Neprilysin Role for regulating Hair Cycle

Abstract

In most mammals, each hair follicle undergoes a cyclic process of growing, regressing and resting phases (anagen, catagen, telogen, respectively) called the hair cycle. Various biological factors have been reported to regulate or to synchronize with the hair cycle. Some factors involved in the extracellular matrix, which is a major component of skin tissue, are also thought to regulate the hair cycle. We have focused on an enzyme that degrades elastin, which is associated with skin elasticity. Since our previous study identified skin fibroblast elastase as neprilysin (NEP), we examined the fluctuation of NEP enzyme activity and its expression during the synchronized hair cycle of rats. NEP activity in the skin was elevated at early anagen, and decreased during catagen to telogen. The expression of NEP mRNA and protein levels was modulated similarly. Immunostaining showed changes in NEP localization throughout the hair cycle, from the follicular epithelium during early anagen to the dermal papilla during catagen. To determine whether NEP plays an important role in regulating the hair cycle, we used a specific inhibitor of NEP (NPLT). NPLT was applied topically daily to the dorsal skin of C3H mice, which had been depilated in advance. Mice treated with NPLT had significantly suppressed hair growth. These data suggest that NEP plays an important role in regulating the hair cycle by its increased expression and activity in the follicular epithelium during early anagen.

Introduction

As the hair cycle is one of the intrinsic and cyclic systems of regenerating tissue, the mechanism of its regulation is intriguing with respect to tissue reconstruction. There are many biological factors which have been reported to regulate or to synchronize with the hair cycle. Those factors can be divided into several classifications, such as hormones, growth factors, enzymes and transcription factors. Examples of enzymes include urokinase, ornithine decarboxylase, γ-glutamyl transpeptidase, alkaline phosphatase, hydroxysteroid dehydrogenase , adenyl-cyclase, aryl hydrocarbon hydroxylase , aromatase, glutathione s-transferase and nexin 1, a serine protease inhibitor. Since the hair cycle might be considered as a process of tissue regeneration, we thought that regulation of the extracellular matrix (ECM) could well affect the hair cycle. With regard to the ECM, proteoglycans have been well investigated and associated with the hair cycle. However, only a few matrix degrading enzymes have been reported to be associated with the hair cycle so far, such as type IV collagenase, matrix metalloproteinase (MMP)-2 and TIMP-1. Since we previously identified neprilysin (NEP) as dermal fibroblast elastase, we focused on the potential role of NEP in the regulation of the hair cycle.

Neprilysin is a cell surface metalloprotease, which is also known as neutral endopeptidase (NEP; EC 3.4.24.11), CD10, CALLA and enkephalinase. It is expressed in various tissues including the central and the peripheral nervous systems, normal and neoplastic lymphoid cells, and adrenal glands. It is also expressed in normal skin such as eccrine glands and sebaceous glands, in cultured keratinocytes and fibroblasts, as well as in hair follicles and hair tumors. NEP can degrade a wide variety of bioactive peptides, for example enkephalins, bradykinin, neurotensin, substance P, CGRP, natriuretic peptide [26], fMet-Leu-Phe, endothelin, and galanin. We previously showed that NEP also has elastase activity and plays important roles during intrinsic and UV-induced skin aging. We now report the role of NEP in regulating the hair cycle.

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Baseline Cortisol Levels measurement with hair in Dogs

Abstract

Cortisol measurements of hair are becoming a valuable tool in monitoring chronic stress. To further validate this approach in domestic dogs, we compared the variability of cortisol immunoreactivity in hair with that in saliva and feces of dogs housed under constant social and physical conditions. Fecal (n = 268), and hair (n = 21) samples were collected over 3 mo from 7 dogs housed in a kennel and kept for training veterinary students in minimally invasive procedures. Salivary samples (n = 181) were collected 3 times daily twice weekly during the last month of the study. Hair and salivary samples were analyzed by enzyme immunoassay and feces by radioimmunoassay. HPLC coupled with tandem mass spectrometry was used to confirm the presence of cortisol in 3 hair samples. Variability of cortisol was compared across sample types by using repeated-measures ANOVA followed by paired t tests. Within dogs, cortisol immunoreactivity was less variable in hair than in saliva or feces. Averaged over time, the variability of fecal samples approached that of hair when feces were collected at least 4 times monthly. As predicted, the stable social and environmental condition of the dogs maintained repeatability over time and supported the hypothesis that data from hair samples reflect baseline cortisol levels. These findings indicate that determining cortisol immunoreactivity in hair is a more practical approach than is using samples of saliva or feces in monitoring the effects of long-term stressors such as social or physical environments and disease progression.

Glucocorticoids are well-established biomarkers of stress in vertebrates, including birds,6 fish, and mammals. Stress—though adaptive in the short term—has been linked with impaired health and ultimately decreased fitness over prolonged periods. Consequently, measuring cortisol or other glucocorticoids over time can reveal how animals respond to prolonged stressors such as changes in their social or physical environments.

Hair is now recognized as a valuable matrix for measuring cortisol in humans and other mammals. In contrast to biologic samples that reflect circulating steroid levels integrated over seconds to hours (for example, serum, saliva, urine, feces), hair integrates steroids over the entire period of hair growth (months to years).Additional advantages are that hair can be collected relatively noninvasively, is easy to store even for long periods, and is not affected by the short-term stress of handling. In a research setting, hair could be shaved at regular intervals to monitor chronic stress. Alternatively, hair could potentially be segmented to provide a retrospective record of cortisol concentrations;this approach would be valuable for documenting the progression of endocrine diseases such as Cushing syndrome in domestic animals presenting with symptoms for the first time and in studying wild animals that can be captured only once.

The currently accepted mechanism for steroid incorporation into growing hair is via the blood vessel that feeds the follicle and potentially from surrounding eccrine and sebaceous glands. Another possibility is that the follicle itself produces cortisol locally in coordination with a broader systemic stress response, in response to localized skin irritation or as part of normal hair follicle functioning. Once inside the follicle, binding of cortisol to the hair fiber is complex and likely involves both melanins and keratin. Regardless of the mechanism, cortisol concentrations in hair have been shown to reflect well-known endocrine patterns. One study in macaques showed a correlation between cortisol levels in hair and saliva. Two other studies have examined the relationship in dogs: one compared cortisol levels in hair and feces and the other compared cortisol data from hair and saliva. In both of these studies hair was collected only once from a single dog and compared with only one other tissue.

The primary objective of the current study was to build on these previous findings by evaluating the variability of hair-based measures of cortisol in a population of dogs with a common environment and diet. We hypothesized that cortisol immunoreactivity in hair would be less variable than that in saliva and feces. In addition, we designed the experiment to allow comparison of data from hair with that of both feces and saliva. We predicted that cortisol immunoreactivity in hair would correlate positively with that in saliva and feces averaged over a corresponding time period. Finally, we used HPLC coupled with tandem mass spectrometry (LC-MS/MS) as a ‘gold standard’ for confirming the presence of cortisol in dog hair.

Resource: http://www.ncbi.nlm.nih.gov