Sensing the environment: regulation of local and global homeostasis by the skin's neuroendocrine system

Abstract

Skin, the body's largest organ, is strategically located at the interface with the external environment where it detects, integrates, and responds to a diverse range of stressors including solar radiation. It has already been established that the skin is an important peripheral neuro-endocrine-immune organ that is tightly networked to central regulatory systems. These capabilities contribute to the maintenance of peripheral homeostasis. Specifically, epidermal and dermal cells produce and respond to classical stress neurotransmitters, neuropeptides, and hormones. Such production is stimulated by ultraviolet radiation (UVR), biological factors (infectious and noninfectious), and other physical and chemical agents. Examples of local biologically active products are cytokines, biogenic amines (catecholamines, histamine, serotonin, and N-acetyl-serotonin), melatonin, acetylocholine, neuropeptides including pituitary (proopiomelanocortin-derived ACTH, beta-endorphin or MSH peptides, thyroid-stimulating hormone) and hypothalamic (corticotropin-releasing factor and related urocortins, thyroid-releasing hormone) hormones as well as enkephalins and dynorphins, thyroid hormones, steroids (glucocorticoids, mineralocorticoids, sex hormones, 7-delta steroids), secosteroids, opioids, and endocannabinoids. The production of these molecules is hierarchical, organized along the algorithms of classical neuroendocrine axes such as hypothalamic-pituitary-adrenal axis (HPA), hypothalamic-thyroid axis (HPT), serotoninergic, melatoninergic, catecholaminergic, cholinergic, steroid/secosteroidogenic, opioid, and endocannbinoid systems. Dysregulation of these axes or of communication between them may lead to skin and/ or systemic diseases. These local neuroendocrine networks are also addressed at restricting maximally the effect of noxious environmental agents to preserve local and consequently global homeostasis. Moreover, the skin-derived factors/systems can also activate cutaneous nerve endings to alert the brain on changes in the epidermal or dermal environments, or alternatively to activate other coordinating centers by direct (spinal cord) neurotransmission without brain involvement. Furthermore, rapid and reciprocal communications between epidermal and dermal and adnexal compartments are also mediated by neurotransmission including antidromic modes of conduction. In conclusion, skin cells and skin as an organ coordinate and/or regulate not only peripheral but also global homeostasis.

Figure 1

Skin senses changes in the environment through cutaneous neuroendocrine system, which computes and translates the received information into chemical, physical and biological messengers that regulate global (A and B) and local (B) homeostasis. These signals travel through humoral, immune or neural pathways to reach the central nervous, endocrine and immune systems as well as other organs. Reproduced with permission from Endocrine Society (Slominski and Wortsman, 2000a).

Figure 2

Skin neuroendocrine system follows the algorithms of classical neuroendocrine or endocrine systems. It also forms a natural platform of signal exchange between internal organs and environment. For this purpose skin cells not only are subjected to neurohormonal regulation but also do produce neuropeptides, biogenic amines, melatonin, opioids, cannabinoids, acetylcholine, steroids, secosteroids as well as growth factors and cytokines. Skin neuroendocrine system also entrains immune cells to act as cellular messengers at distal sites.

Figure 3. Catecholamine synthesis in the skin

The common pathway in the skin requires its consecutive hydroxylations of L-phenylalanine (mediated by phenylalanine hydroxylase (PH)) to L-tyrosine with following hydroxylation by tyrosine hydroxylase (TH) or tyrosinase to produce L-dihydroxyphenylalanine (L-DOPA). L-DOPA is either oxidized to DOPA quinone with following multistep transformation to melanin or serves as a substrate for synthesis of catecholamines. The skin expresses complete enzymatic machinery required for dopamine synthesis (L-amino acid decarboxylase - AAD) and its subsequent conversion into norepinephrine (dopamine β-hydroxylase) and methylation (phenylethanolamine N-methyltransferase) to form epinephrine. An alternative pathway of catecholamine synthesis involves decarboxylation of L-tyrosine to tyramine, which in turn is hydroxylated by TH (and Cyp2D) or dopamine β-hydoxylase to octopamine or dopamine, respectively. Octopamine could be metabolized to norepinephrine by TH. This alternative pathway present in invertebrates remains to be tested in the skin. Catecholamines also undergo oxidation to corresponding quinoinones with further multistep transformation to neuromelanin that is similar to melanogenesis starting from L-DOPA.

