Immunohistochemical study of 3 beta-hydroxysteroid dehydrogenase in dog's perianal sinus.

In this study, the localization of 3 beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in the wall of canine perianal sinus (PS) was determined. The 3 beta-HSD activity was found out both in the cytoplasm of cells, situated in the propria and forming clusters adjacently to apocrine glands and in the cytoplasm of some epithelial cells in apocrine cells' glands. The results obtained about the 3 beta-HSD activity allowed us to propose a role of this enzyme in PS development and possibly, in tumourogenesis.

15-prostaglandin dehydrogenase expression alone or in combination with ACSM1 defines a subgroup of the apocrine molecular subtype of breast carcinoma.

Established histopathological criteria divide invasive breast carcinomas into defined groups. Ductal of no specific type and lobular are the two major subtypes accounting for around 75 and 15% of all cases, respectively. The remaining 10% include rarer types such as tubular, cribriform, mucinous, papillary, medullary, metaplastic, and apocrine breast carcinomas. Molecular profiling technologies, on the other hand, subdivide breast tumors into five subtypes, basal-like, luminal A, luminal B, normal breast tissue-like, and ERBB2-positive, that have different prognostic characteristics. An additional subclass termed "molecular apocrine" has recently been described, but these lesions did not exhibit all the histopathological features of classical invasive apocrine carcinomas (IACs). IACs make up 0.5-3% of the invasive ductal carcinomas, and despite the fact that they are morphologically distinct from other breast lesions, there are presently no standard molecular criteria available for their diagnosis and as a result no precise information as to their prognosis. Toward this goal our laboratories have embarked in a systematic proteomics endeavor aimed at identifying biomarkers that may characterize and subtype these lesions as well as targets that may lead to the development of novel targeted therapies and chemoprevention strategies. By comparing the protein expression profiles of apocrine macrocysts and non-malignant breast epithelial tissue we have previously reported the identification of a few proteins that are specifically expressed by benign apocrine lesions as well as by the few IACs that were available to us at the time. Here we reiterate our strategy to reveal apocrine cell markers and present novel data, based on the analysis of a considerably larger number of samples, establishing that IACs correspond to a distinct molecular subtype of breast carcinomas characterized by the expression of 15-prostaglandin dehydrogenase alone or in combination with a novel form of acyl-CoA synthetase medium-chain family member 1 (ACSM1). Moreover we show that 15-prostaglandin dehydrogenase is not expressed by other breast cancer types as determined by gel-based proteomics and immunohistochemistry analysis and that antibodies against this protein can identify IACs in an unbiased manner in a large breast cancer tissue microarray making them potentially useful as a diagnostic aid.

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.

Nongastric H-K-ATPase in rodent prostate: lobe-specific expression and apical localization.

The molecular basis of active ion transport in secretory glands such as the prostate is not well characterized. Rat nongastric H-K-ATPase is expressed at high levels in distal colon surface cell apical membranes and thus is referred to as "colonic." Here we show that the ATPase is expressed in rodent prostate complex in a lobe-specific manner. RT-PCR and Western blot analyses indicate that rat nongastric H-K-ATPase alpha-subunit (alpha(ng)) mRNA and protein are present in coagulating gland (anterior prostate) and lateral and dorsal prostate and absent from ventral lobe, whereas Na-K-ATPase alpha-subunit is present in all lobes. RT-PCR analysis shows that Na-K-ATPase alpha(4) and alpha(3) and gastric H-K-ATPase alpha-subunit are not present in significant amounts in all prostate lobes. Relatively low levels of Na-K-ATPase alpha(2) were found in lateral, dorsal, and anterior lobes. alpha(ng) protein expression is anteriodorsolateral: highest in coagulating gland, somewhat lower in dorsal lobe, and even lower in lateral lobe. Na-K-ATPase protein abundance has the reverse order: expression in ventral lobe is higher than in coagulating gland. alpha(ng) protein abundance is higher in coagulating gland than distal colon membranes. Immunohistochemistry shows that in rat and mouse coagulating gland epithelium alpha(ng) protein has an apical polarization and Na-K-ATPase alpha(1) is localized in basolateral membranes. The presence of nongastric H-K-ATPase in rodent prostate apical membranes may indicate its involvement in potassium concentration regulation in secretions of these glands.

15-Lipoxygenase-2 expression in benign and neoplastic sebaceous glands and other cutaneous adnexa.

