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Pomari, Elena (2011) Local synthesis and effect of sex steroids in human skin and the hair follicle and hormonal effects on the wound healing response. [Tesi di dottorato]

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Abstract (inglese)

Humans, as others mammals, are able to synthesize steroid hormones from endocrine organs, such as gonads, adrenal gland and placenta. The expression and activity of steroidogenic enzymes in peripheral tissues provide the intracrine production of active androgens and estrogens by transformation of adrenal precursor dehydroepiandrosterone (DHEA). Only human and primates are unique in having large amounts of the inactive adrenal DHEA and especially DHEA-sulfate (DHEA-S). However, adrenal secretion decreases from the age of 30 years in both sexes, thus potentially providing an explanation for part of the mechanisms involved in the pathogenesis of age-related conditions, such as skin aging. In this study, it was determined whether human skin expresses the enzymes required for the local biosynthesis of active sex steroid hormones. Specifically, biopsies of skin and hair follicles (HFs) were collected from female healthy donors, and cultured dermal fibroblasts (DFs) and cultured epidermal keratinocytes (EKs) were established from female and male healthy donors. The mRNA expression of seven key steroidogenic proteins (P450arom, P450scc, P450c17, STS, OATP2B1, 5α-reductase 1 and 5α-reductase 2), and the estrogen receptors (ERα and ERß) and the androgen receptor (AR) were investigated using RT-PCR and qRT-PCR. All samples produced PCR products of the expected size for P450scc, P450c17, STS and 5α-reductase 1. The mRNA expression of 5α-reductase 2 was detected only in HF. EK did not produce the transcript of P450arom and DF did not express OATP2B1. Both ERα and ERß were expressed in EK as well as in DF. In particular, EK showed to strongly express ERß compared to ERα. In contrast, ERα has been detected with higher expression than ERß in DF. Differences in estrogen receptors expression have been demonstrated also in human HFs that have showed strongly expressed ERß in comparison with ERα. On the other hand, all the samples showed AR expression. In order to the possibility of steroids metabolism, a gene array analysis was performed on whole transcriptome of human HFs and cultured EKs after steroid treatment. Three different hormones were used for 24h of incubation: DHEA-S (10µM), testosterone (TST) (50nM) and 17ß-estradiol (E2) (1nM). The global mRNA expression profiling revealed changes in the expression of a large number of genes. In particular, interesting up-regulation of genes involved in inflammation, cell proliferation and differentiation and structural functions. The analysis on EKs was focused on four genes up-regulated with all three steroids investigated: CXCL1, ANGPTL4, TXNIP, and LMNB1. In addition, the in vitro scratch wound assay was used to determine the direct effects of E2, DHEA, DHEA-S and TST on the migration of mechanically wounded human DFs and EKs. All steroids stimulated cell migration, although both DHEA and TST were blocked by an aromatase inhibitor (Arimidex) and DHEA-S by the STS inhibitor. These results indicate an important role for local synthesis of E2 in skin from the circulating precursor DHEAS and expression of its action in the wound healing process. To verify the hypothesis of the local synthesis of E2, the activity of the aromatase, which is the enzyme that catalyzes the conversion of androgen to estrogen, was assessed in EKs and DFs using the tritiated water (3H2O) assay. Specifically, it was investigated whether the aromatase activity and mRNA expression were modulated in cultured skin cells in response to either dexamethasone or mechanical wounding. The analysis revealed that dexamethasone increased the activity and mRNA expression in particular in DFs, in contrast, the mechanically wounded showed in EKs an increase of the aromatase activity at 24h compared to the non-wounded confluent monolayer cells. All data suggest that human skin and individual cells as target and source of active androgens and estrogens. Further studies are required to understand the role of steroids in inflammation, proliferation and differentiation which have implications for hair growth, skin cancer, aging and wound-healing. In particular, a greater understanding of DHEA signalling may help develop wound-healing therapies, without the risks of estrogen therapy, benefiting the elderly and patients with impaired wound-healing.

