Senescent fibroblasts induce moderate stress in lung epithelial cells in vitro
Introduction
The temporal activation of defined signaling molecules, transcription factors, extracellular matrix proteins and their receptors contributes to differentiation and development of the lung epithelium. Direct cell–cell interactions of epithelial with mesenchymal cells and the secretion of a variety of molecules are essential in this complex process. The induction of tracheal buds by pulmonary mesenchyme, which was grafted onto tracheal epithelium, is one of the examples demonstrating the importance of epithelial–mesenchymal interactions in mammals (Alescio and Cassini, 1962). However, the multi-factorial control of epithelial growth and differentiation does not only play a crucial role in developmental processes. In this regard, cell signaling mediated by the fibroblast growth factor (FGF) contributes to cellular function in fetal and adult lung as well (Demayo et al., 2002, McKeehan et al., 1998). In addition to FGF, fibroblasts are a relevant source of other growth promoting factors, cytokines and proteases that can act at a distance within tissues and considerably modify the local tissue microenvironment. The release of those factors strongly depends on tissue localization and functional status of the fibroblasts, which alters during cellular life-span, in response to chronic inflammation or environmental impacts, such as irradiation. In this regard, it has been shown that irradiated fibroblasts of the tumor stroma support the tumor-forming ability of transplanted cells in mice (Barcellos-Hoff and Ravani, 2000), whereas fibroblasts undergoing cellular senescence can alter the epithelial cell differentiation (Parrinello et al., 2005) and cause the oncogenic transformation of pre-neoplastic epithelial cells (Krtolica et al., 2001).
One of the most frequently described effects of fibroblasts or fibroblast-related factors is the induction of growth processes and mediation of cell protection against cellular stress. This is of advantage for stabilizing tissue function and integrity in physiological and pathophysiological conditions (Demayo et al., 2002) but of disadvantage in anti-cancer therapy of solid tumors (Song et al., 2000). However, these data mainly result from experimental studies that used mitotic fibroblasts or isolated factors thereby neglecting age-related changes of the fibroblasts. Therefore, our study focused on the impact of senescent fibroblasts on the induction of mitogenic and cell death-protective signaling pathways in human lung epithelial cells.
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Cell culture
National Cancer Institute (NCI)-H358 lung epithelial cell line and primary embryonic WI-38 lung fibroblasts (ATCC cell bank; Manassas, VA) were used. WI-38 cells undergo replicative senescence after multiple cell passages, that was assessed by reduction of the population doubling and positive cell staining for acid β-galactosidase (Fig. 1A) as previously described (Bartling et al., 2006). H358 cells were stably transfected with the mammalian expression vector pIRES2-enhanced green fluorescent
Impact of fibroblasts on spontaneous epithelial cell death
Direct co-culture with WI-38 lung fibroblast as well as the use of conditioned medium from WI-38 fibroblasts with lung epithelial H358 cells resulted in a diminished amount of spontaneous cell death of H358 cells in vitro. This has been demonstrated by reduced plasma membrane leakage and mitochondrial dysfunction of H358 cells (Fig. 1B and C). However, the protection against mitochondrial dysfunction was less pronounced in response to senescent compared with pre-senescent fibroblasts (Fig. 1C).
Discussion
Replicative senescence limits the proliferation of normal somatic cells in culture causing an irreversible growth arrest and, in contrast to post-mitotic mature cells, multiple changes in their cell function. Those functional changes include the secretion of a variety of soluble molecules, including metalloproteinases, pro-inflammatory cytokines and growth factors (reviewed in Krtolica and Campisi, 2002) that can strictly impair the local tissue homeostasis. On the basis of
Acknowledgements
This project was supported by Wilhelm Roux grant of the Bundesministerium für Bildung und Forschung, BMBF (FKZ7/04) and by the Deutsche Forschungsgemeinschaft, DFG (SFB 598/TPA5).
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