Dr Burkhard Poeggeler, Department of Dermatology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany, Tel.: +49 451 500 2869, Fax: +49 451 500 6595, e-mail: email@example.com
Abstract: Here, we examine the currently available information which supports that the adipokine, leptin, is a major player in the biology and pathology of mammalian skin and its appendages. Specifically, the potent metabolic effects of leptin and its mimetics may be utilized to improve, preserve and restore skin regeneration and hair cycle progression, and may halt or even partially reverse some aspects of skin ageing. Since leptin can enhance mitochondrial activity and biogenesis, this may contribute to the wound healing-promoting and hair growth-modulatory effects of leptin. Leptin dependent intracellular signalling by the Janus kinase 2 dependent signal transducer and activator of transcription 3, adenosine monophosphate kinase, and peroxisome proliferator-activated receptor (PPAR) gamma coactivator/PPAR converges to mediate mitochondrial metabolic activation and enhanced cell proliferation which may orchestrate the potent developmental, trophic and protective effects of leptin. Since leptin and leptin mimetics have already been clinically tested, investigative dermatology is well-advised to place greater emphasis on the systematic exploration of the cutaneous dimensions and dermatological potential of this pleiotropic hormone.
The hormone leptin is best known as a lipostatic signal serving as a key regulator of food intake. Obesity and ageing can lead to increased leptin concentrations which are, due to dysfunctional leptin signalling, however, not associated with reduced food intake and subsequent weight loss. The loss of metabolic control may have broad physiological consequences affecting all body tissues, including its largest organ: the skin. The current Viewpoint article aims at stimulating wider interest within the skin research community in the potent trophic effects that leptin may exert in cutaneous biology. After a brief synthesis of the relevant background on leptin biology, we critically examine the published evidence which suggests that leptin may be a major regulator of skin and hair biology under physiological and pathological conditions, with a potential key role in tissue regeneration events. Exploring the ‘leptin–skin connection’ in more detail, we emphasize the potential role of leptin in wound healing and hair growth control, and discuss particularly intriguing, new perspectives of leptin biology for dermatological research.
The adipokine leptin
Leptin is a 16-kDa protein and as a hormone or cytokine predominantly synthesized and secreted by adipocytes, including those in the subcutis. The discovery of this adipocyte-secreted hormone, which is named after its main biological effect (from the Greek word leptos = lean), challenged the simple paradigm of adipocytes as mere fat storage cells. Subcutaneous and visceral fat is the primary source of this protein encoded by the obese (ob) gene, which, together with other adipokine signals originating from this tissue, is considered to constitute one of the most important endogenous regulators of body weight in particular and energy metabolism in general (1,2). Leptin secretion is positively correlated with body mass and therefore obesity is accompanied by high circulating leptin levels (2).
Investigations in mice with a mutation in the diabetes (db) locus, the gene encoding for the leptin receptor, indicated its decisive role in mediating the central and peripheral functions of leptin (2). Leptin and its most prominent intracellular signal transduction pathway signal transducer and activator of transcription 3 (STAT3) thus play a key role in regulating fat metabolism and body mass (3). The adipokine leptin acts as a metabolic hormone on the hypothalamus to suppress food intake and increase energy expenditure (3).
Multiple isoforms of the leptin receptor (ObR) have been identified (2–4). The Ob receptors are members of the interleukin-6 (IL-6) receptor family of the class I cytokine receptor superfamily (3). ObR is most closely related to gp130, the signal transducing membrane protein of the IL-6 signalling complex (IL-6-SC), the leukaemia inhibitory factor (LIF) receptor and the granulocyte colony-stimulating factor (G-CSF) binding site (2). Ob receptors are generally classified as the short (ObRa, ObRc, ObRd and ObRf), soluble (ObRe) and long (ObRb) forms (2–4). The leptin receptor itself has no intrinsic enzymatic activity and instead, ligand-induced signalling depends on the JAK2. It is generally believed that the long form constitutes the main mediator of the physiological actions of leptin in controlling feeding and energy balance (2–4), because only the long form of the leptin receptor has been shown to be able to activate all known downstream signalling cascades (Fig. 1).
