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The skin barrier protects the body from water loss, allergens, and pathogens. Profilaggrin is produced by differentiated keratinocytes and is processed into filaggrin monomers. These monomers cross-link keratin filaments and are also decomposed to natural moisturizing factors in the stratum corneum for skin hydration and barrier function. Deficits in FLG expression impair skin barrier function and underlie skin diseases such as dry skin and atopic dermatitis. However, intrinsic factors that regulate FLG expression and their mechanisms of action remain unknown. Here, we show that lysophosphatidic acid induces FLG expression in human keratinocytes via the LPAR1 and LPAR5 receptors and the downstream RHO-ROCK-SRF pathway. Comprehensive gene profiling analysis further showed that lysophosphatidic acid not only induces FLG expression but also facilitates keratinocyte differentiation. Moreover, lysophosphatidic acid treatment significantly up-regulated FLG production in a three-dimensional culture model of human skin and promoted barrier function in mouse skin in vivo. Thus, our work shows a previously unsuspected role for lysophosphatidic acid and its downstream signaling in the maintenance of skin homeostasis, which may serve as a novel therapeutic target for skin barrier dysfunction., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Atopic dermatitis (AD) is a common inflammatory skin disorder. Chronic AD lesions present hyperkeratosis, indicating a disturbed desquamation process. KLK7 is a serine protease involved in the proteolysis of extracellular corneodesmosome components, including desmocollin 1 and corneodesmosin, which leads to desquamation. KLK7 is secreted by lamellar granules and upregulated in AD lesional skin. However, despite increased KLK7 protein levels, immunostaining and electron microscopy indicated numerous corneodesmosomes remaining in the uppermost layer of the stratum corneum from AD lesions. We aimed to clarify the discrepancy between KLK7 overexpression and retention of corneodesmosomes on AD corneocytes. Western blot analysis indicated abnormal corneodesmosin degradation patterns in stratum corneum from AD lesions. The KLK activity of tape-stripped corneocytes from AD lesions was not significantly elevated in in situ zymography, which was our new attempt to detect the protease activity more precisely than conventional assays. This ineffective KLK activation was associated with impaired KLK7 secretion from lamellar granules and increased expression of LEKTI in AD. Such imbalances in protease-protease inhibitor interactions could lead to abnormal proteolysis of corneodesmosomes and compact hyperkeratosis. Upregulated expression of LEKTI might be a compensatory mechanism to prevent further barrier dysfunction in AD., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Kallikrein-related peptidase 8 (KLK8) is believed to be involved in the maintenance of skin homeostasis and pathogenesis of inflammatory skin diseases. Although previous studies have shown that KLK8 is expressed around incisional wounds, the exact role of KLK8 in wound healing remains obscure. In the present study, we compared wound healing in wild-type (WT) and Klk8 gene-disrupted (kallikrein-related peptidase 8 knockout, Klk8(-/-)) mouse skin. Wound healing in Klk8(-/-) mice was hampered with defective keratinocyte proliferation, differentiation, and migration in the early stages of wound healing. Compared with the prominent induction of Klk6 and protease-activated receptor 2 (PAR2) messenger RNA, and protein in WT mice after wounding, a much lower increase was observed in Klk8(-/-) skin. After skin wounding in WT mice, increased Klk6 was detected from the upper stratum spinosum to the stratum corneum. Moreover, in WT mice, Klk6 protein was processed. PAR2 was diffusely expressed in the cytoplasm of the stratum spinosum at day 7 post wounding in WT mice. These results suggest that Klk8 is involved in the proliferation and migration of keratinocytes through the upregulation and activation of Klk6 in the early stages of wound healing, and possibly in keratinocyte differentiation associated with the upregulation and activation of PAR2 in the late stages of wound healing.

Epidermal lamellar granules (LGs) are specialized organelles that transport and secrete various molecules, including lipids, proteases, protease inhibitors, and structural proteins, thereby providing a protective barrier against the environment. Abnormalities in LG-related molecules result in severe skin diseases, but their transport mechanisms are poorly understood. We studied the distribution of Rab11, a common GTPase in recycling endosomes, in normal human epidermis. Confocal laser scanning microscopy detected Rab11 immunoreactivity in differentiated epidermal keratinocytes. Staining was strong at the apical side of each cell, a pattern commonly seen in LG-associated molecules. Around the nuclei, Rab11 was colocalized with TGN46, a trans-Golgi network marker. Rab11 was also colocalized with known LG-molecules, namely lymphoepithelial Kazal-type-related inhibitor, corneodesmosine, cathepsin D, and glucosylceramides. Immunoelectron microscopy revealed that Rab11 was widely distributed along TGN and tubular-vesicular structures containing different LG molecules. The present results suggest that Rab11 plays a role in the intracellular trafficking of various types of LG-molecule from the TGN to the cell surface.

Lamellar granules (LG) of the epidermis appear as discrete round or oblong shaped granules in classical transmission electron micrographs, but a recent cryo-transmission electron microscopy study has claimed that LG are in fact branched tubular structures. LG contain various cargoes including lipids, hydrolytic enzymes, and several other proteins. It is not known whether there are any differences in the timing of expression among them and whether they are sorted into the granules individually or collectively. In order to address these questions, we studied the expression of glucosylceramides (GlcCer), cathepsin D (CatD), corneodesmosin (Cdsn), kallikrein (KLK)7, and KLK8 in normal human epidermis using confocal laser scanning microscopy and immunoelectron microscopy. The results were consistent with the model that LG are parts of a branched tubular structure. In this structure, all the components were shown to be distributed as separate aggregates. In the trans-Golgi network (TGN), bulbous protrusions containing GlcCer, Cdsn, KLK7 and KLK8, and small CatD-positive vesicles were observed. The molecules were shown to be delivered to the apical region of granular keratinocytes. This study provides strong evidence for the sequential synthesis and independent trafficking of various LG cargoes, including for the first time CatD and KLK8, from TGN.