Proliferation in the developing intestine is regulated by the endosomal protein Endotubin.

During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTBfl/fl mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.

Identification of novel targets of diabetic nephropathy and PEDF peptide treatment using RNA-seq.

BACKGROUND: Diabetic nephropathy (DN) is a major complication of type1 and type 2 diabetes. Understanding how diabetes regulate transcriptome dynamics in DN is important for understanding the biology of the disease and for guiding development of new treatments. RESULTS: We analyzed the kidney transcriptome of a DN mouse model, D2.B6-Ins2 Akita /MatbJ, before/after treatment with P78-PEDF. Age, weight, and gender-matched mice and wild-type (wt) littermates were treated at 6 weeks (early treatment) or 12 weeks (late treatment) of age for the duration of 6 weeks. Animals were implanted with an osmotic mini pump delivering 0.3 ug/g/day P78-PEDF or vehicle. Using RNA-seq, we identified14,316 transcripts (12,328 coding;1,988 non-coding) that were significant and reliably expressed (FPKM > =1) in diabetic kidneys. Expression of 1,129 (7.9%) including 901 coding genes was altered by diabetes with log2 fold changes (FC) between -86.2 and +86.0 (q < 0.05) compared to wt. Of these, 164 (14.5%) showed increased and 965 (85.5%) decreased expression with FC > 1.5. Coding genes with highest FC in diabetic kidneys include Nhej1 (32.04), Ept1 (8.6), Srd5a2 (-6.55), Aif1 (-6.05), and Angptl7 (-4.71). Early and late stage diabetic groups receiving continuous infusion of P78 showed altered expression of 316/14,316 (2.2%) transcripts, including 121 coding genes compared to non-treated diabetic controls. Of these, 183 were upregulated and 133 downregulated with FC +50.9--93.3 (q < 0.05). P78 reversed diabetes-induced changes in 138/1129 (12.2%) transcripts, including 49/901 (5.44%) coding genes. Nhej1 (-37.94), Tceanc2 (5.76), Ept1 (-4.45), Ugt1a2 (3.03), and Tmsb15l (-3.0) showed the highest FC with treatment. The DNA repair gene, Nhej1 with the greatest FC in diabetic kidneys was completely restored to control levels by both early and late P78 treatments. Expression of other coding genes regulated by diabetes with FC > =(+/-) 1.5 and completely reversed by P78 include Mamdc4, Kdm4b, Tmem252, Selm, and Hpd. RT and QRT-PCR validated expression of gene with FC > (+/-)2.0. Transcriptome changes were also observed between early and late-stage treatments. Precursor non-coding miRNAs showed the highest fold changes in expression in the diabetic and P78 treatment groups. Several diabetic-induced changes were reversed in direction of expression by treatment including Gm24083, GM25953, miR1905, Gm25535, Gm27903, and miR196a1 with FC > =(+/-)20. From Ingenuity pathway analysis (IPA), mitochondrial dysfunction, Nrf-2- mediated oxidative stress and renal injury pathways emerged as key mechanisms in DN. DN-enriching genes in these pathways were reduced in number or regulated in the opposite direction by treatment. CONCLUSIONS: Unique biomarkers and canonical pathways identified in this study may hold the key to understanding mechanisms of DN pathobiology with value for clinical translation. Our data suggest that mitochondrial dysfunction, genotoxicity and oxidative stress are principal events in DN and that P78-PEDF holds promise for its management.

The Endosomal Protein Endotubin Is Required for Enterocyte Differentiation.

BACKGROUND & AIMS: During late embryonic development and through weaning, enterocytes of the ileum are highly endocytic. Defects in endocytosis and trafficking are implicated in neonatal disease, however, the mechanisms regulating trafficking during the developmental period are incompletely understood. The apical endosomal protein endotubin (EDTB) is highly expressed in the late embryonic and neonatal ileum. In epithelial cells in vitro, EDTB regulates both trafficking of tight junction proteins and proliferation through modulation of YAP activity. However, EDTB function during the endocytic stage of development of the intestine is unknown. METHODS: By using Villin-CreERT2, we induced knockout of EDTB during late gestation and analyzed the impact on endocytic compartments and enterocyte structure in neonates using immunofluorescence, immunocytochemistry, and transmission electron microscopy. RESULTS: Deletion of the apical endosomal protein EDTB in the small intestine during development impairs enterocyte morphogenesis, including loss of the apical endocytic complex, defective formation of the lysosomal compartment, and some cells had large microvillus-rich inclusions similar to those observed in microvillus inclusion disease. There also was a decrease in apical endocytosis and mislocalization of proteins involved in apical trafficking. CONCLUSIONS: Our results show that EDTB-mediated trafficking within the epithelial cells of the developing ileum is important for maintenance of endocytic compartments and enterocyte integrity during early stages of gut development.

Postnatal regulation of MAMDC4 in the porcine intestinal epithelium is influenced by bacterial colonization.

The MAM domain-containing 4 (MAMDC4) protein is associated with the unique endocytotic mechanism observed in the intestine of mammals during the immediate postnatal period. Transcriptional expression of MAMDC4 was substantially upregulated at birth in both the piglet jejunum and ileum and its expression decreases after birth. The protein was found localized specifically to the apical region of the luminal epithelium, however, MAMDC4 protein expression was lost at day 10 and 15 in the jejunum and ileum, respectively, and was not associated with "fetal" enterocyte replacement. Although spatial variation in the subcellular localization of Claudin 1 (CLDN1) was noted at day 3, the loss of MAMDC4 at later stages of development did not appear to have any effect on the tight junction structure. Germ-free (GF) piglets and piglets whose gastrointestinal flora consists exclusively of Escherichia coli (EC) or Lactobacillus fermentum (LF) maintained MAMDC4 protein expression to 14 days of age in distal regions of the small intestine whereas those with conventionalized intestinal flora (CV) showed no MAMDC4 protein at this age. MAMDC4 protein expression was most pronounced in the LF and GF colonized piglets which showed staining in the epithelial cells at 75% and 95% of the length of the small intestine, respectively, which matched that of the newborn. In contrast, EC animals showed only a low abundance at these regions as well as a discontinuous staining pattern. Collectively these results suggest that maturation of MAMDC4 expression in the porcine epithelium occurs more rapidly than what is reported in previously studied rodent species. Furthermore, intestinal bacterial colonization is a major regulator of MAMDC4 in a manner specific to bacterial species and independent of enterocyte turnover.