Plausible role of INPP4A dysregulation in idiopathic pulmonary fibrosis

Abstract

INPP4A has been shown to be involved in the regulation of cell proliferation and apoptosis of multiple cell types including fibroblasts. Previous reports from our group have demonstrated the role of inositol polyphosphate 4-phosphatase Type I A (INPP4A) in these functions. Though existing evidences suggest a critical role for INPP4A in the maintenance of lung homeostasis, its role in chronic lung diseases is relatively under explored. In the current study, we made an attempt to understand the regulation of INPP4A in idiopathic pulmonary fibrosis (IPF). Through integration of relevant INPP4A gene expression data from public repositories with our results from in vitro experiments and mouse models, we show that INPP4A is altered in IPF. Interestingly, the direction of the change is dependent both on the disease stage and the region of the lung used. INPP4A was found to be upregulated when analyzed in lung sample representative of the whole lung, but was downregulated in the fibrotic regions of the lung. Similarly, INPP4A was found to be high, compared to controls, only in the early stage of the disease. Though the observed increase in INPP4A was found to be negatively correlated to physiological indices, FVC, and DL_CO, of lung function, treatment with anti-INPP4A antibody worsened the condition in bleomycin treated mice. These contrasting results taken together are suggestive of a nuanced regulation of INPP4A in IPF which is dependent on the disease stage, cellular state and extent of fibrosis in the lung region being analyzed.

Keywords: INPP4A; bleomycin; disease heterogeneity; idiopathic pulmonary fibrosis; transforming growth factor‐β.

FIGURE 1

Inositol Polyphosphate‐4‐Phosphatase Type I A (INPP4A) is upregulated in Idiopathic Pulmonary Fibrosis. Normalized INPP4A gene expression data from control and IPF whole lungs in microarray based GEO datasets namely (a) GSE32537 and (b) GSE53845 and RNA‐sequencing based GEO datasets namely (c) GSE124685 (d) GSE199949 and (e) GSE213001 and (f) GSE134692 (only batch 1 samples were considered for normalizing batch effects). No of subjects in each category is indicated on all graphs as “n.” Information about datasets also provided in Table 2. Statistical significance was calculated using unpaired t‐test for (d) and (f), unpaired t‐test with Welch's correction in (a) and (c), Mann–Whitney test for (b) and (e). Relative expression levels are represented as fragments per kilobase of transcript per million mapped reads (FPKM) or reads per kilobase of exon per million reads mapped (RPKM) or transcript per million (TPM) or counts per million (CPM). The numbers in parentheses are indicative of p‐values for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ctrl, Control; IPF, idiopathic pulmonary fibrosis; UIP, usual interstitial pneumonia.

FIGURE 2

Inositol Polyphosphate‐4‐Phosphatase Type I A (INPP4A) gene expression correlates with forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) in idiopathic pulmonary fibrosis. Pearson correlation coefficients of FVC as a function of lung tissue expression of INPP4A for (a) IPF; n = 157 (b) non‐IPF interstitial lung disease (non‐IPF ILD); n = 82 (c) combined cohort of IPF and non‐IPF ILD patients; n = 239. Pearson correlation coefficients of DL_CO as a function of INPP4A gene expression in lung tissues for (d) IPF; n = 145 (e) non‐IPF ILD; n = 67 and (f) combined cohort of IPF and non‐IPF ILD; n = 212. Data for lung function parameters, percent predicted FVC and percent predicted DL_CO and INPP4A lung tissue expression were extracted from lung tissue research consortium (LTRC) dataset GSE47460. INPP4A square root (sqrt) expression denotes sqrt–transformed hybridisation signal intensities from the mentioned microarray dataset. R ² (where R values denote Pearson correlation coefficient) and p‐values are mentioned in the figure. n indicate number of subjects/samples in each case. ILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis.

FIGURE 3

Inositol Polyphosphate‐4‐Phosphatase Type I A (INPP4A) expression is increased in pulmonary fibrosis. (a) Representative images for immunohistochemical staining performed for INPP4A on lung tissue sections of control and IPF patients. Bronchiolar and alveolar regions of IPF lung tissues were labeled to show INPP4A expression in these regions. Protein of interest can be seen brown colored due to DAB staining and nuclei are labeled blue due to hematoxylin staining. Scale bars indicate 200 μm. Original magnification is 20×. (b) Quantification of Figure 3a. Normalized DAB intensity indicates normalization of total protein expression value by value of number of nuclei in each section/image. Number of fields unbiased and random, for covering the entire lung section with high confidence are mentioned in the figure. (c) Representative immunofluorescence confocal microscopy images used to study INPP4A expression. INPP4A (green), nuclei (blue). Scale bar = 20 μm. Original magnification 20× (d) INPP4A quantification from Figure 3c. Number of fields per sample are indicated. n indicates number of subjects/samples in each case. Statistical significance was calculated using Mann–Whitney test for (b) and unpaired t‐test for (d). The numbers in parentheses are indicative of p‐values for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. IPF, idiopathic pulmonary fibrosis.