Figure 4. Catecholamine catabolism

Catecholamines are deactivated by L-monoamine oxidase (MAO) and Catechol-O-methyltransferase (COMT) leading to synthesis of homovanillic acid (from dopamine) or vanillylmandelic acid from norepinephrine or epinephrine. Alternatively, as shown for dopamine metabolism order of reaction may be change with COMT acting first followed by MAO.

Figure 5. Biochemical pathway of serotonin synthesis and metabolism in the skin

The pathway starts with hydroxylation of tryptophan by tryptophan hydroxylase type 1 or 2 (TPH1 or TPH2) to form 5-hydroxytryptophan (5-TPH; TrpOH). TrpOH can also be produced by non-enzymatic action of UVA and H2O2. Serotonin (5-hydroxytryptamine, 5-HT) derives from 5-TPH by action of L-amino acid decarboxylase - AAD. Serotonin can be acetylated by aralkylamine N-acetyltransferase (AANAT) or N-acetyltransferase (NAT) to produce N-acetylserotonin (NAS) with further methylation by hydroxy-indole-O-methyl transferase (HIOMT) to melatonin. Deactivation of serotonin is catalyzed mainly by MAO with formation of 5-hydroxyindoleacetaldehyde (5-HIAD) which is followed by action of alcohol (AD) or aldehyde dehydrogenase (ADD) with formation of 5-hydroxytryptophanol (5-HTOL) or 5-hydroxyindole-3-acetic acid (5-HIAA), respectively. Alternatively, HIOMT activity may also lead to production of methylated derivatives of serotonin. First step catalyzed by HIOMT leads to formation of 5-methoxytryptamine 5-MT and subsequent action of MAO results in 5-metoxyindoleacetaldehyde (5-MIAD). Finally, AD or ADD facilitates synthesis of 5-methoxytryptophol (5-MTOL) or 5-methoxyindole-3-acetic acid (5-MIAA), respectively. HIOMT was found also to catalyze conversion of 5-HIAA to 5-MIAA. Melatonin by action of MAO can be metabolized to 5-methoxytryptamine (5-MT), thus entering the pathway leading to 5-MTOL or 5-MIAA formation.

Figure 6. Expression of TPH, serotonin (5-HT) and serotonin transporter (5-HTT) in skin cells

Panels A-H show immunocytochemical detection of 5-HT (B, C), 5-HTT (E, F) and TPH (H) in fixed cells using corresponding antibody at dilution 1:5,000 (Antibody against 5-HT, Diasporin Corp., Stillwater, MN) or 1:1,000 (antibodies against TPH and 5-HTT, Chemicon, Temecula, CA). A, D, G: negative controls incubated with secondary antibody only. I. Western blot showing detection of 5-HTT in membranous (ppt) but not cytosolic (sup) fractions from human melanoma (HuMel), HaCaT keratinocytes (HaCaT) and ATt-20 pituitary (ATT-20) cells. For technical details of immunocytochemistry, or western blot assay see (Slominski et al., 2005d).

Figure 7. Melatoninegic system in the skin

TPH1 Western blot insert in the panel a is of approximately 50kD (arrowhead) that is processed and/or degraded to lower molecular weight species (asterisk). It is immunolocalized in the epidermis (ES), hair follicle (ORS), eccrine glands (EG), showing the highest expression in melanocytes (arrows) (panels a and b). 5-hydroxytryptophan is further decarboxylated by aromatic amino acid decarboxylase (AAD). AANAT (enzyme acetylating serotonin) is expressed in cells of epidermal, dermal and adnexal compartments (E, BV, EG and hair follicle structures in panel c on the left). Immunocytochemical localization of melatonin-like immunoreactivity is shown in panel d on the right (upper E, BV and MC). Immunocytochemistry was performed on human skin biopsies: E – epidermis, D – dermis, BV-blood vessel, EG - eccrine gland, HF ORS - hair follicle outer root sheath, FP – hair follicle papilla; MX – hair follicle matrix, MC – mast cells. For technical details see (Slominski et al., 2005d). Reproduced with permission from the publisher (Slominski et al., 2008a).