The short forms ObRa and ObRc, which are abundantly expressed on central nervous micro vessels, are assumed to act as transport proteins for leptin across the blood brain barrier (2,4). Furthermore, ObRa may be involved in removal and degradation of leptin (2,4). The soluble form of the receptor, ObRe, which is lacking a transmembrane section, may constitute a leptin binding variety that mediates the bioavailability of leptin (2,4).
Leptin-dependent STAT3 and adenosine monophosphate kinase (AMPK) signalling can induce and orchestrate peroxisome proliferator-activated receptor (PPAR) gamma coactivator (PGC) and PPAR signalling to support mitochondrial function and integrity (5–11). The mitochondrial iron sulphur cluster N2 in complex I and the cytochrome-c-oxidase unit 5A in complex IV seem to constitute major targets of leptin in stimulating and maintaining mitochondrial energy metabolism and oxidative phosphorylation (7,10,11).
When contemplating the role of leptin in skin biology, it is important to keep in mind that central and peripheral signalling induced by leptin can greatly differ. For example, in the hypothalamus, AMPK activity is downregulated by leptin, reducing neuronal activity and feeding behavior, whereas in subcutaneous and visceral adipocytes as well as in other peripheral tissues AMPK activity is enhanced to stimulate fat burning, energy metabolism and metabolic activity (12–15).
With advancing age, progressive leptin resistance (characterized by downregulation and dysfunction of STAT3, AMPK, PGC and PPAR signalling) severely compromises the regeneration of mitochondria and thereby contributes greatly to the age-related deterioration of energy metabolism (6,7,12–18). Conversely, activation of the endogenous leptin signalling pathways and related transcription factors which enhance mitochondrial proliferation and metabolic capacity can restore and even reverse age-dependent deleterious changes such as mitochondrial dysfunction and the universal bioenergetic decline (10,11,19–22).
The trophic and protective signalling by leptin appear to converge on mitochondrial metabolism. Leptin is a very potent endogenous mitochondrial metabolism modifier that can greatly enhance activity and efficacy of oxygen and energy utilization (6,7,10,11,22). Leptin, therefore, has been proposed to play a key role in metabolic adaptation and regulation (6,7,10–15). The profound effects of leptin in supporting, improving and maintaining mitochondrial physiology by enhancing electron flow, proton potential and oxidative phosphorylation at near physiological concentrations in the nanomolar range (10,11,22) designate leptin to serve as a key metabolic regulator that can act as a trophic and protective factor of unique potency – in addition to its well established neuroendocrine functions as a chief controller of food intake (7,10–15).
The spectrum of leptin functions
Circulating leptin produced by adipose tissue communicates the levels of fat stores in the periphery to the central nervous system (CNS) in order to limit food intake and permit energy expenditure (2,3). The hormone acts specifically via its receptor and specific intracellular signalling pathways in the periphery and the CNS to regulate and maintain energy balance and metabolism (Table 1). Signalling by leptin can thereby play a decisive role in metabolic control and orchestrates adaptive responses that sense the nutritional state of the organism and the availability of endogenous energy resources (2,3,23).
Table 1. Major intracellular signal transduction pathways, mitochondrial mediators, and target genes of leptin
AMPK, adenosine monophosphate kinase; CART, cocaine and amphetamine related transcript; C-FOS, cellular FBJ (a virus named after its discoverers, Finkel, Biskins, and Jinkins) murine osteosarcoma viral oncogene homolog; ERK1/2, extracellular signal-regulated kinase 1/2; JAK2, Janus kinase 2; PGC, gamma coactivator; PI3K, phosphatidylinositol-3 phosphate kinase; POMC, pro-opiomelanocortin; PPAR, peroxisome proliferator-activated receptor; SOCS3, suppressor of cytokine signalling 3; STAT3, signal transducer and activator of transcription.
In obesity, leptin resistance with downregulated STAT3 signalling is associated with enhanced body weight resulting in metabolic inefficiency, with all the features of premature ageing (18). A future regenerative medicine may be based on preserving, restoring and supporting the metabolic control by leptin (3,6,7,10–12,23). Enhanced leptin signalling can retard the progression of ageing (7) and a cutaneous gene therapy approach using leptin expressing keratinocyte grafts was highly successful in correcting leptin deficiency with the ob/ob phenotype (24), whereas overexpression of leptin in keratinocytes of transgenic mice in which leptin cDNA overexpression was driven by keratin K5 gene regulatory sequences resulted in a lack of specific skin phenotype but induction of early leptin resistance (25).
