[ES] Nuestra investigación se centra en comprender los mecanismos moleculares que median las acciones de los glucocorticoides (GCs) en la fisiopatología de la piel mediante el análisis funcional del receptor de GCs (GR) y el receptor de mineralocorticoides (MR), dos proteínas altamente relacionadas estructural y funcionalmente, que actúan como factores de transcripción dependientes de ligando. Nuestros datos previos demuestran que GR juega un papel central en el desarrollo de la piel; en la edad adulta, tanto GR como MR actúan como mediadores anti-inflamatorios en enfermedades cutáneas (Sevilla et al. 2013; Boix et al. 2016). No obstante, desconocíamos si los receptores ejercían funciones cooperativas o antagónicas en la epidermis. Esta tesis doctoral se ha centrado en la generación y caracterización de ratones con inactivación específica en la epidermis de GR y MR (ratones double knock-out o DKO). Al nacer, los DKO mostraron un fenotipo cutáneo con diferenciación epidérmica defectuosa y un estado inflamatorio único caracterizado por infiltrados inmunes epiteliales y alteraciones en la expresión génica, similar a las lesiones psoriáticas. Este fenotipo fue mucho más severo que el de los KO individuales (ratones GR epidermal KO o GREKO y MR epidermal KO o MREKO), pero se resolvió espontáneamente a partir del día post-natal 3. En la edad adulta, la piel DKO mostró un aumento en el grosor epidérmico, similar al de los KO individuales. Todos los ratones KO mostraron una mayor susceptibilidad a la inflamación aguda respecto a los controles (CO), que no se contrarrestó de forma efectiva por un tratamiento tópico con GCs. Además, los ratones DKO mostraron una mayor susceptibilidad a la psoriasis inducida por imiquimod respecto a los KO individuales. El aumento de la respuesta inflamatoria en los DKO era consistente con un aumento significativo de la actividad de AP-1 y NF-kappaB en queratinocitos DKO respecto a los CO o KO individuales. En conjunto, nuestros datos demuestran que GR y MR epidérmicos actúan de manera cooperativa para contrarrestar la inflamación de la piel, durante el desarrollo y la edad adulta, y que ambos son necesarios para una respuesta transcripcional óptima y una actividad terapéutica de los GCs. Los tratamientos prolongados con dosis farmacológicas de GCs producen defectos como la atrofia cutánea, similar a la que tiene lugar durante el envejecimiento cronológico, que correlaciona con un aumento de los niveles locales endógenos de GCs. Este trabajo ha abordado las consecuencias fenotípicas de la pérdida epidérmica de MR durante el envejecimiento cronológico y los mecanismos involucrados. Los ratones MREKO de 13 meses de edad fueron resistentes a la atrofia epidérmica pero mostraron un menor grosor dérmico y depósito de colágeno, en parte debido a una disminución de la actividad SMAD2/3 respecto a la piel de ratones CO. Además, el tejido adiposo subcutáneo (dWAT) se engrosó 2.5 veces en MREKO vs CO a los 13 meses, con hiperplasia e hipertrofia de adipocitos. Estos cambios se desencadenaron, al menos en parte, a través de alteraciones en la señalización mediada por GCs, y la activación de WNT/beta-catenina inducida por señales paracrinas epidérmicas que condujeron al aumento de expresión de Pparg. Estos resultados demuestran un papel crucial de MR epidérmico en la regulación del cross-talk entre compartimientos durante el envejecimiento cronológico de la piel. / [CA] La nostra investigació se centra en comprendre els mecanismes moleculars que regulen les accions dels glucocorticoides (GCs) en la fisiopatologia de la pell mitjançant l'anàlisi funcional del receptor de GCs (GR) i el receptor de mineralocorticoides (MR), dues proteïnes altament relacionades estructural i funcionalment, que actuen com a factors de transcripció dependents de lligant. Els nostres resultats previs demostren que GR juga un paper central en el desenvolupament de la pell; en l'edat adulta, tant GR com MR actuen com a mediadors antiinflamatoris en malalties cutànies (Sevilla et al. 2013; Boix et al. 2016). No obstant, desconeixíem si els receptors exercien funcions cooperatives o antagòniques en l'epidermis. Aquesta tesi doctoral s'ha centrat en la generació i caracterització de ratolins amb inactivació específica