15-Lipoxygenase-2 has a limited tissue distribution in epithelial tissues, with mRNA detected in skin, cornea, lung, and prostate. It was originally cloned from human hair rootlets. In this study the distribution of 15-lipoxygenase-2 was characterized in human skin using immunohistochemistry and in situ hybridization. Strong uniform 15-lipoxygenase-2 in situ hybridization (n = 6) and immunostaining (n = 16) were observed in benign cutaneous sebaceous glands, with expression in differentiated secretory cells. Strong 15-lipoxygenase-2 immunostaining was also observed in secretory cells of apocrine and eccrine glands. Variable reduced immunostaining was observed in skin-derived sebaceous neoplasms (n = 8). In the eyelid, Meibomian glands were uniformly negative for 15-lipoxygenase-2 in all cases examined (n = 9), and sebaceous carcinomas apparently derived from Meibomian glands were also negative (n = 12). The mechanisms responsible for differential expression in cutaneous sebaceous vs eyelid Meibomian glands remain to be established. In epidermis, positive immunostaining was observed in the basal cell layer in normal skin, whereas five examined basal cell carcinomas were negative. Thus, the strongest 15-lipoxygenase-2 expression is in the androgen regulated secretory cells of sebaceous, apocrine, and eccrine glands. This compares with the prostate, in which 15-lipoxygenase-2 is expressed in differentiated prostate secretory cells (and reduced in the majority of prostate adenocarcinomas). The product of 15-lipoxygenase-2, 15-hydroxyeicosatetraenoic acid, may be a ligand for the nuclear receptor peroxisome proliferator activated receptor-gamma, which is expressed in sebocytes, and contribute to secretory differentiation in androgen regulated tissues such as prostate and sebaceous glands.

A novel aspartyl proteinase from apocrine epithelia and breast tumors.

GCDFP-15 (gross cystic disease fluid protein, 15 kDa) is a secretory marker of apocrine differentiation in breast carcinoma. In human breast cancer cell lines, gene expression is regulated by hormones, including androgens and prolactin. The protein is also known under different names in different body fluids such as gp17 in seminal plasma. GCDFP-15/gp17 is a ligand of CD4 and is a potent inhibitor of T-cell apoptosis induced by sequential CD4/T-cell receptor triggering. We now report that GCDFP-15/gp17 is a protease exhibiting structural properties relating it to the aspartyl proteinase superfamily. Unexpectedly, GCDFP-15/gp17 appears to be related to the retroviral members rather than to the known cellular members of this class. Site-specific mutagenesis of Asp(22) (predicted to be catalytically important for the active site) and pepstatin A inhibition confirmed that the protein is an aspartic-type protease. We also show that, among the substrates tested, GCDFP-15/gp17 is specific for fibronectin. The study of GCDFP-15/gp17-mediated proteolysis may provide a handle to understand phenomena as diverse as mammary tumor progression and fertilization.

Atrial natriuretic peptide and guanylin-activated guanylate cyclase isoforms in human sweat glands.

The ultracytochemical localization of membrane-bound guanylate cyclases A and C, stimulated by atrial natriuretic peptide and guanylin respectively, has been studied in human sweat glands. The results showed that the peptides stimulated guanylate cyclases A and C in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was present on the plasma membranes and on intracellular membranes involved in the secretory mechanism. In eccrine glands, the cells of the excretory duct also presented enzymatic activity on the plasma membranes. In both glands, myoepithelial cells, surrounding the secretory cells, exhibited only guanylate cyclase A activity. These localizations of enzymatic activity suggest a role for both atrial natriuretic peptide and guanylin in regulating glandular secretion.

The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol.