Although the subcutis with its massive stores of adipocytes represents the dominant morphological component of mammalian skin, subcutaneous adipocytes in contrast to their visceral counterparts have been previously investigated mainly in the biological context of energy storage, physiological buffer or thermoregulation and not as a tissue from which important neuroendocrine signals and metabolic regulators like leptin can originate (26,27). Since then, this outdated view has rapidly evolved into a much more complex perspective by the fast growing fields of adipobiology and adiposcience (26) suggesting that the skin and the hair follicle are a source and target of the auto-, para- and endocrine effects of adipokines such as leptin (26–29). Skin regeneration and hair growth are affected by leptin-dependent signalling, which induces enhanced cell proliferation mediated by STAT3 (28,29). These observations indicate that leptin may exert important biological effects well extending its classical role as an endogenous endocrine regulator of food consumption (Fig. 2).
Leptin and the skin
Reportedly, leptin is predominantly synthesized in adipocytes, including subcutaneous adipocytes (26). However, synthesis of leptin and its receptors has also been detected in human and mice fibroblasts and keratinocytes (28–33). In fact, leptin and its full length receptor is present in the human epidermis at the gene and protein level (28,34,35). Leptin and leptin receptor expression in the human skin was validated and confirmed by real-time quantitative PCR (35).
Using the polyclonal Ob (A-20) sc-842 antibody provided by Santa Cruz Biotechnology as well as adequate positive and negative controls, strong leptin-like immunoreactivity can indeed be observed in acetone-fixed cryosections of human scalp skin. With this immunostaining method, the most prominent leptin-like immunoreactivity is seen in keratinocytes of the basal and suprabasal layer of the epidermis, while lower-intensity specific immunoreactivity is seen in endothelial cells, fibroblasts and adipocytes of the dermis (Fig. 3).
To our knowledge, no immunohistochemical characterization of the canonical non-existent expression of functional ObRb in db/db or leptin in ob/ob mice were performed as negative controls for the expression of these peptides in the rodent skin.
Leptin is produced in significant amounts by cultured human fibroblasts (31), and its synthesis and release can be further stimulated by insulin (31), indicating that the latter may regulate fibroblast-derived leptin secretion (31). Elegant studies using human skin-mice chimeras have demonstrated that leptin synthesized and secreted from transplanted human skin can contribute significantly to the levels of systemically circulating hormone indicating that this tissue pool may not only exert local auto- and paracrine, but also systemic endocrine effects (28). Numerous studies have unequivocally confirmed that leptin and STAT3 are linked to cell differentiation, proliferation, migration and survival in the skin with pronounced effects on angiogenesis, blood flow and tissue perfusion (30,36–49). Leptin is a potent modulator of innate and adaptive immunity and may upregulate antimicrobial defenses in human skin, e.g. by stimulating the expression of human β-defensin-2 expression (50). Leptin has also been shown to induce the expression of interleukins in the skin, particularly that of interleukin-8 (IL-8) (51,52). The diversity of leptin-dependent signalling in the skin is illustrated by the fact that the hypoxia-inducible factor-1α, which controls the expression of multiple different genes, including that of key regulators of angiogenesis and wound healing (49), is also upregulated by leptin (49).
Local leptin synthesis and secretion is strongly upregulated after skin injury (28), and leptin-deficient animals showed severely impaired and delayed wound healing (24,28,36). In vitro, leptin exerts a specific mitogenic response in keratinocytes that may be responsible for the proliferative processes induced by leptin in skin in vivo (28,36,47,48). Leptin can also act as a potent angiogenic factor on endothelial cells and may thereby promote wound healing-associated angiogenesis (26,36,38–40,49). Auto- and paracrine stimulation by leptin stimulates keratinocyte proliferation and epithelialization as well as fibroblast proliferation and collagen synthesis, resulting in accelerated wound repair and skin regeneration (28,30,32,53). Leptin deficiency and insulin resistance are associated with a prominent impairment of wound healing in the skin (18,24,36,48,49). Correction of metabolic disorders such as hyperlipidaemia and hyperglycaemia is associated with a normalization of the response to leptin and insulin, with complete recovery of the ability of the skin to regenerate and repair (24,36,48,49).