en l'epidermis de GR i MR (ratolins double knock-out o DKO). En néixer, els DKO van mostrar un fenotip cutani amb diferenciació epidèrmica defectuosa i un estat inflamatori únic caracteritzat per infiltrats immunes epitelials i alteracions en l'expressió gènica, similar a les lesions psoriàtiques. Aquest fenotip va ser molt més sever que el dels KO individuals (ratolins GR epidermal KO o GREKO i MR epidermal KO o MREKO), però es va resoldre espontàniament a partir del dia post-natal 3. En l'edat adulta, la pell DKO va mostrar un augment en el gruix epidèrmic, similar al dels KO individuals. Tots els ratolins KO van mostrar una major susceptibilitat a la inflamació aguda en comparació als controls (CO), que no va ser contrarestada de manera efectiva per un tractament tòpic amb GCs. A més, els ratolins DKO van mostrar una major susceptibilitat a la psoriasis induïda per imiquimod respecte als KO individuals. L'augment de la resposta inflamatòria en els DKO era consistent amb un augment significatiu de l'activitat d'AP-1 i NF-kappaB en queratinòcits DKO respecte als CO o KO individuals. En conjunt, les nostres dades demostren que GR i MR epidèrmics actuen de manera cooperativa per contrarestar la inflamació de la pell, durant el desenvolupament i l'edat adulta, i que tots dos són necessaris per a una resposta transcripcional òptima i una activitat terapèutica dels GCs. Els tractaments prolongats amb dosis farmacològiques de GCs produeixen defectes com l'atròfia cutània, similar a la que té lloc durant l'envelliment cronològic, que correlaciona amb un augment dels nivells locals endògens de GCs. Aquest treball ha abordat les conseqüències fenotípiques de la pèrdua epidèrmica de MR durant l'envelliment cronològic i els mecanismes involucrats. Els ratolins MREKO de 13 mesos d'edat van ser resistents a l'atròfia epidèrmica però van mostrar un menor gruix dèrmic i dipòsit de col¿lagen, en part a causa d'una disminució de l'activitat SMAD2/3 respecte a la pell de ratolins CO. A més, el teixit adipós subcutani (dWAT) es va engrossir 2.5 vegades en MREKO vs CO als 13 mesos, amb hiperplàsia i hipertròfia d'adipòcits. Aquests canvis es van desencadenar, almenys en part, a través d'alteracions en la senyalització mediada per GCs, i l'activació de WNT/beta-catenina induïda per senyals paracrines epidèrmiques que van conduir a l'augment d'expressió de Pparg. Aquests resultats demostren un paper crucial de MR epidèrmic en la regulació del cross-talk entre compartiments durant l'envelliment cronològic de la pell. / [EN] Our research focuses on understanding the molecular mechanisms that mediate the actions of glucocorticoids (GCs) in skin pathophysiology through functional analysis of the GC receptor (GR) and the mineralocorticoid receptor (MR), two highly related structural and functionally proteins, which act as ligand-dependent transcription factors. Our previous data show that GR plays a central role in skin development; in adulthood, both GR and MR act as anti-inflammatory mediators in skin diseases (Sevilla et al. 2013; Boix et al. 2016). However, we did not know if the receptors exerted cooperative or antagonistic functions in the epidermis. This doctoral thesis has focused on the generation and characterization of mice with specific inactivation in the epidermis of GR and MR (double knock-out or DKO mice). At birth, DKO show a skin phenotype with defective epidermal differentiation and a unique inflammatory state characterized by epithelial immune infiltrates and alterations in gene expression, similar to psoriatic lesions. This phenotype was much more severe than that of individual KO (GR epidermal KO or GREKO and MR epidermal KO or MREKO mice), but resolved spontaneously from postnatal day 3. In adulthood, DKO skin showed an increase in epidermal thickness, similar to that of individual KO. All KO mice showed greater susceptibility to acute inflammation compared to controls (CO), which was not effectively counteracted by topical treatment with GCs. Furthermore, DKO mice show a greater susceptibility to imiquimod-induced psoriasis relative to individual KO. The increased inflammatory response in DKO was consistent with a significant increase in AP-1 and NF-kappaB activity in DKO keratinocytes relative