Figure 8. Pathways of melatonin degradation

The indolic pathway involves 6-hydroxylation of melatonin [I] by CYP1A1, CYP1A2 or CYP1B1 (1) to 6-hydroxymelatonin [II]. Melatonin deacetylase (2) produces 5-methoxytryptamine [III] that is oxidized by monoamine oxidase (3) to form 5-methoxyindoleacetaldehyde [IV], which is converted to 5-methoxyindole acetic acid [V] by aldehyde dehydrogenase (4) or to 5-methoxytryptophol [VI] by alcohol dehydrogenase (5). In the kynuric pathway, melatonin can be converted non-enzymatically to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) [VII] in the process that may include generation of 3-hydroxymelatonin [VIII], 2-hydroxymelatonin [IX], melatonin 2-indolinone [X], 3-hydroxymelatonin 2-indolinone [XI], and melatonin dioxetane [XII] as intermediates. Enzymes or pseudoenzymes (Enz) are involved in melatonin conversion to AFMK. Different pathways can lead to the conversion of AFMK [VII] to AMK [XIII] (6). Reproduced with permission from the publisher (Slominski et al., 2008a).

Figure 9. Phenotypic effects of melatonin in the skin

Exogenous or endogenously synthesized melatonin can regulate skin cell phenotype via interaction with melatonin receptors. Non-receptor actions are mediated via an interaction with intracellular proteins such as NQO2 or calmodulin. Melatonin and its metabolites can act as direct scavengers of reactive oxygen and nitrogen species (ROS and RNS) and affect mitochondrial functions. Direct effects are shown by solid lines and multiple reactions/signaling are shown by dashed arrows lines. Reproduced from (Slominski et al., 2008a) with permission from the publisher.

Figure 10

CRF related signaling in the skin regulates protective and homeostatic functions of the skin. The specificity of the effect is defined either by local production of the molecule (CRF, Urc1, Urc2 or Urc3) or the type of the receptor expressed (CRF1 vs. CRF2). Reprinted from (Slominski, 2009b) with permission from the publisher

Figure 11. CRF1 and CFR2 receptors and their alternative splicing variants

Upper panel: Human CRF1 gene consists of 14 exons and due to alternative splicing at least ten isoforms can be generated with seven found in human skin (Pisarchik and Slominski, 2001, Slominski et al., 2006c). Coding exons are shown in blue and none coding exons due to frame shift followed by in-frame premature stop codon are showed as white squares. Lower panel: CRF2 gene contains 15 exons and at least three alternative transcription start codons. Due to alternative splicing at least three main isoforms can be created (CRF1α, β, γ) and four additional isoforms could be synthesized from full length mRNA by employing alternative start codons (CRF2α1, β1a, β1b, γ1 as shown on the top of the panel). Exon numbers are marked on the top of each panel. Transmembrane segments of 7TM are shown as squares (dashed line with number I to VII). See the text for appropriate citations.

Figure 12. Regulation of CRF signaling by CRF1 isoforms

CRF1 gene contains 14 exons and only one isoform of receptor - CRF1β (also called pro-CRF1) is coded by all exons. CRF1 transcript is also subjected to alternative splicing resulting in at least 8 isoforms. Recent studies showed that expression and/or co-expression of CRF1 isoforms is responsible for modulation of CRF1 signaling mediated by main CRF1α or alternative CRF1β isoform. Soluble isoforms (CRF1e and h) were also found to stimulate CRF or modify Urc signaling when co-expressed with CRF1α. ‘Minus’ sign indicates inhibition of CRF signaling on different levels including: fast mRNA decay (CRF1e), dimerization and subsequent intercellular retention resulting in most probable premature receptor degradation (CRF1α with CRF1d, CRFf or CRFg), decoy receptor mechanism (CRF1h and e when secreted), agonist binding impairment (CRF1c) or G-protein binding inhibition (CRF1d). For details see (Zmijewski and Slominski, 2010a). Reproduced with permission from the publisher.

