Vitamin C Stimulates Epidermal Ceramide Production by Regulating Its Metabolic Enzymes

Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 mug/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased de novo ceramide synthesis pathway via serine palmitoyltransferase and ceramide synthase activations. Moreover, sphingosine-1-phosphate (S1P) hydrolysis pathway by action of S1P phosphatase was also stimulated by vitamin C supplementation, contributing, in part, to enhanced ceramide production. However, activity of sphingomyelinase, a hydrolase enzyme that converts sphingomyelin to ceramide, remained unaltered. Taken together, we demonstrate that vitamin C stimulates ceramide production in keratinocytes by modulating ceramide metabolic-related enzymes, and as a result, could improve overall epidermal barrier function.

Analysis of ceramide metabolites in differentiating epidermal keratinocytes treated with calcium or vitamin C

Ceramides (Cer) comprise the major constituent of sphingolipids in the epidermis and are known to play diverse roles in the outermost layers of the skin including water retention and provision of a physical barrier. In addition, they can be hydrolyzed into free sphingoid bases such as C18 sphingosine (SO) and C18 sphinganine (SA) or can be further metabolized to C18 So-1-phosphate (S1P) and C18 Sa-1-phosphate (Sa1P) in keratinocytes. The significance of ceramide metabolites emerged from studies reporting altered levels of SO and SA in skin disorders and the role of S1P and Sa1P as signaling lipids. However, the overall metabolism of sphingoid bases and their phosphates during keratinocyte differentiation remains not fully understood. Therefore, in this study, we analyzed these Cer metabolites in the process of keratinocyte differentiation. Three distinct keratinocyte differentiation stages were prepared using 0.07 mM calcium (Ca2+) (proliferation stage), 1.2 mM Ca2+ (early differentiation stage) in serum-free medium, or serum-containing medium with vitamin C (50 microL/mL) (late differentiation stage). Serum-containing medium was also used to determine whether vitamin C increases the concentrations of sphingoid bases and their phosphates. The production of sphingoid bases and their phosphates after hydrolysis by alkaline phosphatase was determined using high-performance liquid chromatography. Compared to cells treated with 0.07 mM Ca2+, levels of SO, SA, S1P, and SA1P were not altered after treatment with 1.2 mM Ca2+. However, in keratinocytes cultured in serum-containing medium with vitamin C, levels of SO, SA, S1P, and SA1P were dramatically higher than those in 0.07- and 1.2-mM Ca2+-treated cells; however, compared to serum-containing medium alone, vitamin C did not significantly enhance their production. Taken together, we demonstrate that late differentiation induced by vitamin C and serum was accompanied by dramatic increases in the concentration of sphingoid bases and their phosphates, although vitamin C alone had no effect on their production.

Effects of Calcium on the Epidermis in a Skin Organ Culture / 대한피부과학회지

BACKGROUND: Calcium plays a role in the proliferation and differentiation of keratinocytes. In a normal situation, the calcium concentration forms a gradient across the epidermal layers. Calcium is sparse in the basal layer and spinous layer. Skin organ culture is a useful model for conducting research on various aspects of skin biology. Skin organ culture systems are used for defining factors that affect homeostasis when elucidating the modulatory effects of biologic response modifiers, drugs and physical agents on the skin and also when studying complex aspects of cutaneous biology in normal and diseased skin. OBJECTIVE: In this study, we investigated the effects of extracellular calcium on the epidermis in a skin organ culture. METHODS: We compared the skin organ culture patterns under various culture conditions (calcium 0.1, 0.7, 1.4 and 2.0 mM). RESULTS: H&E staining showed different phenotypes according to the calcium concentration and IHC also showed different phenotyes compared to that of keratin 10, involucrin, filaggrin, loricrin and PCNA. CONCLUSION: As a result, we concluded that the calcium gradient is also an important factor in skin organ culture to maintain the vivo-like environment and the appropriate calcium concentration is 1.4 mM.

Expression Profiling of Calcium Induced Genes in Cultured Human Keratinocytes

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. To examine the gene expression profile in calcium-induced keratinocyte differentiation, we constructed a normalized cDNA library using mRNA isolated from these calcium-treated keratinocytes. After sequencing about 10,000 clones, we were able to obtain 4,104 independent genes. They consisted of 3,699 annotated genes and 405 expressed sequence tags (ESTs). Some were the genes involved in constituting epidermal structures and others were unknown genes that are probably associated with keratinocytes. In particular, we were able to identify genes located at the chromosome 1q21, the locus for the epidermal differentiation complex, and 19q13.1, another probable locus for epidermal differentiation-related gene clusters. One EST located at the chromosome 19q13.1 showed increased expression by calcium treatment, suggesting a novel candidate gene relevant to keratinocyte differentiation. These results demonstrate the complexity of the transcriptional profile of keratinocytes, providing important clues on which to base further investigations of the molecular events underlying keratinocyte differentiation.