to CO or individual KO. Taken together, our data show that epidermal GR and MR act cooperatively to counteract skin inflammation, during development and adulthood, and that both are required for optimal transcriptional response and therapeutic activity of GCs. Prolonged treatments with pharmacological doses of GCs produce defects such as cutaneous atrophy, similar to that which occurs during chronological aging, which correlates with an increase in endogenous local levels of GCs. This work has addressed the phenotypic consequences of epidermal loss of MR during chronological aging and the mechanisms involved. The 13-month-old MREKO mice were resistant to epidermal atrophy but displayed reduced dermal thickness and collagen deposition, in part due to a decrease in SMAD2 3 activity relative to the skin of CO mice. In addition, the subcutaneous adipose tissue (dWAT) thickened 2.5 times in MREKO vs CO at 13 months, with hyperplasia and hypertrophy of adipocytes. These changes were triggered, at least in part, through alterations in GC-mediated signaling, and the activation of WNT/beta-catenin induced by epidermal paracrine signals that led to increased expression of Pparg. These results show a crucial role for epidermal MR in the regulation of the cross-talk between compartments during chronological skin aging. / Este trabajo ha sido realizado con el apoyo económico de los proyectos de investigación que se enumeran a continuación: SAF2014-59474-R, SAF2017-88046-R. Judit Bigas Corominas ha disfrutado de una beca predoctoral FPI (BES2015-072722) otorgada por el Ministerio de Economía y Competitividad, asociada al proyecto SAF2014-59474-R. Agradecemos el apoyo de COST ADMIRE BM-1301 y NuRCaMeIn (SAF2015-71878-REDT y SAF2017-90604-REDT). / Bigas Corominas, J. (2020). Contribuciones relativas de los receptores de glucocorticoides y mineralocorticoides en la biología cutánea [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/156214

Among the 33 human adhesion G-protein-coupled receptors (aGPCRs), a unique subfamily of GPCRs, only ADGRF4, encoding GPR115, shows an obvious skin-dominated transcriptomic profile, but its expression and function in skin is largely unknown. Here, we report that GPR115 is present in a small subset of basal and in most suprabasal, noncornified keratinocytes of the stratified epidermis, supporting epidermal transcriptomic data. In psoriatic skin, characterized by hyperproliferation and delayed differentiation, the expression of GPR115 and KRT1/10, the fundamental suprabasal keratin dimer, is delayed. The deletion of ADGRF4 in HaCaT keratinocytes grown in an organotypic mode abrogates KRT1 and reduces keratinocyte stratification, indicating a role of GPR115 in epidermal differentiation. Unexpectedly, endogenous GPR115, which is not glycosylated and is likely not proteolytically processed, localizes intracellularly along KRT1/10-positive keratin filaments in a regular pattern. Our data demonstrate a hitherto unknown function of GPR115 in the regulation of epidermal differentiation and KRT1.

No / Chromatin structure and spatial interactions between proximal and distal gene regulatory elements, including gene core promoters and enhancers, are important in the control of gene transcription. In this issue, Oh et al. characterized an AP-1-dependent enhancer at the epidermal differentiation complex locus that establishes spatial interactions with numerous gene promoter regions at that locus.

No / Although p53 has long been known as the “guardian of the genome” with a role in tumor suppression in many tissues, the discovery of two p53 ancestral genes, p63 and p73, more than a decade ago has triggered a considerable amount of research into the role of these genes in skin development and diseases. In this review, we primarily focus on mechanisms of action of p53 and p63, which are the best-studied p53 family members in the skin. The existence of multiple isoforms and their roles as transcriptional activators and repressors are key to their function in multiple biological processes including the control of skin morphogenesis, regeneration, tumorigenesis, and response to chemotherapy. Last, we provide directions for further research on this family of genes in skin biology and pathology.

