Söbia Kauser

Serotoninergic and melatoninergic systems are fully expressed in human skin

We investigated the cutaneous expression of genes and enzymes responsible for the multistep conversion of tryptophan to serotonin and further to melatonin. Samples tested were human skin, normal and pathologic (basal cell carcinoma and melanoma), cultured normal epidermal and follicular melanocytes, melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and fibroblasts from dermis and follicular papilla. The majority of the samples showed simultaneous expression of the genes for tryptophan hydroxylase, arylalkylamine N‐acetyltransferase (AANAT), and hydroxyindole‐Omethyltransferase (HIOMT). The products of AANAT activity were identified by RP‐HPLC with fluorimetric detection in human skin and in cultured normal and malignant melanocytes and immortalized keratinocytes; HIOMT activity was detected in human skin, keratinocytes, and melanoma cells. N‐acetylserotonin (NAS) was detected by RP‐HPLC in human skin extracts. NAS identity was confirmed further by LC/MS in keratinocytes. In conclusion, we provide evidence that the human skin expresses intrinsic serotonin and melatonin biosynthetic pathways.

Functional activity of serotoninergic and melatoninergic systems expressed in the skin.

We tested the expression of genes coding receptors of a cutaneous serotoninergic/melatoninergic system in whole human skin and in normal and pathologic cultured skin cells. Evaluation of serotonin (5HT), melatonin (MT), and melatonin‐related receptors (MRR) showed expression of the isoforms 5HT2B, 5HT7, and MT1 genes in almost all the tested samples. Expression of other isoforms was less prevalent; 5HT2C, MRR, and MT2 were rarely detected. We also found novel isoforms for MT2, MRR, and 5HT2B and documented the process of RNA editing for 5HT2C. Testing for functional activity of these receptors with serotonin and melatonin (10−14 to 10−10 M) showed variable effects depending on cell type and culture conditions. Thus, serotonin stimulated proliferation of melanocytes in medium deprived of growth factors, while inhibiting cell growth in the presence of growth factors. Melatonin inhibited both apoptosis of HaCaT keratinocytes incubated in serum‐free media, and proliferation of cells cultured in medium supplemented with serum. Melatonin also increased the numbers of viable fibroblasts incubated in serum free medium. N‐acetylserotonin (NAS) and 5 methoxytryptamine (5MTT) were generally without effect on cell proliferation, with the exception of an inhibition of melanocyte proliferation at the higher 5MTT concentration of 10−10 M. Thus, skin cells represent a true target for the products of the serotoninergic/melatoninergic cutaneous pathway with their actions modulating cell proliferation or viability.

Corticotropin releasing hormone and the skin.

Cotricotropin-releasing hormone (CRH) and related peptides are produced in skin that is dependent on species and anatomical location. Local peptide production is regulated by ultraviolet radiation (UVR), glucocorticoids and phase of the hair cycle. The skin also expresses the corresponding receptors (CRH-R1 and CRH-R2), with CRH-R1 being the major receptor in humans. CRH-R1 is expressed in epidermal and dermal compartments, and CRH-R2 predominantly in dermal structures. The gene coding for CRH-R1 generates multiple isoforms through a process modulated by UVR, cyclic adenosine monophosphate (cAMP) and phorbol 12-myristate 13-acetate. The phenotypic effects of CRH in human skin cells are largely mediated by CRH-R1alpha through increases in concentrations of cAMP, inositol triphosphate (IP3), or Ca2+ with subsequent activation of protein kinases A (PKA) and C (PKC) dependent pathways. CRH also modulates the activity of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-kappaB), activator protein 1 (AP-1) and cAMP responsive element binding protein (CREB). The cellular functions affected by CRH depend on cell type and nutritional status and include modulation of differentiation program(s), proliferation, viability and immune activity. The accumulated evidence indicates that cutaneous CRH is also a component of a local structure organized similarly to the hypothalamo-pituitary-adrenal axis.