The Role of Nkx2.5 in Keratinocyte Differentiation

BACKGROUND: Nkx2.5 is a homeodomain-containing nuclear transcription protein that has been associated with acute T-lymphoblastic leukemia. In addition, Nkx2.5 has an essential role in cardiomyogenesis. However, the expression of Nkx2.5 in the skin has not been investigated. OBJECTIVE: In an attempt to screen the differentially regulated genes involved in keratinocyte differentiation, using a cDNA microarray, we identified Nkx2.5 as one of the transcription factors controlling the expression of proteins associated with keratinocyte differentiation. METHODS: To investigate the expression of Nkx2.5 during keratinocyte differentiation, we used a calcium-induced keratinocyte differentiation model. RESULTS: RT-PCR and Western blot analysis revealed that the expression of Nkx2.5, in cultured human epidermal keratinocytes, increased with calcium treatment in a time-dependent manner. In normal skin tissue, the expression of Nkx2.5 was detected in the nuclei of the keratinocytes in all layers of the epidermis except the basal layer by immunohistochemistry. In addition, the expression of Nkx2.5 was significantly increased in psoriasis and squamous cell carcinoma, but was barely detected in atopic dermatitis and basal cell carcinoma. CONCLUSION: These results suggest that Nkx2.5 may play a role in the change from proliferation to differentiation of keratinocytes and in the pathogenesis of skin disease with aberrant keratinocyte differentiation.

An Update of the Defensive Barrier Function of Skin

Skin, as the outermost organ in the human body, continuously confronts the external environment and serves as a primary defense system. The protective functions of skin include UV-protection, anti-oxidant and antimicrobial functions. In addition to these protections, skin also acts as a sensory organ and the primary regulator of body temperature. Within these important functions, the epidermal permeability barrier, which controls the transcutaneous movement of water and other electrolytes, is probably the most important. This permeability barrier resides in the stratum corneum, a resilient layer composed of corneocytes and stratum corneum intercellular lipids. Since the first realization of the structural and biochemical diversities involved in the stratum corneum, a tremendous amount of work has been performed to elucidate its roles and functions in the skin, and in humans in general. The perturbation of the epidermal permeability barrier, previously speculated to be just a symptom involved in skin diseases, is currently considered to be a primary pathophysiologic factor for many skin diseases. In addition, much of the evidence provides support for the idea that various protective functions in the skin are closely related or even co-regulated. In this review, the recent achievements of skin researchers focusing on the functions of the epidermal permeability barrier and their importance in skin disease, such as atopic dermatitis and psoriasis, are introduced.

An Update of the Defensive Barrier Function of Skin

Skin, as the outermost organ in the human body, continuously confronts the external environment and serves as a primary defense system. The protective functions of skin include UV-protection, anti-oxidant and antimicrobial functions. In addition to these protections, skin also acts as a sensory organ and the primary regulator of body temperature. Within these important functions, the epidermal permeability barrier, which controls the transcutaneous movement of water and other electrolytes, is probably the most important. This permeability barrier resides in the stratum corneum, a resilient layer composed of corneocytes and stratum corneum intercellular lipids. Since the first realization of the structural and biochemical diversities involved in the stratum corneum, a tremendous amount of work has been performed to elucidate its roles and functions in the skin, and in humans in general. The perturbation of the epidermal permeability barrier, previously speculated to be just a symptom involved in skin diseases, is currently considered to be a primary pathophysiologic factor for many skin diseases. In addition, much of the evidence provides support for the idea that various protective functions in the skin are closely related or even co-regulated. In this review, the recent achievements of skin researchers focusing on the functions of the epidermal permeability barrier and their importance in skin disease, such as atopic dermatitis and psoriasis, are introduced.

