Androgenetic alopecia: combing the hair follicle signaling pathways for new therapeutic targets and more effective treatment options.

Introduction: In the past 30 years, only two drugs have received FDA approval for the treatment of androgenetic alopecia reflecting a lack of success in unraveling novel targets for pharmacological intervention. However, as our knowledge of hair biology improves, new signaling pathways and organogenesis processes are being uncovered which have the potential to yield more effective therapeutic modalities. Areas covered: This review focuses on potential targets for drug development to treat hair loss. The physiological processes underlying the promise of regenerative medicine to recreate new functional hair follicles in bald scalp are also examined. Expert opinion: The discovery of promising new targets may soon enable treatment options that modulate the hair cycle to preserve or extend the growth phase of the hair follicle. These new targets could also be leveraged to stimulate progenitor cells and morphogenic pathways to reactivate miniaturized follicles in bald scalp or to harness the potential of wound healing and embryogenic development as an emerging paradigm to generate new hair follicles in barren skin.

Mechanistic insight into the activity of a sulfone compound dapsone on Propionibacterium (Newly Reclassified as Cutibacterium) Acnes-mediated cytokine production.

Acne remains as the most common skin disease in the United States even despite multiple approved topical and systemic medications available. These treatments available over the counter and by prescription can be classified based on comedolytic, antibacterial and anti-inflammatory activities and are often used in combination. Therefore, understanding of the mechanism of action is critical to achieving the best clinical outcome and synergy. One of the newer acne medications with historical data suggesting both antibacterial and anti-inflammatory activity is dapsone. In order to gain mechanistic insight into the anti-inflammatory activity of dapsone in Propionibacterium (a former genus name recently reclassified as "Cutibacterium") (P. acnes)-driven inflammation, we used two human in vitro models: primary human neonatal epidermal keratinocytes and human monocytes (THP-1). We demonstrate that dapsone suppresses production of specific cytokine signatures interleukin (IL)1α and IL8 in human epidermal keratinocytes and IL1ß, IL6, IL8 and tumor necrosis factor-α in THP-1 cells in response to P. acnes. Using THP-1 cell in vitro model, we show that IL1ß and CASP-1 are regulated by dapsone independently of NFκB activity at transcriptional and post-transcriptional levels, respectively.

Time-Restricted Feeding Shifts the Skin Circadian Clock and Alters UVB-Induced DNA Damage.

The epidermis is a highly regenerative barrier protecting organisms from environmental insults, including UV radiation, the main cause of skin cancer and skin aging. Here, we show that time-restricted feeding (RF) shifts the phase and alters the amplitude of the skin circadian clock and affects the expression of approximately 10% of the skin transcriptome. Furthermore, a large number of skin-expressed genes are acutely regulated by food intake. Although the circadian clock is required for daily rhythms in DNA synthesis in epidermal progenitor cells, RF-induced shifts in clock phase do not alter the phase of DNA synthesis. However, RF alters both diurnal sensitivity to UVB-induced DNA damage and expression of the key DNA repair gene, Xpa. Together, our findings indicate regulation of skin function by time of feeding and emphasize a link between circadian rhythm, food intake, and skin health.

The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity.