No / Genome-wide microarray technology has facilitated the systematic discovery of diagnostic biomarkers of cancers and other pathologies. However, meta-analyses of published arrays often uncover significant inconsistencies that hinder advances in clinical practice. Here we present an integrated microarray analysis framework, based on a genome-wide relative significance (GWRS) and genome-wide global significance (GWGS) model. When applied to five microarray datasets on melanoma published between 2000 and 2011, this method revealed a new signature of 200 genes. When these were linked to so-called ‘melanoma driver’ genes involved in MAPK, Ca2+, and WNT signaling pathways we were able to produce a new 12-gene diagnostic biomarker signature for melanoma (i.e., EGFR, FGFR2, FGFR3, IL8, PTPRF, TNC, CXCL13, COL11A1, CHP2, SHC4, PPP2R2C, and WNT4). We have begun to experimentally validate a subset of these genes involved in MAPK signaling at the protein level, including CXCL13, COL11A1, PTPRF and SHC4 and found these to be over-expressed in metastatic and primary melanoma cells in vitro and in situ compared to melanocytes cultured from healthy skin epidermis and normal healthy human skin. While SHC4 has been reported previously to be associated to melanoma, this is the first time CXCL13, COL11A1, and PTPRF have been associated with melanoma on experimental validation. Our computational evaluation indicates that this 12-gene biomarker signature achieves excellent diagnostic power in distinguishing metastatic melanoma from normal skin and benign nevus. Further experimental validation of the role of these 12 genes in a new signaling network may provide new insights into the underlying biological mechanisms driving the progression of melanoma.

No / The skin produces bioactive lipids that participate in physiological and pathological states, including homeostasis, induction, propagation, and resolution of inflammation. However, comprehension of the cutaneous lipid complement, and contribution to differing roles of the epidermal and dermal compartments, remains incomplete. We assessed the profiles of eicosanoids, endocannabinoids, N-acyl ethanolamides, and sphingolipids, in human dermis, epidermis, and suction blister fluid. We identified 18 prostanoids, 12 hydroxy-fatty acids, 9 endocannabinoids and N-acyl ethanolamides, and 21 non-hydroxylated ceramides and sphingoid bases, several demonstrating significantly different expression in the tissues assayed. The array of dermal and epidermal fatty acids was reflected in the lipid mediators produced, whereas similarities between lipid profiles in blister fluid and epidermis indicated a primarily epidermal origin of suction blister fluid. Supplementation with omega-3 fatty acids ex vivo showed that their action is mediated through perturbation of existing species and formation of other anti-inflammatory lipids. These findings demonstrate the diversity of lipid mediators involved in maintaining tissue homeostasis in resting skin and hint at their contribution to signaling, cross-support, and functions of different skin compartments. Profiling lipid mediators in biopsies and suction blister fluid can support studies investigating cutaneous inflammatory responses, dietary manipulation, and skin diseases lacking biomarkers and therapeutic targets.

Nowdays there is a plethora of evidence for H₂O₂-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H₂O₂ and O₂̇⁻, yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H₂O₂ in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H₂O₂ in the mM range. H₂O₂-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H₂O₂. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H₂O₂, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H₂O₂ contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to α/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H₂O₂-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45α leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H₂O₂-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