Human apocrine and sebaceous glands function to secrete lipids, predominantly triglycerides, fatty acids, cholesterol and its esters, and, in the sebaceous gland, squalene. The enzymes that catalyze the important regulatory steps in cholesterol and fatty acid biosyntheses, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase, respectively, were therefore studied in isolated human skin appendages, and their relevant kinetic parameters determined. The enzyme activities that were observed can account for previously described rates of incorporation of radiolabeled substrates into the appropriate lipids by glands in vitro. Reduced enzyme activities following homogenization in the presence of fluoride indicated that both of these enzymes in skin appendages are inactivated by phosphorylation. The activity of the enzyme known to catalyze this phosphorylation, the AMP-activated protein kinase, was also measured. Compactin was shown to inhibit HMG-CoA reductase in homogenates of these appendages. Conversely, incubation of whole sebaceous glands with compactin resulted in the stimulation of enzyme activity, which suggests that these appendages can respond to diminishing cholesterol levels. The effect of exogenous low density lipoprotein and 25-hydroxycholesterol on HMG-CoA reductase activity from skin appendages was investigated. HMG-CoA reductase activity in both apocrine and sebaceous glands was reduced following incubation with either low density lipoprotein or 25-hydroxycholesterol. Low density lipoprotein receptor and lipoprotein lipase mRNA expression was also detected in skin appendages. These results indicate that apocrine and sebaceous glands have the capacity to sequester dietary cholesterol and fatty acids that may have important implications for the understanding of both acne and axillary odor.

Predominance of type I 5alpha-reductase in apocrine sweat glands of patients with excessive or abnormal odour derived from apocrine sweat (osmidrosis).

High levels of 5alpha-reductase activity have been detected in human apocrine glands, and the concentration of dihydrotestosterone has been found to be higher than that of testosterone in the nuclear fraction of the skin of patients who suffer from excessive or abnormal odour derived from apocrine sweat (osmidrosis). Although these results suggest that 5alpha-reductase may play a central role in the action of androgens in the apocrine gland, the isozyme responsible is not known. We therefore assayed 5alpha-reductase type I and type II activity and mRNA expression in isolated apocrine glands from four patients with osmidrosis. When we incubated gland homogenates with [3H]testosterone, we found that the biochemical properties of the apocrine gland enzyme were consistent with those of type I 5alpha-reductase: at substrate concentrations of both 50 nmol/L and 1 micromol/L, the optimum pH was in the range 6.0-7.5, and the apparent Km was 21.1 micromol/L. The apocrine gland enzyme was inhibited by MK386, a specific inhibitor of type I 5alpha-reductase, in a dose-dependent manner, but it was hardly affected by finasteride, a specific inhibitor of type II isozyme, in that a nanomolar concentration of finasteride produced only a slight inhibition. Reverse transcriptase-polymerase chain reaction showed that the apocrine gland expressed type I 5alpha-reductase mRNA exclusively, except for a faint band of type II isozyme in a few preparations. These data indicate that the type I isozyme is the predominant form of 5alpha-reductase in the apocrine gland and may play a central role in the anabolic activity of androgens, as reported for the sebaceous gland. In addition, a small amount of type II isozyme may be expressed by mesenchymal cells that surround the apocrine glands and also contribute to their development.

Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes.

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.

Ultrastructural localization of alkaline phosphatase activity in human eccrine and apocrine sweat glands.

Alkaline phosphatase (ALP) is a membrane-bound enzyme that catalyzes the hydrolysis of inorganic and organic monophosphate esters at alkaline pH. Although the functions of ALP are poorly understood, it is believed to be involved in membrane transport. Because little is known about the functions and distribution of ALP in the sweat glands, we studied the localization of ALP in human sweat glands with light and electron microscopic enzyme cytochemistry. In eccrine sweat glands, ALP was restricted to the cell membranes of intercellular canaliculi. Luminal cell membranes of secretory cells that are in continuity with intercellular canaliculi did not show ALP activity. These results suggest that ALP participates in the production of primary sweat at intercellular canaliculi. In apocrine sweat glands, basal cell membranes of secretory cells and myoepithelial cell membranes that were in apposition with each other showed ALP activity, where as no activity was seen in eccrine sweat glands. These differences in the distribution of ALP in myoepithelial cells between eccrine and apocrine sweat glands might be related to the functional differences of these sweat glands. ALP histochemistry could help to diagnose and to determine the direction of differentiation in sweat gland tumors.

Demonstration of NADPH-diaphorase (NO-synthase) in the apocrine and eccrine skin glands of domesticated mammals.

The study demonstrates a strong enzyme histochemical and immunohistochemical reaction staining for NADPH-diaphorase/NO-synthase in the secretory cells of the apocrine glands in the hairy skin, and the eccrine glands in the foot pads of domesticated mammals. The results obtained are discussed in view of a regulatory action of the NO generated by these enzyme activities, implying a direct influence of NO on the contractile properties of glandular myoepithelial cells. In this way, a basic and simple mechanism to couple secretion production and secretion extrusion can be proposed.

