Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development.

Caspase-14 is a seemingly non-apoptotic caspase involved in keratinocyte differentiation and cornification of the skin. Keratin-19 is an epithelial marker and a potential marker of epidermal stem cells that is expressed during human fetal skin development. We examined the immunohistochemical expression of caspase-14 in relation to CK-19 in the human fetal skin during development and perinatally, to assess their role in human skin maturation. Skin samples were received at autopsy. In the fetal epidermis, caspase-14 was predominantly expressed in the more differentiated layers, gradually disappearing from the basal layer toward term. By contrast, keratin-19 expression gradually decreased with epidermal maturation through gestation (rho = -0.949; p = 0.0001) and was a marker of the germinative layers. Keratin-19 was preserved in scarce basal cell nests at term and postnatally. Caspase-14 and keratin-19 were inversely expressed in the differentiating epidermal layers through gestation (p < 0.0001). Concerning the appendages, in hair follicles and sebaceous glands, caspase-14 located preferentially in the more differentiated layers of the inner root sheath, whereas keratin-19 was expressed in the outer sheath. Eccrine sweat glands showed a variable pattern of caspase-14 and keratin-19 expression. In conclusion, caspase-14 emerged as a marker of human skin differentiation during development, while keratin-19 marked the germinative epithelial layers in the fetal epidermis and appendages and possibly the nests of epidermal stem cells.

Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum.

We have previously presented evidence that two human kallikrein-related peptidases, KLK5 (hK5, stratum corneum tryptic enzyme, SCTE) and KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), which are abundant in the stratum corneum, may be involved in desquamation. Since we had noted that not all trypsin-like activity in the plantar stratum corneum could be ascribed to KLK5, we set out to identify other skin proteases with similar primary substrate specificity. Here we describe purification of a protease identified as KLK14 from plantar stratum corneum, and show that this enzyme may be responsible for as much as 50% of the total trypsin-like activity in this tissue, measured as activity towards a chromogenic substrate cleaved by a wide variety of enzymes with trypsin-like specificity. This was in spite of very low levels of KLK14 protein compared to KLK5 and KLK7. KLK14 could be detected by immunoblotting in normal superficial stratum corneum of all individuals examined. The majority of KLK14 in the plantar stratum corneum is present in its catalytically active form. KLK14 could be immunohistochemically detected in sweat ducts, preferentially in the intraepidermal parts (the acrosyringium), and in sweat glands. The role played by this very efficient protease under normal and disease conditions in the skin remains to be elucidated.

Peptidylarginine deiminase isoforms are differentially expressed in the anagen hair follicles and other human skin appendages.

Peptidylarginine deiminases (PAD) catalyze the conversion of arginine residues to citrullines. Five isoforms are known that present distinct tissue locations. In the epidermis, like in the skin, only PAD1, 2, and 3 are expressed. Their pattern of expression in skin appendages is not known. Here, confocal microscopy analysis using highly specific antibodies demonstrated that PAD1 and 3 are expressed in human anagen hair follicles, PAD1 and 2, in arrector pili muscles and sweat glands, whereas no PAD were detected in sebaceous glands. PAD1 was detected in the cuticle and the Huxley layer of the inner root sheath (IRS), and in the companion layer. PAD3 was localized in the medulla, and in the three layers of the IRS. Using anti-modified citrulline antibodies, we also showed that deiminated proteins appeared in the lower part of the IRS, first in the Henle layer, then in the cuticle, and finally in the Huxley layer. Our data demonstrate that PAD3 is the enzyme that deiminates trichohyalin in the medulla and the Henle layer, indicate that PAD1 and 3 are involved in the hair follicle program of differentiation, and suggest a role for PAD1 and 2 in the physiology of sweat glands and arrector pili muscles.

Impaired 11-beta hydroxysteroid dehydrogenase type 2 activity in sweat gland ducts in human essential hypertension.