3 domaines cellulaires définissent l'épiderme de la queue de Ciona intestinalis. Le domaine des lignes médianes, donne naissance à deux structures différenciées larvaires, la nageoire et système nerveux périphérique. Il a été montré que les voies de signalisation BMP et FGF sont respectivement les inducteurs des lignes médianes ventrale et dorsale. Ce travail a consisté en la caractérisation des mécanismes moléculaires, situés à l'interface des signaux inducteurs et des processus de différenciation, définissant l'identité cellulaire des lignes médianes. L'identification des relations épistatiques reliant 7 facteurs de transcription impliqués dans ce processus suggère un fonctionnement en réseau hiérarchisé et place Msxb comme gène clé. Cette hiérarchie est validée par: un atlas d'expression spatio-temporelle, la perte de fonction de Msxb, l'analyse des régions cis-régulatrices de la transcription. Nous avons identifié 2 types d'enhancers, contrôlant respectivement les gènes précoces et tardifs du réseau. L'expression précoce de Msxb dans les précurseurs dorsaux est controlée par un enhancer distinct régulé par la voie FGF relayée par Otx et Nodal. Cette signature transcriptionnelle est retrouvée dans les enhancers de gènes co-exprimés, et chez l'orthologue de Msxb chez une autre ascidie. Enfin, nous montrons que la partie la plus postérieure des lignes médianes constitue un troisième compartiment, déployant un programme génétique distinct. Ce travail nous renseigne sur la structure, les mécanismes moléculaires de formation des lignes médianes, l'existence d'une signature transcriptionnelle évolutivement conservée pour le gène clé de l'acquisition de ce destin cellulaire. / Ciona intestinalis tail epidermis has 8 rows of cells defining 3 domains. One of them, the midline domain, gives rise to differentiated cells which form the larval fin and part of peripheral nervous system. Previous work has shown that BMP and FGF signalling are the inducers of ventral and dorsal midlines respectively. My work consisted in the identification of molecular events which lead epidermal cells to adopt midline fate, from induction to tail differentiation. We identified 7 transcription factors involved in this process. Identification of epistatic relationships suggest that these genes are in a hierarchical network where Msxb is a key gene. This hierarchy is validated by 1) a spatio-temporal expression atlas, 2) loss of function of Msxb, 3) cis-regulatory regions analysis for each network gene. We identified 2 types of enhancers, one capable to decouple ventral / dorsal signals used by early genes, and the other used by later genes, acting as a global response in both midlines. We showed that the early expression of Msxb in dorsal precursors is controled by a distinct enhancer, regulated by FGF9/16/20 via Otx and Nodal. This transcriptional signature is found in enhancers of co-expressed genes and in Msxb orthologue in another ascidian. Finally, we showed the most posterior part of the midlines is controlled by a distinct genetic program than the one used in dorsal and ventral midlines. This work gives insight into midlines structure, the mechanisms involved in their formation and a conserved transcriptional signature for the key gene involved in midline cell fate.

Le récepteur à l'IGF1 (IGF-1R) ainsi que ses voies de régulation sous-jacentes ont été largement étudié pour leur importance au cours du développement et leur rôle mitotique sur divers types cellulaires. A l'échelle de la peau, l'IGF-1R contribue à l'homéostasie épidermique et est souvent associé au compartiment basal pour son effet pro-prolifératif. Très peu d'études ont montré son implication au niveau de la différenciation épidermique et celles-ci présentent des résultats contradictoires. Au cours du vieillissement, la peau s'amincit et la barrière épidermique présente des défauts de perméabilité. Parallèlement, l'activité de l'IGF- 1R, maximale à l'adolescence, diminue avec l'âge. Cette étude a contribué à éclaircir le rôle de l'IGF-1R sur la différenciation épidermique et à montrer un lien entre vieillissement, perte en qualité de la peau et diminution d'activité de l'IGF-1R. L'élaboration de modèles 2D et 3D mimant le vieillissement par diminution d'activité de l'IGF-1R, nous a permis de confirmer le rôle mitotique de l'IGF-1R sur les kératinocytes et les progéniteurs et de démontrer son effet régulateur sur la différenciation épidermique par augmentation ou diminution de ces marqueurs. L'IGF-1R renforce l'adhésion cellulaire sur différentes matrices, impliquant de possibles interactions avec les intégrines α6 et β1. Ces résultats ont été corrélés aux observations de biopsies de peaux jeunes et âgées. Nous avons aussi montré que l'IGF-1R conférait un état sénescent aux cellules soumises à de fortes doses d'H2O2. Ces travaux montrent ainsi que l'IGF-1R est nécessaire pour le processus de différenciation épidermique et pour en assurer sa protection face au stress oxydant / Insulin-like growth factor 1 receptor (IGF-1R) and its signaling pathway have been widely studied for their growth promoting role on many cell types and their implication in development. On skin, the IGF-1R function has been associated to basal proliferation and contributes to epidermal morphogenesis, but very little is known