RESUMO
Sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) pumps belong to the family of Ca2+-ATPases responsible for the maintenance of calcium in the endoplasmic reticulum. In epidermal keratinocytes, SERCA2-controlled calcium stores are involved in cell cycle exit and onset of terminal differentiation. Hence, their dysfunction was thought to provoke impaired keratinocyte cohesion and hampered terminal differentiation. Here, we assessed cultured keratinocytes and skin biopsies from a canine family with an inherited skin blistering disorder. Cells from lesional and phenotypically normal areas of one of these dogs revealed affected calcium homeostasis due to depleted SERCA2-gated stores. In phenotypically normal patient cells, this defect compromised upregulation of p21(WAF1) and delayed the exit from the cell cycle. Despite this abnormality it failed to impede the terminal differentiation process in the long term but instead coincided with enhanced apoptosis and appearance of chronic wounds, suggestive of secondary mutations. Collectively, these findings provide the first survey on phenotypic consequences of depleted SERCA-gated stores for epidermal homeostasis that explain how depleted SERCA2 calcium stores provoke focal lesions rather than generalized dermatoses, a phenotype highly reminiscent of the human genodermatosis Darier disease.
Assuntos
Vesícula/patologia , ATPases Transportadoras de Cálcio/genética , Doença de Darier/genética , Epiderme/patologia , Animais , Vesícula/genética , Adesão Celular , Ciclo Celular , Diferenciação Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Doença de Darier/patologia , Cães , Retículo Endoplasmático/metabolismo , Epiderme/química , Humanos , Junções Intercelulares/genética , Queratinócitos/química , Queratinócitos/patologia , Antígeno Ki-67/análise , Masculino , Mutação , Fenótipo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Regulação para CimaRESUMO
Ca(2+) signaling in cells is largely governed by Ca(2+) diffusion and Ca(2+) binding to mobile and stationary Ca(2+) buffers, including organelles. To examine Ca(2+) signaling in cardiac atrial myocytes, a mathematical model of Ca(2+) diffusion was developed which represents several subcellular compartments, including a subsarcolemmal space with restricted diffusion, a myofilament space, and the cytosol. The model was used to quantitatively simulate experimental Ca(2+) signals in terms of amplitude, time course, and spatial features. For experimental reference data, L-type Ca(2+) currents were recorded from atrial cells with the whole-cell voltage-clamp technique. Ca(2+) signals were simultaneously imaged with the fluorescent Ca(2+) indicator Fluo-3 and a laser-scanning confocal microscope. The simulations indicate that in atrial myocytes lacking T-tubules, Ca(2+) movement from the cell membrane to the center of the cells relies strongly on the presence of mobile Ca(2+) buffers, particularly when the sarcoplasmic reticulum is inhibited pharmacologically. Furthermore, during the influx of Ca(2+) large and steep concentration gradients are predicted between the cytosol and the submicroscopically narrow subsarcolemmal space. In addition, the computations revealed that, despite its low Ca(2+) affinity, ATP acts as a significant buffer and carrier for Ca(2+), even at the modest elevations of [Ca(2+)](i) reached during influx of Ca(2+).