The enzyme 11-beta hydroxysteroid dehydrogenase type 2 plays a major role in blood pressure regulation. It metabolizes glucocorticoid hormones into derivatives with low affinity for the mineralocorticoid receptor, preventing its permanent occupancy by circulating cortisol, which is 100- to 1000-fold more abundant than aldosterone in the plasma. Inactivating mutations of the enzyme result in severe hypertension, as seen in children with apparent mineralocorticoid excess syndrome. In patients with essential hypertension, however, attempts to evidence enzyme deficiency have been inconclusive. In this pilot study, its catalytic activity was measured directly in aldosterone-sensitive sweat gland ducts collected from skin biopsy samples of 10 male normotensive subjects and 10 subjects with essential hypertension (more than 140 to 90 mm Hg) with no sign of hypermineralocorticism. Isolated ducts were assayed for nicotinamide-dinucleotide-dependent dehydrogenase activity (transformation of tritiated corticosterone into tritiated-11 dehydrocorticosterone, as measured by high-pressure liquid chromatography). Hypertensive patients exhibited significantly lower 11-beta hydroxysteroid dehydrogenase type 2 activity (9.7+/-4.7 femtomoles per 3 mm length of duct and per 10 minutes incubation, median+/-SD) than did normotensive subjects (15.9+/-2.6). Such defect was undetectable using the classical urinary corticosteroid metabolism indexes, probably because of compensatory mechanisms. Relations between these findings and blood pressure levels should benefit from direct enzyme measurements in the vasculature. In conclusion, this cross-sectional study points to partial 11-beta hydroxysteroid dehydrogenase type 2 deficiency as a novel feature of essential hypertension, which should stimulate search for new signaling pathways and therapeutical targets.

PACAP activated adenylate cyclase in human sweat glands. An ultracytochemical study.

The ultracytochemical localization of adenylate cyclase (AC) was studied after stimulation with pituitary adenylate cyclase activating peptide (PACAP) in human sweat glands. PACAP stimulated AC in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was associated with membranes involved in the secretory mechanism. In both glands, the cells of the excretory duct and myoepithelial cells presented AC activity. These localizations of enzymatic activity suggest a role for PACAP in regulating glandular secretion.

Carbonic anhydrase activity and sweat gland morphology in trained and untrained Standardbred trotters.

Sweat gland morphology and carbonic anhydrase (CA) distribution was studied after exercise in trained and untrained horses using a histochemical technique and light microscopic image analysis. Three trained and 3 untrained Standardbred trotters performed an exercise test (20 min trot at 6 m/s with 5 min walk at 1.8 m/s in the beginning and end) on a high-speed treadmill at 35 degrees C. Skin biopsies were taken before exercise and after trot. The fluid loss after exercise was 10, 12 and 12 g/kg bwt in the untrained horses and 4, 6 and 11 g/kg in the trained. Trained horses had a larger cell area than untrained after exercise, which might be related to an increase in secretory capacity. The area of the cell occupied by CA was independent of training status, but increased with exercise in both groups. The CA activity was higher in untrained animals and increased after exercise in both groups. The change in CA during exercise might be a response to an increasing demand for HCO3- secretion during sweat formation. Therefore, the sweat gland undergoes morphological changes due to stimuli such as heat, exercise and training, but species differences are evident. To our knowledge, no one has previously studied the influence of training on the morphology of the equine sweat gland.

The distribution of choline acetyltransferase- and acetylcholinesterase-like immunoreactivity in the palmar skin of patients with palmoplantar pustulosis.

