Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine

Abstract

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

Fig. 1.

Depletion of PtdIns(3,5)P₂ in PIPKIII-deficient ES cells. (A) Defective PtdIns(3,5)P₂ production. PipkIII^+/+ (WT) and PipkIII^neo/neo (KO) ES cells were labeled with [³H]-inositol for 48 h, followed by treatment (or not) for 10 min with 10 mM TEA. Lipids were extracted, and PtdIns(3,5)P₂ levels were determined by HPLC. PtdIns(3,5)P₂ levels are expressed as a percentage of total phosphoinositide levels and are the mean ± SEM (n = 3). (B) Increased PtdIns(3)P and normal PtdIns(4)P, PtdIns(3,4)P₂, and PtdIns(4,5)P₂. WT and KO ES cells were labeled with [³²P]-orthophosphate under steady-state conditions. Lipids were extracted, and the indicated phosphoinositides were analyzed by HPLC and plotted in chromatograms. PtdIns(5)P and PtdIns(3,4,5)P₃ were undetectable under the experimental conditions used. Similar results were obtained in five replicate experiments (Fig. S3). (C) Loss of PIPKIII results in vacuolation. Untreated KO ES cells (i) show the same vacuolation phenotype as WT ES cells that were transfected with an shRNA against PipkIII (ii); transfected with a dominant-negative K1831E PIPKIII expression vector (iii); or treated with 800 nM YM-201636 (PIPKIII inhibitor) (iv). Cells were subjected to differential interference contrast (DIC) imaging. (D) PIPKIII expression prevents vacuolation. KO ES cells were cotransfected with a PIPKIII expression vector (pCAGGS PIPKIII) plus an EGFP expression vector (pEGFP). DIC and fluorescent images were captured at 24 h after transfection. Arrows indicate a transfected, PIPKIII-expressing KO ES cell. Results shown are representative of at least three independent experiments.

Fig. 2.

Defective embryogenesis in the absence of PIPKIII. (A) Bright-field stereomicroscopic views of H&E-stained longitudinal sections from control PipkIII^+/+ (WT) or PipkIII^+/neo (Het) and PipkIII^neo/neo (KO) embryos in decidua. VE cells, but not epiblasts, were vacuolated in the mutant embryos at the egg cylinder stage. (B) LAMP1-positive swollen vacuoles (white arrowheads) in the KO VE. Sections of WT and KO E6.5 embryos were subjected to LAMP1 staining to detect lysosomal compartments. (C) Diagram of mouse embryo at E6.0 and E6.5. (D) Reduced endocytosis in the VE. WT and KO E6.2 embryos were isolated from deciduae, freed from the parietal endoderm, labeled with rhodamine-labeled dextran for 15 min, and incubated for 50 min. The stained embryos were fixed and serial optical sections were viewed under a laser scanning confocal microscope. A parasagittal focal plane is shown. (E) Abnormal localization of maternal IgG in VE of KO E6.5 embryos. At low magnification, IgG puncta were reduced in the mutant VE layer compared with the WT, and IgG bound abnormally to the apical surface of KO VE cells (white arrowheads). (E Right) Higher magnification of the Inset areas in Left reveals that most IgG-positive vacuoles in WT VE cells are also positive for LAMP1 (white arrows), whereas the LAMP1-positive, swollen apical vacuoles in KO VE cells (asterisks) contain no IgG. Results shown are representative of at least three independent experiments.

Fig. 3.

Mortality and intestinal abnormalities of VilCrePipkIII^flox/flox mice. (A) Shortened lifespan. Survival curves of VilCrePipkIII^flox/flox (cKO) and control PipkIII^flox/flox or PipkIII^flox/+ (Ctrl) mice are shown. (B) Reduced body weight. Ctrl and cKO littermates were weighed at 3 wk and 5 wk after birth. Results shown are the mean body weight ± SEM (n = 18–50 mice per genotype). (C) Malnutrition. The indicated nutritional parameters were measured in sera from Ctrl and cKO mice (n = 4 per genotype). (D) Spontaneous inflammation and fibrosis in the ileum. H&E (Upper) and Azan (Lower; blue) staining of consecutive sections of the ileum from 4-wk-old Ctrl and cKO mice. The mutant displays vacuolation of enterocytes (arrows), lymphocyte infiltration (dashed yellow line), and fibrogenic changes (blue) in the lamina propria and muscularis mucosa. For B and C, *P < 0.05; **P < 0.01 (Student t test). Results shown are representative of at least three independent experiments, except D, where results are representative of at least 50 mice examined per genotype.

Fig. 4.

Altered gene expression pattern and compromised apical membrane structure in the small intestine of VilCrePipkIII^flox/flox mice. (A and B) Enhanced expression of genes related to inflammation (A) and fibrosis (B). The mRNA expression of the indicated genes in intestinal mucosa from PipkIII^flox/flox (Ctrl) and VilCrePipkIII^flox/flox (cKO) mice (three pairs from three independent litters; 6-wk-old males) was examined by using cDNA microarray profiling. The mRNA expression level of each gene was normalized to β-actin mRNA. Results are expressed as fold induction in the cKO over the Ctrl. The up-regulation of the genes was also confirmed by RT–PCR (Insets). For A and B, results shown are representative of at least three independent experiments. (C–F) Abnormal intestinal epithelium. Small intestines from Ctrl and cKO mice were examined by electron microscopy (C) and fluorescent imaging (red) plus DAPI counterstaining (blue) (D–F). (C) A cKO enterocyte shows shortened microvilli and an enlarged vacuole (V) compared with a Ctrl enterocyte. (D) Phalloidin staining reveals decreased F-actin underneath the apical plasma membrane of a cKO enterocyte. (E and F) Mislocalization of apical proteins. The ilea of Ctrl and cKO mice were subjected to immunofluorescent microscopy to detect intracellular localization of DPPIV (E) and IAP (F). cKO cells showed abnormal punctate and cytosolic staining of these proteins. For C–F, results shown are representative of at least five mice examined per genotype.