Inhibition of PI3K prevents the proliferation and differentiation of human lung fibroblasts into myofibroblasts: the role of class I P110 isoforms

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-β (TGF-β)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-β signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-β: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-β-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and β. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-β-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.

Figure 1. Effects of PI3Ks pan-inhibitor LY294002 on TGF-β-induced proliferation of human lung fibroblasts and their differentiation into myofibroblasts.

Ex vivo human lung fibroblasts were cultured in vitro and all cell lines (from five different donors) were used at a passage earlier than eight. Prior to treatment, cells were incubated for 24 hrs in serum-free RPMI medium, then left resting or treated with LY294002, at indicated concentrations, one hour before subsequent TGF-β stimulation (10 ng/ml) in the absence or presence of LY294002. Afterward, cells were incubated for 48 hrs in serum-free medium and then harvested. A) Representative western blot analysis of phosphorylated (Ser⁴⁷³) Akt and pertinent graph with the pAKT/α-tubulin ratios of five separate experiments. B) Results of cell counting by optical microscopy (grey columns) and WST-1 cell proliferation assay (black columns) were represented as % increase over control (untreated cells, assumed as 100%). C) Flow-cytometry analysis of α-SMA expression: mean values (± sd) of Mean Fluorescence Intensity (MFI) are indicated. Plots of one representative experiment are shown in FigureS1. D) Total collagen levels, measured by the Sircol Soluble Collagen Assay, were reported as % increase over control. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a statistically significant difference among relevant groups, is designated with an asterisk.

Figure 2. Expression of PI3Kclass IA p110δ and class IB p110γ in human ex-vivo lung fibroblasts.

A) RT-PCR products of PI3K p110δ run in 2% agarose gel (upper line) and western blot analysis of proteins from four different fibroblast cell lines (medium line). The protein loading control was α-tubulin (bottom line). A commercially available RT-PCR kit and a monoclonal rabbit anti human PI3K p110δ (1∶500) were used as detailed in the Mat and Met section. B) RT-PCR products of PI3K p110γ run in 2% agarose gel and western blot analysis of proteins from the same fibroblast cell lines indicated in panel A. A commercially available RT-PCR kit and a monoclonal mouse anti human PI3Kp110γ (1∶500) were used.

Figure 3. Effects of selective PI3K p110α inhibition.

Ex vivo human lung fibroblasts (from three different donors) were cultured and treated with YM-024 following the protocol indicated in Figure 1. A) Representative western blot analysis of phosphorylated (Ser⁴⁷³) Akt and pertinent graph with pAKT/α-tubulin ratios of three separate experiments. B) Results of cell counting by optical microscopy (grey columns) and WST-1 cell proliferation assay (black columns). C) Flow-cytometry analysis of α-SMA expression: MFI mean values (± sd) of three separate experiments are indicated. Plots of one representative experiment are shown in FigureS2. D) Total collagen levels were reported as % increase over control. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a statistically significant difference among relevant groups, is designated with an asterisk.

Figure 4. Effects of selective PI3K p110β inhibition.

Ex vivo human lung fibroblasts (from three different donors) were cultured, and treated with TGX-221 following the protocol indicated in Figure 1. A) Representative western blot analysis of phosphorylated (Ser⁴⁷³) Akt and pertinent graph with pAKT/α-tubulin ratios of three separate experiments. B) Results of cell counting by optical microscopy (grey columns) and WST-1 cell proliferation assay (black columns) were represented as % increase over control. C) Flow-cytometry analysis of α-SMA expression: MFI mean values (± sd) of three separate experiments are indicated. Plots of one representative experiment are shown in FigureS3. D) Total collagen levels, were reported as % increase over control. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a statistically significant difference among relevant groups, is designated with an asterisk.

Figure 5. Effects of selective PI3K p110δ inhibition.

Ex vivo human lung fibroblasts (from three different donors) were cultured, and treated with IC-87114 following the protocol indicated in Figure 1. A) Representative western blot analysis of phosphorylated (Ser⁴⁷³) Akt and pertinent graphic with pAKT/α-tubulin ratios of three separate experiments. B) Results of cell counting by optical microscopy (grey columns) and WST-1 cell proliferation assay (black columns) were represented as % increase over control. C) Flow-cytometry analysis of α-SMA expression: MFI mean values (± sd) of three separate experiments are indicated. Plots of one representative experiment are shown in FigureS4. D) Total collagen levels were reported as % increase over control. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a statistically significant difference among relevant groups, is designated with an asterisk.

Figure 6. Effects of selective PI3K p110γ inhibition.

Ex vivo human lung fibroblasts (from three different donors) were cultured, and treated with AS-2524224 following the protocol indicated in Figure 1. A) Representative western blot analysis of phosphorylated (Ser⁴⁷³) Akt and pertinent graph with pAKT/α-tubulin ratios of three separate experiments. B) Results of cell counting by optical microscopy (grey columns) and WST-1 cell proliferation assay (black columns) were represented as % increase over control. C) Flow-cytometry analysis of α-SMA expression: MFI mean values (± sd) of three separate experiments are indicated. Plots of one representative experiment are shown in FigureS5. D) Total collagen levels, were reported as % increase over control. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a statistically significant difference among relevant groups, is designated with an asterisk.

Figure 7. Effects of specific PI3K p110α gene suppression by siRNA.

Ex vivo human lung fibroblasts (from three different donors) were kept resting or transfected with small interfering RNAs (siRNAs) specifically targeting p110α (at the concentrations of 20 and 50 nM) or with negative control (50 nM), for 24 hrs in serum-free RPMI medium, afterward they were kept resting or stimulated with TGF-β (10 ng/ml) for 24 hrs in 2% FBS medium. A) Representative western blot analysis of p110α, phosphorylated (Ser⁴⁷³) Akt and GAPDH expression. with means of p110α-/GAPDH and pAKT/α-GAPDH ratios calculated in three separate experiments B) Results of cell counting by optical microscopy. Medians and standard deviations of at least three separate counts in triplicate wells for three separate experiments are reported. C) Representative Western blot analysis of α-SMA expression. and pertinent graph with means of α-SMA/GAPDH ratios calculated in three separate experiments. D) Total soluble collagen levels in one ml of culture medium, measured by the Sircol Soluble Collagen Assay, were reported as micrograms for ml. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a significant difference, is designated with an asterisk.

Figure 8. Effects of specific PI3K p110γ gene suppression by siRNA.

Ex vivo human lung fibroblasts (from three different donors) were kept resting or transfected with small interfering RNAs (siRNAs) specifically targeting p110γ (at the concentration of 20 and 50 nM) or with negative control (50 nM), for 24 hrs in serum-free RPMI medium, afterward they were kept resting or stimulated with TGF-β (10 ng/ml) for 24 hrs in 2% FBS medium. A) Representative western blot analysis of p110α, phosphorylated (Ser⁴⁷³) Akt and GAPDH expression. with means of p110γ-/GAPDH and pAKT/α-GAPDH ratios calculated in three separate experiments B) Results of cell counting by optical microscopy. Medians and standard deviations of at least three separate counts in triplicate wells for three separate experiments are reported. C) Representative Western blot analysis of α-SMA expression and pertinent graph. Means of α-SMA/GAPDH ratios calculated in three separate experiments are indicated. D) Total soluble collagen levels in one ml of culture medium, measured by the Sircol Soluble Collagen Assay, were reported as micrograms for ml. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a significant difference, is designated with an asterisk.