Proopiomelanocortin (POMC), the ACTH/ melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin‐1 receptor (MC‐1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melano‐cytes in culture. Much lower levels of α‐MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and α‐MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone conver‐tases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and α‐MSH, and this was enhanced in response to corti‐cotrophin‐releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably trans‐fected with the MC‐1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, α‐MSH, and β‐MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC‐1R highlight the importance of POMC processing as a key regulatory event in the skin.—Rousseau, K., Kauser, S., Pritchard, L. E., Warhurst, A., Oliver, R. L., Slominski, A., Wei, E. T., Thody, A. J., Tobin, D. J., White, A. Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and mela‐nocytes and stimulates melanogenesis. FASEB J. 21, 1844–1856 (2007)

Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles.

Abstract: Study of the involvement of the hair follicle papilla in hair growth regulation was greatly facilitated by the isolation and cultivation of this tiny cluster of fibroblast‐like cells in the rat vibrissae and in the human hair follicle. While isolation of the hair follicle papilla from the former is relatively straightforward, the current method to isolate the much smaller human hair follicle requires significant skill. Thus, the routine initiation of primary cultures of human scalp hair follicle papilla cells requires significant training, time, and commitment.

Human hair follicle and epidermal melanocytes exhibit striking differences in their aging profile which involves catalase.

in the DNFB-applied skin. We assume that this distinct migratory activity may be due to the fact that not all T cells in the LNs after DNFB sensitization were DNFB specific. In contrast, almost all TNCB-sensitized T cells actively migrated when DNFB was applied. In addition, we showed reverse motile activities in the TNCB-painted skin. Moreover, skin-infiltrating T cells colocalized with APCs. These findings suggest that skin-infiltrating T cells may actively scan for antigens, and when they meet APCs carrying their specific antigens, they stop migrating and stably interact with APCs. Our results also indicate that in the elicitation phase of CHS, hapten seems to be presented mainly by APCs in the dermis. Additional detailed studies are needed to clarify which subset of dermal APCs is essential for hapten presentation and whether epidermal Langerhans cells contribute.

In situ and in vitro evidence for DCoH/HNF-1α transcription of tyrosinase in human skin melanocytes

Human epidermal melanocytes hold the full capacity for autocrine de novo synthesis/regulation/recycling of the essential cofactor 6-tetrahydrobiopterin (6BH(4)) for conversion of L-phenylalanine via phenylalanine hydroxylase to L-tyrosine and for production of L-Dopa via tyrosine hydroxylase to initiate both pigmentation and catecholamine synthesis in these neural crest-derived cells. Earlier we have demonstrated pterin-4a-carbinolamine dehydratase (PCD) mRNA and enzyme activities in epidermal melanocytes and keratinocytes. This protein dimerises also the transcription factor hepatocyte nuclear factor 1 (HNF-1), leading to activation of multiple genes. This study demonstrates for the first time DCoH/HNF-1 alpha expression and transcriptional activity in human epidermal melanocytes in vitro and in situ and identified tyrosinase, the key enzyme for pigmentation, as a new transcriptional target. Specific binding of DCoH/HNF-1 complex to the human tyrosinase promoter was confirmed by gel shift analysis. These results provide a novel mechanism in the regulation of skin pigmentation.

In vivo and in vitro evidence for autocrine DCoH/HNF‐1α transcription of albumin in the human epidermis

Abstract:  The presence of albumin in the human epidermis has been reported more than a decade ago, but until now, it was assumed that this protein is synthesized in the liver and transported to the avascular skin. To our knowledge, transcription of albumin in the human epidermis was never considered. In this report, we present for the first time evidence for autocrine synthesis of albumin in the human epidermis in keratinocytes in situ and in vitro. Using double immunofluorescence labelling, we identified that albumin colocalized together with its transcription factor PCD/DCoH/HNF‐1α in suprabasal keratinocytes in human full‐thickness skin sections and in keratinocytes cultured in serum‐free medium. Moreover, albumin and HNF‐1α protein expression was confirmed by Western blotting in undifferentiated and differentiated keratinocytes as well as in human epidermal suction blister roof extracts. Reverse‐transcriptase polymerase chain reaction analysis from human epidermal keratinocytes and epidermal suction blister roofs revealed the transcription of albumin. Using in vivo fluorescence excitation spectroscopy at the surface of human skin, we confirmed albumin as a major constituent yielding a λmax at 295 nm, which was assigned to the single tryptophan 214 fluorophore in this protein. This in vivo result is in agreement with albumin concentrations of 10−3 M, underlining the importance of this protein in epidermal homeostasis.