FIGURE 4

Inositol Polyphosphate‐4‐Phosphatase Type I A (INPP4A) expression is increased in bleomycin induced pulmonary fibrosis in mice. (a) Representative images for immunohistochemical staining performed on lung tissue sections to determine INPP4A expression in vehicle (PBS) and bleomycin (Bleo) treated mice. Interstitial or alveolar lung regions shown here. Protein of interest appeared brown due to DAB staining and nuclei were labeled blue due to hematoxylin staining. Scale bars indicate 100 μm. Original magnification is 20×. (b) Quantification of Figure 4a. Normalized DAB intensity indicates normalization of total protein by number of nuclei in the field. Number of fields imaged per sample are indicated in the figure. DAB: 3,3′‐Diaminobenzidine. (c) Representative immunofluorescence confocal microscopy images of vehicle/PBS (n = 3) and bleomycin treated mice (n = 4) lung tissue sections stained with anti‐INPP4A (red) and nuclear stain, DAPI (blue). Original magnification is 20× (d) Quantification of Figure 4c (e) Cellular or tissue level INPP4A expression in total lung protein (TLP) of vehicle and bleomycin treated mice, determined by ELISA. Statistical significance was calculated using Mann–Whitney test in (b) and (d) and using unpaired t‐test in (d). The numbers in parentheses are indicative of p‐values for statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

INPP4A gene dysregulation is highest in non‐fibrotic or lesser fibrotic IPF tissues. Box plot representing median and interquartile ranges using data mined from RNA sequencing based datasets for (a) INPP4A expression in control alveolar septae, IPF alveolar septae and IPF fibroblastic foci isolated using laser microdissection in GEO dataset GSE169500. (b) INPP4A gene expression levels in selected lung tissue regions of non‐disease control donors and IPF patients; in GEO dataset GSE213001. Base and apex denote samples derived from base (end‐stage) and matched lung apices (pre‐terminal disease) lung regions from controls and IPF patients. (c) INPP4A expression in GEO dataset GSE199949. Sample labelling indicates macroscopically non‐fibrotic central (IPF central) and fibrotic peripheral (IPF peripheral) areas. Donor lung, non‐IPF biopsies are labeled similarly as central (Ctrl central) and peripheral (Ctrl peripheral). (d) INPP4A expression in isolated lung lobes of IPF categorized as control/early‐stage IPF (IPF1), progressive IPF2 (IPF2), and severe/end‐stage IPF (IPF3) based on micro CT and Ashcroft staining (histological staining) from GEO dataset GSE124685 (e) Differential expression of INPP4A in control and IPF lung biopsy and transplant samples in microarray dataset GSE24206. Additional information about datasets used here in Table 3. Statistical significance was calculated using one‐way ANOVA with Tukey's multiple comparison's test for (a), Kruskal–Wallis test for (b), unpaired t‐test for (c), one way ANOVA with Dunnett's multiple comparison test for (d) and one way ANOVA for (e). RPKM: reads per kilobase million; TPM: Transcript per million. FPKM: Fragments per Kilobase of transcript per Million mapped reads. The numbers in parentheses are indicative of p values for statistical significance between the marked groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non‐significant. n indicates no of subjects in each case.

FIGURE 6

INPP4A Expression is reduced in TGF‐β1 induced myofibroblasts. (a) Western blot analysis of alveolar epithelial cells (A549) treated with vehicle (Veh) or TGF‐β1 for 120 h.; total cell lysates were extracted and analyzed for expression of E‐cadherin, vimentin and INPP4A using immunoblotting. β‐Actin was used as an internal control. Densitometric quantification of Figure 6a for (b) E‐Cadherin and (c) Vimentin and (d) Total INPP4A (sum of cytoplasmic INPP4A that is, 107KDa and nuclear INPP4A that is, 120KDa band intensities). (e) INPP4A levels in TGF‐β1 treated rat precision cut lung slices (PCLS), GSE120679. Relative expression levels are represented as counts per million (CPM). The numbers in parentheses are p values indicating statistical significance calculated using unpaired t‐test in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data shown are means ± SD. Minimum number of independent experiments in each case is three.

FIGURE 7

Secretory INPP4A is protective in mice with bleomycin induced pulmonary fibrosis. (a) Measurement of secretory/extracellular INPP4A levels in bleomycin treated mice by measuring INPP4A expression in bronchoalveolar lavage (BAL) fluid determined by ELISA. n represents number of mice in each case. (b) Schematic of experimental protocol used for anti‐INPP4A antibody treatment in bleomycin treated mice with pulmonary fibrosis (c) Representative images for H&E stained sections for different groups as mentioned. Original magnification is 10×. Scale bars represent 200 μm (d) TGF‐ β1 cytokine levels in TLP prepared from lungs of mice treated with isotype or anti‐ INPP4A antibody as measured by ELISA (e) Amount of soluble collagen in the lungs of mice treated with isotype or anti‐INPP4A antibody measured by Sircol assay (f) Lung elastance measurement after treatment with anti‐INPP4A or isotype antibody in mice induced with bleomycin (g) Total leukocyte count (TLC) in mice given bleomycin followed by anti‐INPP4A antibody treatment. TLP = total lung protein. Statistical significance was calculated using Mann–Whitney test for (a), Kruskal–Wallis test for (c) and (f), one way ANOVA with Tukey's multiple comparisons test for (d) and Mann–Whitney test for (e). The numbers in parentheses are indicative of respective p values as marked between the groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant. Number of animals is five or more in each group as indicated by dots. Data shown are means ± SD.