PI3K–AKT pathway mediates growth and survival signals during development of fetal mouse lung
Introduction
The serine/threonine protein kinase protein kinase B (also known as AKT) and the mitogen-activated protein kinase (MAPK) signalling pathways are involved in multiple cellular functions, such as cell proliferation, differentiation, and survival (Su and Karin, 1996, Rameh and Cantley, 1999). AKT activity is regulated by phosphatidylinositide (PI) 3-kinase (PI3K) (James et al., 1996). Phosphorylation of Thr-308 and Ser-473 is required for AKT activity (Alessi et al., 1996). After AKT is activated, it dissociates from the membrane and enters the nucleus. A number of proteins are likely to be phosphorylated in the cytoplasm and the nucleus (Lawlor and Alessi, 2001). MAPK-specific inhibitor PD98059 has been shown to reduce MAPK (ERK1/2) signalling (Dudley et al., 1995). Activation of AKT was inhibited by PI3K inhibitors LY294002 and wortmannin (Ui et al., 1995, Vlahos et al., 1994).
Peptide growth factors such as fibroblast growth factors (FGFs) and bone morphogenetic protein-4 (BMP-4), as well as morphogens such as sonic hedgehog and retinoids, mediate signalling in the lung (Minoo, 2000). FGFs, which activate the PI3K–AKT and MAPK pathways, are critical to the development of many branched organs and act to promote branching of the kidney, lung, mammary gland, prostate and salivary gland (Davies, 2002). However, the relationship between the signaling pathways and lung development has not been fully elucidated.
In the fetal hamster lung, cell proliferation activity is high in the early period and becomes decreased with increased expression of a cyclin-dependent kinase inhibitor, p27Kip1 (Ikoma et al., 2001). Downstream effectors of PI3K and ERK activation such as Rac1 and Cdc42 stimulate expression of cyclin D1, a key regulator of the mammalian cell cycle (Ammii and Panettieri, 2001). AKT acts to promote activation of cyclin-dependent kinases by reducing the expression of inhibitors such as p21Cip1 and by stabilizing protein levels of cell cycle activators such as cyclin D1 (Lawlor and Alessi, 2001, Diehl et al., 1998).
In the lung, cellular differentiation is complex, with several morphologically distinct cell types comprising the airway epithelium (Bohinski et al., 1994). The primitive early embryonic epithelium can be induced to express specific peripheral cell lineages and the lung epithelial cell lineages become restricted to specific region of the airway in later gestation (Warburton et al., 2000). Cell differentiation is determined by various transcription factor systems (Warburton et al., 2000). It was reported that a family of lung-specific gene promoters that included the surfactant apoproteins and Clara cell secretory protein gene promoters were specifically activated by thyroid transcription factor-1 (TTF-1) (Bohinski et al., 1994). Neuroendocrine differentiation depends on a network of basic helix-loop-helix factors (Ito et al., 2000). FGF-7 induced the expressions of surfactant apoprotein C (SPC) and TTF-1, and enhanced respiratory epithelial cell differentiation and proliferation (Tichelaar et al., 2000). However, signalling via the PI3K–AKT pathway to transcription factors remains unclear.
Apoptosis has been demonstrated to occur at several stages of lung development (Kresch et al., 1998), but the significance and regulatory mechanisms of apoptosis in the development of the lung have not been fully investigated. The incidence of apoptotic cells in fetal lungs is relatively low in the epithelium and mesenchyme. According to Stiles et al. (2001), the occurrence of terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive cells is rare in the ealier stage, high (up to 10%) at the transition from the psuedoglandular stage to the canalicular stage, rare (0.1%) again, and then increases up to 0.7% at the transition from the canalicular stage to the saccular stage. In an explant culture experiment of fetal rat lungs, the MAPK pathway was demonstrated to be important in maintaining cell survival (Kling et al., 2002). AKT is reported to directly phosphorylate and inhibit the caspase proteases, key executioners of apoptosis (Lawlor and Alessi, 2001), but it is not clear how the PI3K–AKT pathway is involved in lung cell apoptosis during development.
The present study examined the expression of AKT and MAPK during mouse lung development focusing on the phosphorylation status of these molecules. Moreover, we examined the roles of AKT and MAPK during lung development using a mouse lung organ culture system. Inhibition studies of PI3K and MAPK, using inhibitors LY294002 and wortmannin for PI3K and PD98059 for MAPK, were also done to reveal the function of the signal pathways. Through these experiments, we demonstrated the significance of the PI3K–AKT signalling pathway in lung development.
Section snippets
Animals
ICR mice were purchased from Japan SLC (Shizuoka, Japan). To obtain fetal lungs, mice were mated during the night, and the day of the discovery of the vaginal plug was counted as embryonic day (E) 0. For the present studies, lungs of gestational days 12–18 (E12, E14, E16, E18) of ICR mice were used. For organ culture, lungs from E11.5 mice were used and cultured for 72 h.
Immunohistochemistry and Western blotting analysis of embryonic mouse lungs
Immunohistochemical detection in embryonic lung in vivo was performed. Six pairs of fetal lungs at E14 and E18 were fixed in a
Expression of AKT/pAKT, ERK/pERK, GSK/pGSK, cyclin D1, p21Cip1 and SPC in embryonic mouse lungs
We performed the Western blotting for AKT/pAKT and ERK/pERK in embryonic lungs at 12, 14, 16 and 18 days (Fig. 1). Expression of AKT and ERK were abundant and unchanged in embryonic lungs during development. However, pAKT and pERK were the highest at E12 and decreased gradually thereafter, suggesting that pAKT and pERK play important roles in the earlier stage of lung development. We also examined expression of one of downstream proteins of the PI3K–AKT signalling pathway, GSK/pGSK. pGSK
Discussion
In the present study we surveyed expression profiles of AKT, MAPK and their phosphorylated forms in fetal developing lungs of mice, and compared them with those of cell proliferation regulators, cyclin D1 as a cell proliferation activator and p21Cip1 as a repressor, and with a cell differentiation marker, SPC. pAKT and pERK were expressed more in the earlier stages of fetal lung development, and decreased before birth. Their decreasing expression pattern is parallel to the changes in cell
Acknowledgements
The authors thank H. Mitsui and T. Suzuki for their excellent technical assistance. This study is supported partly by the grant from the Smoking Research Foundation of Japan and from Research Promotion Grant (Umehara Fund) of Yokohama Medical Research Foundation.
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