF in relation to the stage of cell differentiation and in rapidly growing or activated keratinocytes. Received: 22 April 1996