PI3K p110γ overexpression in idiopathic pulmonary fibrosis lung tissue and fibroblast cells: in vitro effects of its inhibition

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease whose molecular pathogenesis remains unclear. In a recent paper, we demonstrated a key role for the PI3K pathway in both proliferation and differentiation into myofibroblasts of normal human lung fibroblasts treated with TGF-β. In this research, we assessed the expression of class I PI3K p110 isoforms in IPF lung tissue as well as in tissue-derived fibroblast cell lines. Moreover, we investigated the in vitro effects of the selective inhibition of p110 isoforms on IPF fibroblast proliferation and fibrogenic activity. IHC was performed on normal and IPF lung tissue. Expression levels of PI3K p110 isoforms were evaluated by western blot and flow cytometry analysis. Fibroblast cell lines were established from both normal and IPF tissue and the effects of selective pharmacological inhibition as well as specific gene silencing by small interfering RNAs were studied in vitro. No significant differences between normal and IPF tissue/tissue-derived fibroblasts were observed for the expression of PI3K p110 α, β and δ isoforms whereas p110γ was more greatly expressed in both IPF lung homogenates and ex vivo fibroblast cell lines. Myofibroblasts and bronchiolar basal cells in IPF lungs exhibited strong immunoreactivity for p110γ. Positive staining for the markers of proliferation proliferating cell nuclear antigen and cyclin D1 was also shown in cells of fibrolastic foci. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as α-SMA expression in IPF fibroblasts. Our data suggest that PI3K p110γ isoform may have an important role in the etio-pathology of IPF and can be a specific pharmacological target.

Common pathways in idiopathic pulmonary fibrosis and cancer

“…Furthermore, both p110c pharmacological inhibition and gene silencing were able to significantly inhibit proliferation, as well as a-smooth muscle actin expression in IPF fibroblasts. These data suggest that the PI3K p110c isoform may have an important role in the pathogenesis of IPF and can be a specific pharmacological target [54]. In this regard, it has already been reported that oral administration of a p110c inhibitor significantly prevents bleomycin-induced pulmonary fibrosis in rats [55].…”

Effects of thymosin β4 and its N-terminal fragment Ac-SDKP on TGF-β-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis

“…Primary normal human lung fibroblast cell lines were established as previously described [14]. IPF lung fibroblast cell lines were established from explanted organs from IPF patients who had received a lung transplant.…”

“…qPCR was performed by using the commercially available Taqman assays for human a-SMA (ACTA2), COL-I (COL-IA1) and the housekeeping GAPDH gene (QIAGEN, Germany) following the manufacturer's instructions. Amplification conditions were identical for all genes: 94 C, 5 min and 45 cycles of two steps: 95 C, 15 s and 60 C, 30 s. Relative quantification of target gene levels was performed by comparing DCt as described elsewhere [15,16].…”

The role of Endoplasmic Reticulum (ER) stress in pulmonary fibrosis

“…The activation and differentiation of fibroblasts into apoptosisresistant, ECM-producing myofibroblasts is a critical factor towards the development of IPF. The accumulation of myofibroblasts within pathologic lesions called fibroblast foci (FF) is a hallmark of IPF lungs, and their fatal apoptosis-resistance promotes their expansion and the consecutive exaggerated ECM deposition in IPF lungs [43,234,235]. Transforming growth factor-beta (TGF-β) is a major stimulus of ECM production in fibroproliferative diseases [236].…”

“…In conclusion, a cytoprotective (adaptive) ER stressresponse takes places in contractile protein-expressing myofibroblasts, presumably in order to restore and maintain ER proteostasis under conditions of elevated ECM protein production [232,233,238], and maybe also to allow the continuous production of various survivalrelated proteins, such as survivin, PI3Kγ, and members of the inhibitor of apoptosis (IAP) protein family which maintain the apoptosis-resistance of myofibroblasts and which all have been reported to be upregulated in myofibroblastic cell populations in experimental lung fibrosis and IPF in vivo [234,235,249].…”