Acute Modulations in Stratum Corneum Permeability Barrier Function Affect Claudin Expression and Epidermal Tight Junction Function via Changes of Epidermal Calcium Gradient

Tight junction (TJ) is recognized as a second barrier of the skin. Altered expression of TJ proteins in various skin diseases characterized by the abnormal permeability barrier such as psoriasis suggests that TJ could be affected by stratum corneum (SC) barrier status. However, the physiological relationship between SC and TJ barrier remains to be investigated. Therefore, we examined the effect of SC barrier disruption on the expression of TJ proteins, claudin (Cldn)-1 and Cldn-4, and TJ barrier function in hairless mouse skin. We also investigated whether the alterations in epidermal Ca2+ affected TJ proteins expression in vivo. Repeated tape-stripping induced a sequential change of the expression and function of TJ. As early as 15-30 minutes after tape-stripping, downregulation of Cldn-1 and Cldn-4 immunoreactivity and protein level without change in mRNA level was found. This was accompanied by the abnormal leakage of lanthanum. However, by 1 hour Cldn-1 and Cldn-4 immunolocalization recovered along with normalized lanthanum permeation pattern. Moreover, the mRNA and protein levels of Cldn-1 and Cldn-4 were increased by 1 to 6 hours after tape-stripping. Inhibition of calcium loss by immersion of barrier-disrupted skin into a high Ca2+ solution prevented the dislocation of Cldn-1 and Cldn-4. Occlusion of barrier-disrupted skin delayed the restoration of Cldn-1 and Cldn-4. Our results suggest that the alteration of epidermal Ca2+ gradient caused by SC barrier perturbation affects the TJ structure and function and the faster recovery of TJ as compared to the SC barrier may imply the protective homeostatic mechanism of skin barrier.

The Effect of Cold on the Skin Barrier / 대한피부과학회지

BACKGROUND: Cold is considered an important environmental factor on the skin's barrier function. It is also known that cold can disrupt the barrier's permeability function. Historically irritant contact dermatitis develops frequently amongst fish processing workers. This means that cold may disrupt the skin barrier function. However, there are only a few studies of the effect of cold on the skin barrier. OBJECTIVE: The aim of our study was to investigate the effects of exposure to the cold on the skin's barrier permeability, calcium gradient and cornified enveloped proteins. METHODS: Hairless mice were classified into four groups as follows; tape-stripping and exposure to cold (4degrees C) for 5 hours group (Group 1), exposure to cold (4degrees C) for 5 hours after tape-stripping and occlusion with Gore-Tex group (Group 2), exposure to cold (4degrees C) for 5 hours after tape-stripping and applying with petrolatum group (Group 3), and tape-stripping and exposure at room temperature (25degrees C) for 5 hours group (Group 4). Next, rate of barrier recovery was measured, and skin of cold exposure site was taken for immunohistochemistry of cornified enveloped protein, calcium inon capture chemistry and in situ hybridization. RESULTS: Rate of barrier recovery of Group 1 was measured at -56.33%, the most delayed rate of all groups. Rate of barrier recovery of other groups were measured as follows; Group 2 (20%), Group 3 (55%) and Group 4 (41.78%). In calcium-ion capture cytochemistry, Group 1 appeared to absent calcium deposit in the entire epidermis. Group 3 appeared to the highest density of calcium in entire epidermis. In immunohistochemical staining of cornified enveloped proteins including involucrin, loricrin and filaggrin, Group 1 resulted in the lowest expression of cornified enveloped proteins. Of all groups, Group 3 appeared to have the highest expressionof cornified enveloped proteins. In situ hybridization of loricrin and filaggrin, expression were similar with results of immunohistochemical staining. CONCLUSION: Our results provide that cold impairs the skin's barrier permeability by having a negative effect on recovery of calcium gradient and differentiation of cornifed enveloped proteins. But occlusion with vapor permeable membranes such as Gore-Tex or petrolatum could improve conditions of skin barrier impaired by cold by restoring the recovery rate of calcium gradient and differentiation of cornified enveloped protein.

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.