Localization of Cyclooxygenase Isozymes in Skin Wound Healing in Mouse / 대한해부학회지

Cyclooxygenase (COX)-1 and -2 expressions in the incisional wound healing of mouse skin were determined by immunohistochemistry and Western blot analysis. By Western blotting, compared to normal skin, COX-2 activity was increased at days 1, 4, 8, and 12 and was maximal at 4 day after incisional wound of mouse skin whereas COX-1 was barely detectable. In normal skin, COX-1 immunostaining was observed among the basal cells of epidermis whereas COX-2 immunostaining was detected in the more differentiated, suprabasal keratinocytes. At 1~4 days after wound, COX-2 staining was particularly prominent in the inflammatory cells, and at day 8, many macrophage-like cells were stained positively. COX-2 immunoreactive fibroblast, macrophage-like cells, and newly formed vascular endothelial cells were increased in number at 12 days after incision. These data suggest that COX-2 is constitutively expressed, just as is COX-1, in epidermis and is associated with keratinocyte differentiation. In addition, these findings support the well-established role for COX-2, the prostaglandins that they generate, as mediators of inflammatory response.

The Effects of Change in Epidermal Calcium Gradient on Keratinocyte Differentiation / 대한피부과학회지

BACKGROUND: The cornified cell envelope(CE) which is formed during the terminal differentiation of keratinocytes, is a specialized structure which forms a structurally and functionally complete permeability barrier. OBJECTIVE: The purpose of our study is to investigate the effects of changes in the calcium ions on keratinocyte differentiation, especially in the expression of CE protein. METHODS:The permeability barrier of hairless mice was disrupted by tape-stripping and then exposed to the air or occluded with a water-vapor impermeable membrane, and iontophoresis was done without permeability barrier perturbation. Skin specimens were prepared for ion capture cytochemistry and immunohistochemistry with anti-K5, anti-K10, anti-K6, anti-involucrin and anti-loricrin. RESULTS: The calcium gradient which disappeared after tape-stripping was restored at 36 h after tape-stripping with air exposure and at 60 h after tape-stripping with occlusion. The change in calcium ions produced by both positive and negative iontophoresis showed recovery at 6 h. Expression of basal K5 showed a slight decrease and expression of suprabasal K10 showed an increase at 12 h with air exposure after tape-stripping, tape stripping with occlusion, and iontophoresis. Expression of K6 appeared at 12 h after tape-stripping and then in the whole epidermis at 36 h with air exposure after tape-stripping and tape stripping with occlusion and focally appeared in the stratum granulosum and stratum spinosum after iontophoresis. Expression of involucrin was increased at 12 h with air exposure after tape-stripping and iontophoresis and was extended to the lower spinous layers in tape-stripping with occlusion. Expression of loricrin in air exposure after tape-stripping, tape-stripping with occlusion and iontophoresis was similar to that of normal skin. CONCLUSION: The changes in calcium ions without permeability barrier perturbation are related to the expression of CE protein. It is thought that calcium ions in the epidermis have an important role in the terminal differentiation of keratinocytes.

The Effects of Change in Epidermal Calcium Gradient on Keratinocyte Differentiation / 대한피부과학회지

BACKGROUND: The cornified cell envelope(CE) which is formed during the terminal differentiation of keratinocytes, is a specialized structure which forms a structurally and functionally complete permeability barrier. OBJECTIVE: The purpose of our study is to investigate the effects of changes in the calcium ions on keratinocyte differentiation, especially in the expression of CE protein. METHODS:The permeability barrier of hairless mice was disrupted by tape-stripping and then exposed to the air or occluded with a water-vapor impermeable membrane, and iontophoresis was done without permeability barrier perturbation. Skin specimens were prepared for ion capture cytochemistry and immunohistochemistry with anti-K5, anti-K10, anti-K6, anti-involucrin and anti-loricrin. RESULTS: The calcium gradient which disappeared after tape-stripping was restored at 36 h after tape-stripping with air exposure and at 60 h after tape-stripping with occlusion. The change in calcium ions produced by both positive and negative iontophoresis showed recovery at 6 h. Expression of basal K5 showed a slight decrease and expression of suprabasal K10 showed an increase at 12 h with air exposure after tape-stripping, tape stripping with occlusion, and iontophoresis. Expression of K6 appeared at 12 h after tape-stripping and then in the whole epidermis at 36 h with air exposure after tape-stripping and tape stripping with occlusion and focally appeared in the stratum granulosum and stratum spinosum after iontophoresis. Expression of involucrin was increased at 12 h with air exposure after tape-stripping and iontophoresis and was extended to the lower spinous layers in tape-stripping with occlusion. Expression of loricrin in air exposure after tape-stripping, tape-stripping with occlusion and iontophoresis was similar to that of normal skin. CONCLUSION: The changes in calcium ions without permeability barrier perturbation are related to the expression of CE protein. It is thought that calcium ions in the epidermis have an important role in the terminal differentiation of keratinocytes.