- Deciphering the non-apoptotic cellular functions controlled by the nuclear form of the pro-apoptotic BAX protein during lung fibrosis.
Brayer S 1,2,3,4, Joannes A 1,2,3,4, Marchal Sommé J 1,2,3,4, Crestani B1,2,3,4,5 and Mailleux AA 1,2,3,4
(1) INSERM U1152, Paris, France. (2) DHU FIRE, Paris, France. (3) Labex Inflamex, Paris, France. (4) Univ Paris Diderot, Sorbonne Paris Cité, France. (5) APHP, Hôpital Bichat, Service de Pneumologie, Paris, France.
Introduction: Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix (ECM) proteins and fibroblasts in the distal airways. An imbalance in apoptosis may participate in the development of lung fibrosis. It becomes evident that the BCL-2 apoptotic regulatory protein family also mediates a wide range of non-apoptotic cellular functions. The proapoptotic BAX protein was reported in the nuclei of non-apoptotic lung cells in vitro. Whether the nuclear form of BAX could be involved in non-apoptotic function during lung fibrogenesis is still unknown.
Methods: BAX nuclear localization was assayed by immunohistochemistry in lung cell types in vitro and in paraffin lung sections from control and IPF patients. We next investigated whether nuclear BAX could be implicated in basic cellular functions in non-dying cells. First, we assayed the effects of BAX knock down on basic cellular functions such as proliferation in A549 cells and Human lung fibroblasts (HLF). We also investigated the effect of BAX knock down on basal myofibroblastic differentiation in HLF. But this approach will not distinguish between cytoplasmic and nuclear functions. For this reason, we next investigated whether overexpression of BAX constructs either preferentially targeted or excluded from nucleus would elicit the opposite phenotypic effects than BAX siRNA in A549 cells and primary HLF.
Results: We showed that BAX was in close proximity to the chromatin in A549 cells and HLF. In contrast with control distal airways, BAX was detected in the nuclei of the hyperplastic epithelial cells and in the fibroblasts organized in foci in IPF patients. In vitro, we showed that decreasing total BAX protein level with siRNA was sufficient to impact cell proliferation in A549 cells and primary HLF. BAX knock down also increased basal myofibroblastic differentiation in primary HLF. Conversely, a BAX construct preferentially targeted to the nucleus generally elicited the opposite phenotype than BAX siRNA suggesting strongly that nuclear BAX was involved in these non-apoptotic functions in vitro.
Conclusions: Our study established a strong link between the nuclear localization of the pro-apoptotic BAX protein and key basic cellular functions involved in fibrogenesis.