Identificación de nuevos mecanismos fisiopatológicos en el Síndrome de Kindler

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología MolecularEl Síndrome de Kindler (SK), es una genodermatosis caracterizada clínicamente por la presencia de ampollas acrales, fotosensibilidad y envejecimiento prematuro, atrofia extensiva de la piel y una elevada predisposición a desarrollar carcinomas de la piel y mucosas. La proteína afectada es la kindlina-1 (codificada por el gen FERMT1), un componente de las adhesiones focales de los queratinocitos basales. Las mutaciones en este gen pueden explicar las características clínicas provocadas por la falta de adhesión entre la dermis y la epidermis, pero no aquellas no relacionadas con la fragilidad cutánea (envejecimiento prematuro, fotosensibilidad y predisposición a padecer tumores de piel). El objetivo principal de esta tesis doctoral es el estudio de las bases patológicas de estas manifestaciones, que están firmemente asociadas con alteraciones en el estado redox celular, generado principalmente por las especies reactivas de oxígeno (ROS). Al caracterizar el papel del estrés oxidativo en la patogénesis de la enfermedad, se observó que los queratinocitos de pacientes con SK presentaban un desequilibrio importante en su estado redox. Por un lado, exhibían un estado pro-oxidativo observable mediante el ratio GSSG/GSH (glutatión oxidado/glutatión reducido), así como una menor capacidad de síntesis de novo de este glutatión, que es una de las principales defensas celulares frente a las ROS. Por otra parte, las células SK mostraban un daño oxidativo acentuado en los lípidos de membrana, lo que sugiere la cronicidad del fenómeno. Este estado pro-oxidativo también lo presentaba la línea celular de queratinocitos HaCaT shFERMT1, en la que se ha silenciado parcialmente el gen FERMT1, comportándose de forma análoga a los queratinocitos de pacientes SK respecto a la gestión de las ROS. Además, los análisis de la ultraestructura y función de los principales orgánulos productores de ROS, las mitocondrias, revelaron en los pacientes SK pronunciadas anormalidades morfológicas y funcionales. Al evaluar la fotosensibilidad a la luz ultravioleta, se observó que tanto los queratinocitos de pacientes SK como la línea celular HaCaT shFERMT1 presentaban una mayor fotosensibilidad que sus respectivos controles, la cual se evidenció a través de una menor viabilidad celular. Los análisis transcriptómicos realizados, apoyaron estos datos al revelar la expresión aberrante de algunos genes implicados en la replicación y la reparación del daño al ADN en células SK, eventos que alterados y en paralelo potencian la disminución de la tasa de viabilidad celular. Además de estas alteraciones, los análisis transcriptómicos mostraron múltiples genes desregulados en rutas oncogénicas que podrían favorecer el desarrollo de cáncer de piel en los pacientes SK. Estos datos, en conjunto, señalan bases patológicas novedosas implicadas en las manifestaciones clínicas no relacionadas, a priori, con adhesión presentes en esta genodermatosis. Por último, se generó un modelo de ratón humanizado de SK, que reproducía las principales características histopatológicas de la enfermedad. Además, en este modelo se llevó a cabo la corrección de la enfermedad mediante terapia génica, demostrando que al restaurar la expresión de la proteína kindlina-1 se restauraba el fenotipo fisiológico de la piel.Kindler Syndrome (KS) is a rare heritable skin disorder clinically characterized by acral skin blisters, atrophy of skin, photosensitivity, premature ageing and a high risk of mucocutaneous malignancies. KS results from recessive loss-of-function mutations in the FERMT1 gene that encodes the protein Kindlin-1, a component of the focal adhesions in epithelial cells. However the complex phenotype of KS cannot be exclusively explained based on the adhesive function of Kindlin-1. Therefore, the mechanisms responsible for its clinical features such as photosensitivity, premature ageing and cancer are still unknown. The main goal of this study was to analyze the pathological basis of these clinical features of KS that are not related to cutaneous fragility but to the altered cellular redox state, produced mainly by reactive oxygen species (ROS). Our results, obtained with patient derived keratinocytes and HaCaT shFERMT1 (a keratinocyte cell line with FERMT1 partially silenced), followed a clear trend regarding their oxidative status and mitochondrial alterations. On one hand, they exhibited a pro-oxidative status pointed out by the GSSG/GSH ratio (oxidized glutathione/reduced glutathione), a lower capability to synthesize this glutathione (one of the main cellular defenses against ROS) and also, a high oxidative damage of membrane lipids in KS cells, which suggests a chronic phenomenon. On the other hand, mitochondria were studied, since they are the main source of endogenous ROS. KS patients’ cells showed morphological and functional alterations which are consistent with dysfunctional mitochondria. Our results suggest that alterations of the redox balance and mitochondria in KS may be a potential explanation of the premature skin ageing, photosensitivity and cancer prone phenotype of these patients. Furthermore, our viability assays showed a decrease after ultraviolet light exposure in KS keratinocytes and HaCaT shFERMT1 viability. Using transcriptomic analysis we observed that KS keratinocytes also have a number of altered genes involved in DNA replication and in damage repair. Taking together these data, with the fact that UVB is the main exogenous ROS source and that KS keratinocytes have dysfunctional mitochondria and alterations in the redox balance, we have a possible explanation for the higher risk to develop skin cancer in KS patients. Besides, in the transcriptomic analysis we found that there are several oncogenic genes altered in KS keratinocytes. Finally, we were able to generate a humanized mouse model for KS, which reproduces the main histopathological features of the disease. Also, we could restore collagen IV expression and the physiological phenotype of skin by gene therapy when FERMT1 was reestablished in KS keratinocytes

Oxidative stress and mitochondrial dysfunction in Kindler syndrome

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease. [Methods]: Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.[Results]: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state. [Conclusions]: This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.FL was supported by grants from Instituto de Salud Carlos III (PI11/01225) and Comunidad de Madrid (S2010/BMD- 2359; SKINMODEL). MDR was supported by grants from the Science and Innovation Ministry of Spain SAF2010-16976), Comunidad de Madrid (S2010/BMD-2420; CELLCAM), GENEGRAFT - contract N° HEALTH-F2-2011-261392 and CIBERER ACCI 13-714/172.04. MG is supported in part by ERA-NET grant: E-Rare-2 (SpliceEB). EZ was in part supported by a fellowship from CIBERER and SAF2010-16976.Peer reviewe