Yonsei Med J.  2013 Mar;54(2):523-528. 10.3349/ymj.2013.54.2.523.

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

• Baek JH
• Lee SE
• Choi KJ
• Choi EH
• Lee SH

• KMID: 1503920
• DOI: http://doi.org/10.3349/ymj.2013.54.2.523

Abstract

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.

Keyword

Tight junction; claudin-1; claudin-4; calcium gradient; stratum corneum permeability barrier

MeSH Terms

Animals
Calcium/*metabolism
Claudin-1/genetics/*metabolism
Claudin-4/genetics/*metabolism
Epidermis/metabolism/*physiology
Female
Gene Expression Regulation
Mice
Mice, Hairless
Permeability
RNA, Messenger/metabolism
Tight Junctions/metabolism/*physiology
Claudin-1
Claudin-4
RNA, Messenger
Calcium

Figure

• 

  Fig. 1 Effect of acute permeability barrier disruption on the expression and localization of TJ proteins, Cldn-1 and Cldn-4 in murine epidermis. Skin samples were taken at 15 min, 30 min, 1 h, 3 h, and 6 h after tape-stripping. Frozen sections (5 µm) were immunostained with Cldn-1 and Cldn-4 primary antibodies (Zymed Laboratories, San Francisco, CA, USA) and an FITC conjugated donkey anti-rabbit IgG, secondary antibody (Santa Cruz, CA, USA) and examined by confocal microscopy. Magnification ×400 (A). The levels of mRNA for Cldn-1 (B) and Cldn-4 (C) were determined using real-time PCR and normalized to that of β-actin. Cldn-1 and Cldn-4 protein expression was determined by western blot analysis of tape-stripped murine epidermis (D and E). β-actin was used as a loading control. *p<0.05 for Cldn-1 and Cldn-4 mRNA of tape-stripped epidermis compared with that of untreated control. TJ, tight junction; Cldn, claudin. FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction.

• 

  Fig. 2 Effect of acute permeability barrier disruption on the inside-out barrier function of TJ in murine epidermis. Skin samples were taken from untreated skin and barrier-disrupted skin at 30 min and 1 h after tape-stripping. Freshly obtained skin biopsies were submerged en bloc in 4% colloidal lanthanum nitrate and post-fixed in osmium tetroxide and examined by transmission electron microscope. Insets show the enlarged view of upper stratum granulosum (SG) and stratum corneum (SC) layer. Bars: 2 µm. TJ, tight junction.

• 

  Fig. 3 Effect of epidermal Ca2+ gradient modulation on the expression and localization of Cldn-1 and Cldn-4 in murine epidermis. Skin samples were taken at 15 min, 30 min, 1 h, 3 h, and 6 h after tape-stripping (TS) with/without immersion in calcium containing solution or occlusion. Frozen sections (5 µm) were immunostained with Cldn-1 (A) and Cldn-4 (B) primary antibodies (Zymed Laboratories, San Francisco, CA, USA) and an FITC conjugated donkey anti-rabbit IgG, secondary antibody (Santa Cruz, CA, USA) and examined by confocal microscopy. Magnification ×400. FITC, fluorescein isothiocyanate; Cldn, claudin.

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