Figure 13

Localization of the CRF1 isoform tagged with GFP in human adult ARPE-19 cells. Adult retinal pigment epithelium cells (ARPE-19) as alternative to melanocyte model of pigment producing cells showed similar intracellular distribution of the CRF1 isoform to that described previously in HaCaT keratinocytes (Zmijewski and Slominski, 2009b) and ATT-20 pituitary cells (Zmijewski and Slominski, 2009c). Isoforms CRF1α (Panels a and b) and CRF1c (Panel c) with full length 7-TM are found predominantly within cell membrane. CRF1 isoforms with defects (CRF1d – Panel d, CRF1f – Panel f, CRF1g – Panel g) within 7-TM region show intracellular localization. The soluble isoforms (CRF1e – Panel e, CRF1h – Panel h) are predominant inside the cells. The isoform CRF1e (Panel e) is the only isoform found inside the nucleus (similarly as GFP alone – Panel i). ARPE-19 were transfected with constructs caring CRF1 isoforms fused with GFP (Zmijewski and Slominski, 2009c) and images (as Z stacks) were collected with Zeiss LSM 510 laser scanning microscope (Zeiss, Germany). On the bottom and right sides of Panels b, d, f, g, e and h cross sections (from Z stacks) were shown to emphasize three-dimensional localization of CRF isoforms. On Panel a Z stack projection (average intensity) of APRE-19 cells overexpressing CRF1α is shown to emphasize presence of this isoform on the cell surface. The controls are represented by ARPE cells transfected with GFP alone (Panel i).

Figure 14

Scheme of steroidogenic pathway.

Figure 15. Organization of the systemic HPA axis

Modified from figure 1 published in (Slominski, 2007)

Figure 16. HPA algorithm is expressed in normal human melanocytes

A. CRH stimulates POMC gene expression, and ACTH production with attendant stimulation of cAMP. B. Cortisol production is enhanced by addition of progesterone and/or IBMX (inhibitor of phosphodiesterase). C. Cortisol is identified by LC/MS² in melanocytes (standard = A, C; conditioned media from melanocytes = B, D). D. CRH stimulates cortisol production that is dependent on POMC expression and CRF1 signaling Reproduced from (Slominski et al., 2005e) with permission from the American Physiological Society.

Figure 17. Skin stress response system can activate the central HPA with its direct homeostatic, metabolic and phenotypic consequences

We hypothesized that global responses (on the organism level) to UVR initiated in the skin and involving simultaneously activation of sensory receptors and local production of humoral messages (Slominski, 2005a, Slominski and Wortsman, 2000a, Slominski et al., 2008b). These signals are either delivered by ascending nerve routes to the brain or by circulation to hypothalamus to activate CRF production in the PVN; CRF then would enter portal circulation with subsequent activation of CRF1 in the pituitary. Skin humoral signals can also enter directly the pituitary from the circulation. The final outcome for these signaling processes is stimulation of ACTH release and of POMC activity. Alternatively, although less likely, the cutaneous factors can bypass this axis and enter adrenal gland directly from the circulation. The net effect of all of these processes is release of cortisol/corticosterone and induction of steroidogenesis with subsequent metabolic and homeostatic effects.

Figure 18. Proposed evolution of stress response system

Reproduced with permission from the publisher (Slominski, 2007)

Figure 19. UVB induced production and transformation of 7-dehydrocholestrol

Reprinted from (Slominski, 2009a) with permission from publisher

Figure 20

UVB induced transformation of pregna- or androsta-steroidal 5,7-dienes. hν, Reprinted from (Slominski, 2009a) with permission from publisher

Figure 21

Structure and functions of an equivalent of the hypothalamic-pituitary thyroid axis (HPT) in the skin.

Figure 22

Wavelength-dependent UV stimulation of β-END expression in epidermal layer of human skin. CY™3 positive (red) signals correspond to β-END immunoreactivity (methods described in Skobowiat et al., 2011).

Figure 23

Hypothetical pathway of addictive properties of the cutaneous neuroendocrine system induced by UV stimulation.