Epidermal Barrier in Human Fetus and Newborn / 대한피부과학회지

BACKGROUND: The lipids of the stratum corneum, which originate from polar lipid precursors provided by the cells of the stratum granulosum via the exocytosis of lamellar bodies, with cornified cell envelope form competent epidermal barrier structurally and functionally. The ontogeny of the epidermal barrier is not clearly defined because of difficulty of sampling and methodology which defines epidermal lipids. OBJECT: From ultrastructural observation of skin samples obtained from human fetuses and newborn on serial developmental timings, we tried to clarify the sequential development of epidermal barrier. METHODS: Skin samples were obtained from 13 human fetuses from EGA(estimated gestational age) 10 to 23wks and 2 newborns. Specimens were observed by fluorescent confocal microscopy with nile red to identify the distribution of epidermal lipids, by transmission electron microscope with lanthanum to investigate the functional permeability barrier, with RuO4 to observe the intercellular lipid bilayer and morphology of lamellar bodies, with ion capture cytochemistry to investigate the formation of epidermal calcium gradient. RESULT: In nile red stain, the amount of epidermal lipid increased during fetal period. At EGA 23wks, the lipid distribution revealed linear and continuous pattern. In lanthanum tracer study, the electron dense tracer permeated all the intercellular space of the epidermis up to periderm and subepidermal space until EGA 21wks. At EGA 23wks, the tracer permeated intercellular space of epidermis weakly. It might be predicted that incomplete epidermal barrier is present at this time. In RuO4 stain, precursor of lamellar body was observed at EGA 15wks, and intercellular lipid bilayer was observed at EGA 16wks. As gestation increases, there was a steady increase in epidermal lipid bilayers. In ion capture cytochemistry, epidermal calcium gradient was first observed in follicular epidermis at EGA 20wks, and in interfollicular epidermis at EGA 23wks. From these results, it is concluded that the basic structures of epidermal barrier are formed at EGA 23wks, but it is not complete, and epidermal barrier arises first from follicular epidermis.

The Change of Epidermal Calcium Gradient: Confocal Laser Scanning Microscopic Approach / 대한피부과학회지

BACKGROUND: Ion capture cytochemistry(the potassium oxalate pyroantimonate method), semi- quantitatively, and proton probe X-ray microanalysis, quantitatively, have been applied to investigate the epidermal calcium distribution. OBJECTIVE: In this study, we examined the epidermal calcium distribution with confocal laser scanning microscopy(CLSM) in an attempt to evaluate the possibility of another method in epidermal calcium study. METHODS: The change of epidermal calcium distribution after barrier perturbation with tape stripping was investigated with CLSM and was compared to the results of ion capture cytochemistry. RESULTS: The calcium distribution pattern in normal murine epidermis demonstrated by CLSM show a normal calcium gradient, from a low level of calcium ions in the basal and spinous layer, followed by a progressive increase with a level of calcium ions reaching its maximal density within the outer stratum granulosum. Disruption of the epidermal barrier with tape stripping induced an immediate loss of the calcium gradient and the calcium gradient after 36h was almost normalized, in parallel with the recovery of barrier function. CONCLUSION: These results show that calcium gradient in murine epidermis after tape-stripping is restored by 36h and CLSM study can be used as a new method in epidermal calcium study.

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.

ULTRASTRUCTURAL LOCALIZATION OF EPIDERMAL CALCIUM GRADIENT BY ION CAPTURE CYTOCHEMISTRY / 解剖学报

Objective To delineate the epidermal calcium ions distribution in normal and acetone-treated nude mouse skin. Methods acetone was applied on the nude mouse skin for 20*!min with cotton balls. The ion capture cytochemistry, i.e., the potassium oxalate-pyroantimonate method was employed to localize calcium ions in nude mouse epidermis for ultrastructural examination. Results Ultrastructural examinations demonstrate that abundant calcium ions displayed within the epidermis, with a low content of calcium in the basal and spinous layers, followed by a progressive increase with calcium content and reaching its maximal density within the outer stratum granulosum. Whereas, application of acetone to the nude mouse skin caused the loss of the epidermal calcium gradient.Conclusion The present results suggest that a calcium gradient exists within normal murine epidermis. Moreover, the ion capture cytochemistry is a potentially powerful investigative tool for the demonstration of epidermal ionic environment.