Androgen metabolism by isolated human axillary apocrine glands in hidradenitis suppurativa.

Androgen metabolism was investigated in normal human apocrine glands and in those isolated from age-matched patients with hidradenitis suppurativa. Axillary glands were isolated by shearing and androgen interconverting enzyme activities were measured in cell-free homogenates by incubation with [3H] dehydroepiandrosterone, [3H] androstenedione and [3H] testosterone. The activities (pmol/mg protein/min: mean + SEM) of 3 beta-hydroxysteroid dehydrogenase delta 4-5-isomerase (10.0 +/- 1.2 vs. 5.3 +/- 0.5: n = 5) and 17 beta-hydroxysteroid dehydrogenase (58.1 +/- 4.5 vs. 35.7 +/- 5.2: n = 5) were significantly lower (P less than 0.005) in hidradenitis suppurativa, whereas 5 alpha-reductase activity (12.5 +/- 2.3 vs. 12.5 +/- 2.0: n = 5) was similar. This report suggests that hidradenitis suppurativa cannot be attributed to exaggerated activities of end-organ androgen interconverting enzymes.

Immunohistochemical demonstration of peptidylarginine deiminase in human sweat glands.

Human skin is known to contain protein-bound citrulline. This is the product of enzymatic deimination of arginine residues catalyzed by peptidylarginine deiminase. We probed frozen sections of human skin with a rabbit antiserum raised to rat skeletal muscle peptidylarginine deiminase using the avidin-biotin-peroxidase complex technique. This led us to interesting findings. No staining was observed in epidermis, inner root sheaths of hair follicles, sebaceous glands, and hair erector muscle. However, we noticed specific staining of the cytoplasm of secretory and myoepithelial cells of both eccrine and apocrine sweat glands. The procedure also stained neoplastic cells present in specimens dissected from extramammary Paget's disease. The data mean that peptidylarginine deiminase may be used as a new marker in the classification of skin neoplasms showing sweat gland differentiation. Possible localization of multiple types of peptidylarginine deiminases in human skin is discussed.

Immunohistochemical study of lysozyme in human apocrine glands.

The localization of lysozyme in human apocrine glands was studied by adopting the avidin-biotin-peroxidase complex method. The results showed that the glands were enriched with lysozyme. The apical portion of secretory cells was most heavily stained. Eccrine glands did not stain for lysozyme. Although apocrine glands have been regarded as having no apparent function in man, it is suggested in the present report that they may have an excretory bactericidal role.

Expression of gamma-glutamyl transpeptidase in normal and neoplastic epithelial cells of human skin: a marker for distinguishing malignant epithelial tumours.

The distribution of gamma-glutamyl transpeptidase (GGT) activity was studied histochemically in benign and malignant epithelial tumours of human skin. We found that GGT activity in normal skin was confined to the secretory portion of the eccrine and apocrine glands and to the inner root sheath of the hair follicles. In Bowen's disease and actinic keratosis, GGT activity was noted focally in areas where atypical cells were observed. In extramammary Paget's disease, GGT activity was found only in large round cells scattered among GGT negative epidermal cells. No GGT activity was observed in basal cell epitheliomas or benign epithelial tumours, while squamous cell carcinoma and eccrine porocarcinoma exhibited intense GGT activity. Our study suggests that GGT may be useful as a histochemical marker for distinguishing malignant tumours from benign epithelial tumours in human skin.

The immunohistochemical localization of lysozyme in human axillary apocrine glands.

Lysozyme has been observed in intraluminal secretory products of apocrine glands in specimens of normal human axillary skin. Lysozyme was also observed in an occasional apocrine secretory cell, as well as in leukocytes within vascular lumina and dermal histiocytes. Lysozyme was not observed in sebaceous glands, eccrine glands, or cells of the epidermis. These observations support an epithelial origin of cutaneous lysozyme and suggest a means of further characterization of the origin and/or differentiation of tumors of appendageal origin.

Activity of 17 beta-hydroxysteroid dehydrogenase in various tissues of human skin.

The activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) was assayed in various tissues microdissected from the freeze-dried human skin of fourteen subjects. The apocrine sweat gland, sebaceous gland and hair follicle possessed a high activity of 17 beta-HSD. The enzyme activity was negligible in the epidermis, except that the scalp epidermis showed much the same activity as the hair follicle. The demis showed variable activity because of contamination with other components.