Leptin and the hair follicle
Strong leptin and leptin receptor mRNA and protein expression were detected in murine fetal hair follicles, suggesting that leptin may already be involved in the control of hair follicle morphogenesis (54,55), even though this remains to be confirmed. The development of epithelial structures such as hair follicles may be dependent on leptin signalling since phosphorylated components of active intracellular signalling cascades were consistently observed (29,33). Leptin and its receptor are also expressed by human hair follicles (33). Investigating several human follicular papilla cell lines, Iguchi et al. detected that these, but not neonatal human dermal fibroblasts, expressed leptin mRNA and produced significant amounts of leptin in vitro. By immunohistochemistry and in situ hybridization, both leptin protein and mRNA were found in the hair matrix and in the inner root sheath as well as in the follicular dermal papilla. The intracytoplasmic signal sequence of the leptin receptor was detected in the follicular papilla fibroblasts by immunohistochemistry, while the long isoform of the leptin receptor transcripts were found in the investigated human follicular papilla cell lines. We have recently reinvestigated the presence of leptin-like immunoreactivity in human hair follicles and can largely confirm these findings: High levels of leptin expression were seen in the dermal papilla, the matrix keratinocytes, and the inner and outer root sheath, as well as in the connective tissue sheath of human scalp follicles (Fig. 4a,b).
Interestingly, e.g. interleukin-1 beta, tumor necrosis factor alpha, interferon-gamma, epidermal growth factor (EGF), basic fibroblast growth factor, and transforming growth factor beta1, but not vascular endothelial growth factor, hepatocyte growth factor (HGF), keratinocyte growth factor, and insulin-like growth factor 1, significantly downregulated leptin production by these human hair follicle papilla fibroblast lines (33). This suggests autocrine functions of leptin signalling in human hair biology, which appear to be under the control of a number of mediators that are well-appreciated to play a role in hair growth control.
Leptin may even play a critical role in hair cycle control. This has been suggested most recently in a meeting report of hair phenotype analyses in leptin receptor-deficient db/db mice (29): Whereas the back skin pelage hair follicles of 5-week-old wildtype mice were already in late stages of the first anagen phase, entry into this phase of hair growth was delayed in the leptin-receptor deficient mice until postnatal day-40. These authors also found that the anagen phase of hair growth in mice can be induced by leptin and biologically active leptin fragments through specific receptor-mediated mechanisms (29).
It has been demonstrated that leptin induces STAT3-dependent signalling in human keratinocytes (29). Although transgenic mice whose epidermal and follicular keratinocytes lack functional STAT3 are viable and display seemingly normal skin and hair follicle morphology, both, hair follicle cycling and wound healing are severely compromised (41–44): The mutant mice show sparse hair and develop skin ulcers with age. Skin regeneration and keratinocyte migration in STAT3-deficient mice is retarded, just like their hair cycle progression. Spontaneous ulcer development and sparse hair in STAT3-disrupted mice may be due to an inability of bulge-derived epithelial hair follicle stem cells and/or their progeny to migrate, which is an essential requirement for normal anagen development (41–44). Though STAT3 signalling is utilized by other mediators besides leptin such as cytokines of the IL-6 family like IL-6, IL-11, LIF, ciliary neurotrophic factor (CNTF), oncostatin M and cardiotropin1 and non-cytokine ligands such as EGF, platelet derived growth factor (PDGF), HGF, and G-CSF (41), this strikingly resembles the phenotype of leptin receptor-deficient db/db mice (29).
Skin cancer and other skin diseases
Impaired anagen induction in STAT3-deficient mice goes along with pronounced dermal fibrosis and subcutaneous atrophy, associated with dense inflammatory cell infiltrates in the subcutis (41–44). This raises the question whether insufficient leptin-dependent signalling may be involved in fibrosing dermatoses, such as scleroderma. A decreased serum level of leptin has indeed been observed in patients with systemic sclerosis (56). Intriguingly, STAT3-disrupted mice have a lower incidence of skin cancer, whereas transgenic animals with a constitutive active form of STAT3 may develop squamous cell carcinoma with a shorter latency (44), possibly due to the pro-proliferative response to leptin. Also, individuals at a higher risk of developing psoriasis may at least initially suffer from an enhanced leptin dependent STAT3 signalling, and exposure to enhanced levels of leptin may trigger or aggravate psoriasis, because of the proinflammatory effects of leptin (34,35,44). It is a common misconception, however, that hyperleptinemia is associated with enhanced leptin signalling. In fact, high leptin levels are often associated with a diminished response to the hormone as in leptin resistance (18).