Abstract (italiano)

Gli umani, come altri mammiferi, sintetizzano ormoni steroidei da organi endocrini come le gonadi, la surrenale e la placenta. L’espressione e attività di enzimi steroidogenici a livello di tessuti periferici provvedono alla formazione intracrina di androgeni ed estrogeni attivi a partire dal precursore deidroepiandrosterone (DHEA). Solamente umani ed alcuni primati secernono alti livelli di DHEA e DHEA-S. Tuttavia con l’età si presenta in entrambi i sessi una riduzione della secrezione adrenalica che si riflette in un notevole declino della produzione periferica di ormoni sessuali. Questa diminuita formazione è potenzialmente correlata alla patogenesi di malattie che incorrono con la vecchiaia tra cui anche alcune condizioni della pelle. Nel presente studio è stata valutata l’espressione dell’mRNA di sette proteine chiave della steroidogenesi (P450arom, P450scc, P450c17, STS, OATP2B1, 5α-riduttasi 1 e 5α-riduttasi 2), oltre che dei recettori di estrogeni (ERα e ERß) ed androgeni (AR), in biopsie di pelle e follicoli piliferi derivati da donatrici donne sane e in fibroblasti dermici e cheratinociti epidermici primari di donatori femmine e un maschio sempre sani. L’analisi condotta con RT-PCR e qRT-PCR ha rilevato in particolare che solo i follicoli esprimono 5α-riduttasi 2, i cheratinociti non presentano espressione dell’aromatasi (P450arom) e i fibroblasti non risultano esprimere il trasportatore OATP2B1. Dall’altra parte, entrambi i recettori ERα e ERß e quello per androgeni sono espressi nelle cellule della pelle e nei follicoli piliferi. In particolare ERß risulta con più forte espressione in cheratinociti e follicoli piliferi rispetto a ERα e il contrario in fibroblasti. In relazione quindi alla possibilità di una locale formazione ed azione di ormoni sessuali nella pelle, è stato investigato l’effetto in vitro di tre steroidi sull’espressione genica di follicoli piliferi e cheratinociti ottenuti da pelle facciale di donne sane. L’intero trascrittoma è stato analizzato dopo un’incubazione di 24 ore con DHEA-S (10µM), testosterone (TST) (50nM) e 17ß-estradiolo (E2) (1nM). In particolare sono state individuate modulazioni dell’espressione di geni coinvolti in funzioni come infiammazione, proliferazione e differenziamento cellulare, e di struttura. L’analisi condotta su cheratinociti è stata focalizzata su quattro geni sovra-espressi da tutti e tre i diversi trattamenti steroidei: CXCL1, ANGPTL4, TXNPI e LMNB1. Dati gli effetti e funzioni implicati dagli ormoni in esame, è stato valutato in vitro l’effetto di E2, DHEA, DHEA-S e TST sulla migrazione di fibroblasti e cheratinociti mediante scratch wound assay. L’analisi ha rilevato l’accelerazione della migrazione di entrambi i tipi cellulari in presenza di tutti gli steroidi in esame. In particolare, la combinazione di un inibitore dell’aromatasi (Arimidex) e uno della steroide solfatasi (STX64) hanno determinato l’inibizione dell’effetto stimolatorio rispettivamente di DHEA e TST, e di DHEA-S. I risultati suggeriscono un importante ruolo della formazione locale di E2 nella pelle a partire dal precursore DHEA (-S) e della sua azione nel processo di wound healing. Per verificare tale ipotesi della sintesi locale di estradiolo, è stata investigata l’attività dell’enzima catalizzante la conversione di androgeno ad estrogeno, l’aromatasi. Le misurazioni dell’attività aromatasica sono state eseguite con il metodo dell’acqua triziata. Specificatamente, l’esame è stato condotto su fibroblasti e cheratinociti umani e individuando l’effetto su attività ed espressione dell’mRNA dell’enzima dopo trattamento con desametasone o eseguendo mechanical scratch wound. Nel primo caso, il desametasone ha indotto stimolazione sia di attività che di espressione del trascritto. Mentre nel secondo caso, è stato rilevato un incremento in cheratinociti dopo 24 ore dallo scratch. In generale questo studio suggerisce che la pelle umana, oltre che separatamente fibroblasti e cheratinociti, e follicolo pilifero, siano target e fonte degli ormoni sessuali. Ulteriori studi saranno necessari per comprendere il ruolo di estrogeni ed androgeni in meccanismi come infiammazione, proliferazione e differenziamento cellulare, che possono avere implicazioni nella crescita del pelo, cancro della pelle, invecchiamento e wound healing. In particolare, la comprensione del signalling di DHEA aiuterebbe lo sviluppo di terapie per il wound healing riducendo i rischi correlati alla somministrazione degli estrogeni.

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Tipo di EPrint:Tesi di dottorato
Relatore:Colombo, Lorenzo
Correlatore:Dalla Valle, Luisa
Dottorato (corsi e scuole):Ciclo 23 > Scuole per il 23simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:28 Gennaio 2011
Parole chiave (italiano / inglese):pelle, ormoni steroidei sessuali, wound healing, migrazione cellulare skin, sex steroid hormones, wound healing, cell migration
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:3725
Depositato il:01 Ago 2011 10:56
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