The distribution of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in involved skin in patients with palmoplantar pustulosis (PPP) and in normal palmar skin in healthy non-smokers and smokers has been studied by immunohistochemistry, especially in relation to the sweat gland apparatus. The sweat gland and its duct showed ChAT- and AChE-like immunoreactivity (LI) of varying intensity in all three groups and with stronger reactivity than in the epidermis. ChAT-LI was present in the coil and in the duct except in the corneal layer. Smokers and patients with PPP displayed significantly fewer ChAT+ acrosyringia than non-smokers. In the patients with PPP, the granulocytes in the pustules and in the papillary dermis displayed ChAT-LI. Western blot analysis of granulocytes from peripheral blood from healthy donors confirmed the presence of ChAT-like proteins in large amounts in neutrophils and small amounts in eosinophils. AChE-LI of varying intensity was found in all parts of the sweat gland apparatus in all three groups. The strongest AChE-LI in the acrosyringia was seen in the lowest part of the stratum corneum, where the PPP pustules are located. No significant differences in staining pattern or intensity were found between the coils, nerve fibres surrounding the coils or ducts. The number of mast cells in the papillary dermis was about four times larger in the patients with PPP than in the control subjects. AChE-LI was observed in about 25% of the mast cells in non-smoking control subjects and in patients with PPP, but only in 10% of those in the smoking control subjects. Our findings indicate that the (non-neuronal) cholinergic system may be involved in cutaneous inflammatory processes.

Basement membrane zone remodeling during appendageal development in human fetal skin. The absence of type VII collagen is associated with gelatinase-A (MMP2) activity.

Epithelial cell adhesion, migration, and differentiation are controlled by interactions at the basement membrane zone (BMZ). Type VII collagen is the major collagenous component of anchoring fibrils that are essential for the attachment of the epidermis to the dermis. Gelatinase A (MMP-2) is believed to be necessary for the degradation of type VII collagen. In this study we have examined the in vivo distribution of type VII collagen and gelatinase A (Gel A) in the developing human epidermis and its appendages. At 13-15 wk of gestation a marked decrease in type VII collagen immunoreactivity was seen in the BMZ surrounding invading appendageal buds; however, type VII collagen mRNA was strongly expressed in the budding epidermal keratinocytes adjacent to the BMZ. At these stages, Gel A-positive mesenchymal-like cells were found scattered throughout the stroma with numerous Gel A-containing cells in direct contact with the developing appendageal buds. In situ zymography was used to show Gel A-activity in vivo. Gel A-mediated lysis was present at the interface between the appendageal buds and the underlying BMZ. By 20-25 wk of gestational age, immunostaining for type VII collagen protein was absent from the BMZ surrounding the distal portion of invading appendageal epithelial cords of both hair follicles and sweat glands. In contrast, type VII collagen mRNA was present in the basal keratinocytes adjacent to the BMZ surrounding the distal portion of these invading appendageal epithelial cords. At these stages Gel A-positive cells were present in the stroma directly adjacent to the distal portion of developing appendageal cords that lacked type VII collagen. In situ zymography showed zones of Gel A-mediated stromal lysis at the distal portion of developing appendageal cords. Interestingly, no differences were seen in the distribution of type IV collagen in the BMZ of both budding and resting fetal epidermis. These observations suggest that the absence of type VII collagen protein correlates directly with the presence of Gel A-activity at the BMZ. Gel A appears to play a major role in appendageal development and contributes to remodeling of the BMZ during fetal skin morphogenesis.

An immunohistochemical study of beta1,4-galactosyltransferase in human skin tissue.

An immunohistochemical investigation of beta1,4-galactosyltransferase (beta1,4-GalT) on human skin tissue was performed on formalin-fixed paraffin-embedded sections using a monoclonal antibody, MAb8628, which specifically recognizes a protein moiety of human beta1,4-GalT. Distribution of the galactose beta1,4-N-acetylglucosamine (Gal beta1,4GlcNAc)-R epitope was also detected by staining with Ricinus communis agglutinin (RCA) 120. The beta1,4-GalT was observed to be localized at the perinuclear region of epidermal keratinocytes. The fine localization was also observed at the supranuclear region in the cells of apocrine glands, eccrine ducts and glands. The positive staining with RCA 120 was well colocalized with the cells expressing the beta1,4-GalT. An electron microscopic study revealed that positive signals of beta1,4-GalT definitely reside in the Golgi apparatus. No immunoreactivity was observed in any other intracellular structure or on the cell surface. These findings strongly indicated that the beta1,4-GalT is the major enzyme responsible for the Gal beta1,4GlcNAc-R epitope synthesis in human skin tissue.