Historically, work on peripheral circadian clocks has been focused on organs and tissues that have prominent metabolic functions, such as the liver, fat, and muscle. In recent years, skin has emerged as a model for studying circadian clock regulation of cell proliferation, stem cell functions, tissue regeneration, aging, and carcinogenesis. Morphologically, skin is complex, containing multiple cell types and structures, and there is evidence for a functional circadian clock in most, if not all, of its cell types. Despite the complexity, skin stem cell populations are well defined, experimentally tractable, and exhibit prominent daily cell proliferation cycles. Hair follicle stem cells also participate in recurrent, long-lasting cycles of regeneration: the hair growth cycles. Among other advantages of skin is a broad repertoire of available genetic tools enabling the creation of cell type-specific circadian mutants. Also, due to the accessibility of skin, in vivo imaging techniques can be readily applied to study the circadian clock and its outputs in real time, even at the single-cell level. Skin provides the first line of defense against many environmental and stress factors that exhibit dramatic diurnal variations such as solar ultraviolet (UV) radiation and temperature. Studies have already linked the circadian clock to the control of UVB-induced DNA damage and skin cancers. Due to the important role that skin plays in the defense against microorganisms, it also represents a promising model system to further explore the role of the clock in the regulation of the body's immune functions. To that end, recent studies have already linked the circadian clock to psoriasis, one of the most common immune-mediated skin disorders. Skin also provides opportunities to interrogate the clock regulation of tissue metabolism in the context of stem cells and regeneration. Furthermore, many animal species feature prominent seasonal hair molt cycles, offering an attractive model for investigating the role of the clock in seasonal organismal behaviors.

Resting no more: re-defining telogen, the maintenance stage of the hair growth cycle.

The hair follicle (HF) represents a prototypic ectodermal-mesodermal interaction system in which central questions of modern biology can be studied. A unique feature of these stem-cell-rich mini-organs is that they undergo life-long, cyclic transformations between stages of active regeneration (anagen), apoptotic involution (catagen), and relative proliferative quiescence (telogen). Due to the low proliferation rate and small size of the HF during telogen, this stage was conventionally thought of as a stage of dormancy. However, multiple lines of newly emerging evidence show that HFs during telogen are anything but dormant. Here, we emphasize that telogen is a highly energy-efficient default state of the mammalian coat, whose function centres around maintenance of the hair fibre and prompt responses to its loss. While actively retaining hair fibres with minimal energy expenditure, telogen HFs can launch a new regeneration cycle in response to a variety of stimuli originating in their autonomous micro-environment (including its stem cell niche) as well as in their external tissue macro-environment. Regenerative responses of telogen HFs change as a function of time and can be divided into two sub-stages: early 'refractory' and late 'competent' telogen. These changing activities are reflected in hundreds of dynamically regulated genes in telogen skin, possibly aimed at establishing a fast response-signalling environment to trauma and other disturbances of skin homeostasis. Furthermore, telogen is an interpreter of circadian output in the timing of anagen initiation and the key stage during which the subsequent organ regeneration (anagen) is actively prepared by suppressing molecular brakes on hair growth while activating pro-regenerative signals. Thus, telogen may serve as an excellent model system for dissecting signalling and cellular interactions that precede the active 'regenerative mode' of tissue remodeling. This revised understanding of telogen biology also points to intriguing new therapeutic avenues in the management of common human hair growth disorders.

In vivo single-cell detection of metabolic oscillations in stem cells.

Through the use of bulk measurements in metabolic organs, the circadian clock was shown to play roles in organismal energy homeostasis. However, the relationship between metabolic and circadian oscillations has not been studied in vivo at a single-cell level. Also, it is unknown whether the circadian clock controls metabolism in stem cells. We used a sensitive, noninvasive method to detect metabolic oscillations and circadian phase within epidermal stem cells in live mice at the single-cell level. We observe a higher NADH/NAD+ ratio, reflecting an increased glycolysis/oxidative phosphorylation ratio during the night compared to the day. Furthermore, we demonstrate that single-cell metabolic heterogeneity within the basal cell layer correlates with the circadian clock and that diurnal fluctuations in NADH/NAD+ ratio are Bmal1 dependent. Our data show that, in proliferating stem cells, the circadian clock coordinates activities of oxidative phosphorylation and glycolysis with DNA synthesis, perhaps as a protective mechanism against genotoxicity.

Absence of ductal hyper-keratinization in mouse age-related meibomian gland dysfunction (ARMGD).