Given that the skin is now recognized as a metabolically and endocrinologically highly active organ which is not only a prominent target for multiple endocrine signals, but also produces a wide range of hormones, neuropeptides, and neurotransmitters (57), one of the most fascinating challenges in future cutaneous leptin research is to determine the cross-regulation and signalling interactions between leptin and its receptors on the one hand, and additional players in cutaneous (neuro-)endocrinology on the other (58–60). Particularly instructive leads in this respect arise from the hair follicle.
For example, insulin – a major promoter of human hair follicle growth (61) – is a potent modulator of leptin signalling, in the skin, the hair follicle and the brain (31,33,36,62). Also, both the thyroid hormones triiodothyronine (T3) and thyroxine (T4), which have long been appreciated to alter skin physiology, including human hair growth and pigmentation (63), and thyrotropin stimulating hormone (TSH), for which human hair follicles have recently been identified as a direct target (64), often act synergistically with leptin in enhancing mitochondrial energy metabolism similar to thyrotropin releasing hormone, the factor that triggers TSH secretion (65). Furthermore, human hair follicles express prolactin, which regulates hair follicle cycling (66), while systemic prolactin levels are recognized to render target tissues refractory and resistant to leptin-mediated effects (67,68). Since cannabinoids have been shown to act in an antagonistic manner to leptin signalling and are inhibitors of the mitochondrial energy metabolism (69,70), that reduce proliferation and induce apoptosis in the human hair follicle and in human sebocytes (71,72), the regulation of skin physiology by these agents is of great interest and potential clinical importance (69–72). It is therefore reasonable to ask how all these hormones interact with leptin at the skin level.
In order to investigate the full complexity of leptin/ObR-mediated signalling in human skin and its multiple interactions with other hormonal/cytokine systems (31,33,36,58–60,65,67–70), it is critical to study the effects of leptin not only on isolated fibroblasts and keratinoyctes, but also under in situ conditions. For this purpose, serum-free human hair follicle organ culture (63,64,66) and full-thickness human scalp skin organ culture (73) are optimally suited. Ideally, this is complemented by laser capture microdissection and gene profiling approaches (74).
Other directly acting bioenergetic agents and mitochondrial metabolism modifiers, such as carnitines, are now recognized to prevent lipotoxicity and are promising skin protectants and hair growth stimulators, but likely are much less potent than leptin and similar agents (28,29,75–80). It has long been claimed that the trophic and protective effects of leptin are mediated by specific signalling affecting mitochondrial function (5–9,12,14,17), yet studies on leptin-induced mitochondrial signalling (e.g. on members of the mitochondrial Bcl-2 family of apoptosis-regulatory proteins) are only in their infancy (7,10,11). Therefore, we are currently investigating the effects of leptin and leptin mimetics on human skin regeneration and hair growth in appropriate organ culture assays, with emphasis on the molecular mechanisms by which leptin influences mitochondrial function, phosphorylation, and proliferation.
The human skin expresses the genes for corticotrophin-releasing hormone (CRH) and pro-opiomelanocortin (POMC). The cutaneous CRH/POMC expression is highly reactive to common stressors and to the nutritional status that are key modulators of mitochondrial energy metabolism (81,82). Therefore, studies on the interaction of leptin and CRH/POMC signalling in their regulation of skin and hair physiology and pathophysiology should have a high priority.
Leptin and leptin mimetics such as humanin and colivelin have already been successfully administered intranasally, and rapid and sufficient epithelial absorption/uptake as well as intracellular transport with systemic distribution and high bioavailability have been demonstrated (75,76,79,80). Local topical, and systemic nasal administration of such compounds yields effective pharmacological concentrations in the target tissues that exceed the normal endogenous levels by at least three orders of magnitude. Thus, leptin and leptin mimetics now await and deserve systematic exploration as orchestrators of trophic and protective responses in the skin, which are likely to promote skin and hair regeneration and which may retard or even partially reverse selected aspects of skin ageing.