about its involvement on keratinocytes differentiation and the few studies existing depict contradictory results. IGF-1R activity is maximal during teenage and tend to decrease during aging. Aged skin depicts major thinning and defects in permeability of skin barrier. Our work consisted in clarifying IGF-1R role on epidermal differentiation process and emphasized a correlation between aging, loss of skin quality and IGF-1R activity. By building 2D and 3D aging like models with low IGF-1R activity, we confirmed IGF-1R mitogenic role on both basal and progenitor-like keratinocytes. We demonstrated that IGF-1R activity regulated keratinocytes differentiation by either enhancing or slowing down differentiation markers deposition. More importantly, we highlighted the importance of IGF-1R activity for keratinocytes adhesion on both laminin-332 and collagen I/IV coatings, implying possible interactions with α6 and β1 integrins. This relationship was further correlated on skin biopsies of young and aged donors. In a parallel study, we showed that IGF-1R could induce cell senescence under acute H2O2 stress. Taken together, IGF-1R is necessary for the epidermal differentiation process and protects epidermis from acute oxidative stress induced damages

Queratinócitos são um veículo bastante atrativo para a transferência gênica ex vivo e liberação sistêmica uma vez que as proteínas secretadas por estas células podem atingir a circulação via um mecanismo similar ao processo natural. Um eficiente vetor retroviral (LXSN) contendo o gene do hormônio de crescimento de camundongo (mGH) foi utilizado para transduzir queratinócitos humanos primários. Os queratinócitos transduzidos apresentaram um nível de secreção in vitro alto e estável atingindo até 11 g mGH/106 células/dia. Os epitélios formados por estes queratinócitos geneticamente modificados apresentaram, porém, uma queda na taxa de secreção > 80 % quando foram retirados da placa de cultura utilizando um procedimento clássico. A substituição desta metodologia clássica por uma cultura organotípica resolveu completamente este problema. Camundongos anões imunodeficientes (lit/scid) implantados com estes enxertos organotípicos foram acompanhados durante 4 meses, e apresentaram um aumento de peso significativo (P<0,05) nos primeiros 40 dias. Níveis circulatórios de mGH atingiram um pico de 21 ng/mL 1 h após o implante, mas estes níveis rapidamente atingiram níveis basais (~2 ng/mL). Os queratinócitos humanos primários apresentaram portanto altos níveis de expressão in vitro e os maiores níveis circulatórios, porém por um breve período de tempo, reportados até o momento para GH neste tipo de células. Em conjunto com resultados que mostraram uma recuperação considerável da eficiência de secreção de mGH em cultura por enxertos organotípicos retirados dos animais, foram discutidos os fatores que ainda impedem a utilização clínica deste modelo promissor de terapia gênica cutânea. / Keratinocytes are a very attractive vehicle for ex vivo gene transfer and systemic delivery, since proteins secreted by these cells may reach the circulation via a mechanism which mimics the natural process. An efficient retroviral vector (LXSN) encoding the mouse growth hormone gene (mGH) was used to transduce primary human keratinocytes. Organotypic raft cultures were prepared with these genetically modified keratinocytes and were grafted onto immunodeficient dwarf mice (lit/scid). Transduced keratinocytes presented a high and stable in vitro secretion level of up to 11 g mGH/106cells/day. Conventional epidermal sheets made with these genetically modified keratinocytes, however, showed a drop in secretion rates of > 80% simply due to detachment of the epithelium from its substratum. Substitution of conventional grafting methodologies with organotypic raft cultures completely overcame this problem. The stable long-term grafting of such cultures onto immunodeficient dwarf (lit/scid) mice could be followed for more than 4 months, and a significant weight increase (P<0.05) over the control group was observed in the first 40 days. Circulating mGH levels revealed a peak of 21 ng/mL just 1h after grafting, but unfortunately these levels rapidly fell to baseline values (~ 2 ng/mL). mGH-secreting primary human keratinocytes presented the highest in vitro expression and peak circulatory levels reported to date for a form of GH with this type of cells. Together with data showing that excised implants can recover in culture a remarkable fraction of their original in vitro mGH secretion efficiency, the factors that might still hamper the success of this promising model of cutaneous gene therapy are discussed. SUMÁRIO