Meibomian gland dysfunction (MGD) is frequent with aging and is the primary cause of dry eye disease, the most prevalent ocular complaint. We used a novel 3-D reconstruction technique, immunofluorescent computed tomography (ICT), to characterize meibomian gland keratinization and cell proliferation in a mouse model of age-related meibomian gland dysfunction (ARMGD). To visualize the changes associated with ARMGD, 5-month and 2-year old mouse eyelids were 3-D reconstructed by ICT using antibodies to cytokeratin (CK) 1, 5 and 6 and the proliferation marker Ki67. We quantified total gland, ductal and lipid volume from the reconstructions, observing a dramatic decrease in old glands. In young glands, proliferative ductules suggest a potential site of acinar progenitors that were found to be largely absent in aged, atrophic glands. In the aged mouse, we observed an anterior migration of the mucocutaneous junction (MCJ) and an absence of hyper-keratinization with meibomian gland atrophy. Thus, we propose that changes in the MCJ and glandular atrophy through a loss of meibocyte progenitors are most likely responsible for ARMGD and not ductal hyper-keratinization and gland obstruction.

Analysis of gene expression in skin using laser capture microdissection.

Gene expression analysis is a useful tool to study the molecular mechanisms underlying skin development and homeostasis. Here we describe a method that utilizes laser capture microdissection (LCM) to isolate RNAs from localized areas of skin, allowing the characterization of gene expression by RT-PCR and microarray technologies.

The estrogen-responsive Agr2 gene regulates mammary epithelial proliferation and facilitates lobuloalveolar development.

Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2(-/-) mammary epithelial cells. Consequently, mammary glands of Agr2(-/-) females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis.

Brain and muscle Arnt-like protein-1 (BMAL1) controls circadian cell proliferation and susceptibility to UVB-induced DNA damage in the epidermis.

The role of the circadian clock in skin and the identity of genes participating in its chronobiology remain largely unknown, leading us to define the circadian transcriptome of mouse skin at two different stages of the hair cycle, telogen and anagen. The circadian transcriptomes of telogen and anagen skin are largely distinct, with the former dominated by genes involved in cell proliferation and metabolism. The expression of many metabolic genes is antiphasic to cell cycle-related genes, the former peaking during the day and the latter at night. Consistently, accumulation of reactive oxygen species, a byproduct of oxidative phosphorylation, and S-phase are antiphasic to each other in telogen skin. Furthermore, the circadian variation in S-phase is controlled by BMAL1 intrinsic to keratinocytes, because keratinocyte-specific deletion of Bmal1 obliterates time-of-day-dependent synchronicity of cell division in the epidermis leading to a constitutively elevated cell proliferation. In agreement with higher cellular susceptibility to UV-induced DNA damage during S-phase, we found that mice are most sensitive to UVB-induced DNA damage in the epidermis at night. Because in the human epidermis maximum numbers of keratinocytes go through S-phase in the late afternoon, we speculate that in humans the circadian clock imposes regulation of epidermal cell proliferation so that skin is at a particularly vulnerable stage during times of maximum UV exposure, thus contributing to the high incidence of human skin cancers.

Clock genes, hair growth and aging.

Hair follicles undergo continuous cycles of growth, involution and rest. This process, referred to as the hair growth cycle, has a periodicity of weeks to months. At the same time, skin and hair follicles harbor a functional circadian clock that regulates gene expression with a periodicity of approximately twenty four hours. In our recent study we found that circadian clock genes play a role in regulation of the hair growth cycle during synchronized hair follicle cycling, uncovering an unexpected connection between these two timing systems within skin. This work, therefore, indicates a role for circadian clock genes in a cyclical process of much longer periodicity than twenty four hours.

Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2-/- mice.

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2-/- mice that Agr2 plays important roles in intestinal homeostasis. Agr2-/- intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.

Circadian clock genes contribute to the regulation of hair follicle cycling.

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK-regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.

How the skin can tell time.

The mammalian central circadian pacemaker, which is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, synchronizes and entrains clocks found in peripheral tissues. Skin harbors an active circadian clock that is under the influence of the central clock. This clock, which probably operates in most-perhaps all-types of skin cells, may influence the regulation of several circadian physiological phenomena, including cell proliferation.