Tammy Sadler

Germline Mutations and Variants in the Succinate Dehydrogenase Genes in Cowden and Cowden-like Syndromes

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.

Inflammation-induced endothelial-to-mesenchymal transition: a novel mechanism of intestinal fibrosis.

In addition to mesenchymal cells, endothelial cells may contribute to fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). We investigated whether human intestinal microvascular endothelial cells (HIMEC) undergo EndoMT and contribute to fibrosis in human and experimental inflammatory bowel disease (IBD). HIMEC were exposed to TGF-β1, IL-1β, and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) and evaluated for morphological, phenotypic, and functional changes compatible with EndoMT. Genomic analysis was used to identify transcription factors involved in the transformation process. Evidence of in situ and in vivo EndoMT was sought in inflamed human and murine intestine. The combination of TGF-β1, IL-1β and TNF-α, or activated LPMC supernatants induced morphological and phenotypic changes consistent with EndoMT with a dominant effect by IL-1. These changes persisted after removal of the inducing agents and were accompanied by functional loss of acetylated LDL-uptake and migratory capacity, and acquisition of de novo collagen synthesis capacity. Sp1 appeared to be the main transcriptional regulator of EndoMT. EndoMT was detected in microvessels of inflammatory bowel disease (IBD) mucosa and experimental colonic fibrosis of Tie2-green fluorescent protein (GFP) reporter-expressing mice. In conclusion, chronic inflammation induces transdifferentiation of intestinal mucosal microvascular cells into mesenchymal cells, suggesting that the intestinal microvasculature contributes to IBD-associated fibrosis through the novel process of EndoMT.

Cytokine-induced chromatin modifications of the type I collagen alpha 2 gene during intestinal endothelial-to-mesenchymal transition.

Background:Fibrosis of the intestine is currently an irreversible complication of inflammatory bowel disease; yet, little is understood of the underlying pathogenesis and antifibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen, a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (interleukin 1-&bgr;, tumor necrosis factor &agr;, and transforming growth factor &bgr;) induces collagen I alpha 2 (COL1A2) mRNA and protein. Methods:Chromatin immunoprecipitation assays on pure cultures of human intestinal mucosal endothelial cells undergoing EndoMT were performed with antibodies to specific histone modifications and RNA polymerase II. Reverse transcriptase–PCR was used to quantify the levels of Col1A2 and endothelial-specific von Willebrand factor (vWF) mRNA. Results:We showed that cytokines induce selective chromatin modifications (histone 4 hyperacetylation, and hypermethylation of histone 3) and phosphorylated RNA polymerase II at the COL1A2 promoter. Hypoacetylated and hypomethylated histone 3 was detected on the repressed vWF gene. Prolonged exposure to cytokines (16 days) retained hyperacetylation of select lysines in H4 on the COL1A2 promoter. Removal of cytokines after 16 days and continued culture for 10 days showed persistent hyperacetylation at lysine 16 in histone H4. Conclusions:This is the first study to show that COL1A2 gene expression is associated with cytokine-induced, temporally ordered, and persistent chromatin modifications and suggests that these are important determinants of gene expression in EndoMT and intestinal fibrosis.

Chromosome-Associated Protein D3 Promotes Bacterial Clearance in Human Intestinal Epithelial Cells by Repressing Expression of Amino Acid Transporters

BACKGROUND & AIMS Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The Drosophila chromosome-associated protein D3 (dCAP-D3) regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, the products of which heterodimerize to form an amino acid transporter in HT-29 cells after bacterial infection; levels of SLC7A5-SLC3A2 were increased in tissues from patients with UC compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5-SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3-deficient cells. CONCLUSIONS CAP-D3 down-regulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with active UC; strategies to increase its levels might restore mucosal homeostasis to patients with active UC.

Mutual Regulation of TLR/NLR and CEACAM1 in the Intestinal Microvasculature: Implications for IBD Pathogenesis and Therapy

Background Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) displays multiple activities, among which pathogen binding and angiogenesis are particularly prominent. These same functions are also exerted by Toll- and NOD-like receptors (TLRs and NLRs), which are critical mediators of innate immune responses. We investigated whether a functional inter-relationship exists between CEACAM1 and TLRs and NLRs and its potential impact on induction of intestinal angiogenesis. Methods This hypothesis was tested using human intestinal microvascular endothelial cells, a unique cell population exposed to microbial products under physiological and pathological conditions. Results The results show that activation of TLR2/4, TLR4, NOD1, and NOD2 by specific bacterial ligands selectively and differentially upregulates the levels of cellular and soluble CEACAM1 produced by intestinal microvascular endothelial cells. The results also show that CEACAM1 regulates the migration, transmigration, and tube formation of these endothelial cells and mediates vessel sprouting induced by specific TLR and NLR bacterial ligands. Combined, these results demonstrate a close and reciprocal regulatory interaction between CEACAM1 and bacterial products in mediating multiple functions essential to new vessel formation in the gut mucosa. Conclusions A coordinated and reciprocal interaction of CEACAM1 and microbiota-derived factors is necessary to optimize angiogenesis in the gut mucosa. This suggests that a coordination of endogenous and exogenous innate immune responses is necessary to promote intestinal angiogenesis under physiological and inflammatory conditions such as inflammatory bowel disease.

505 Epigenetic Regulation of Inflammation-Induced Endothelial-to-Mesenchymal Transition (EndoMT): A Novel Mechanism of Fibrosis in Inflammatory Bowel Disease (IBD)

BACKGROUND: Intestinal fibrosis is a common and potentially serious complication of inflammatory bowel disease (IBD). In addition to mesenchymal cells, endothelial cells can contribute to fibrosis through the recently described process of EndoMT. We investigated whether i) human intestinal microvascular endothelial cells (HIMEC) can undergo EndoMT and contribute to fibrosis in IBD, and ii) epigenetic changes are involved in mediation of EndoMT.MATERIALS ANDMETHODS: HIMEC exposed to TGF-β1, IL-1β and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) were evaluated for morphological, phenotypic and functional changes compatible with EndoMT. Microarray genomic analysis was used to identify transcription factors controlling the transformation process. Evidence of in situ and In Vivo EndoMT was sought in human and experimental IBD tissues. Chromatin immunoprecipitation (ChIP) assays were performed to identify specific histone modification changes on the promoters of von Willebrand factor (vWF) and collagen1 alpha2 (Col1α2) genes (markers of endothelial cells and fibroblasts, respectively) during EndoMT. RESULTS: The combination of TGF-β1, IL-1β and TNF-α induced morphologic and phenotypic changes in HIMEC consistent with EndoMT, and these were reproduced using supernatants of activated LPMC. Transformed HIMEC exhibited loss of acetylated LDL-uptake, enhanced extracellular matrix secretion, and acquisition of de novo collagen synthesis capacity. These functional alterations persisted at 10 days after removal of the inducing agents, and then still were associated with an approximate 8-fold upregulation of Col1α2 gene expression and 1.6-fold downregulation of vWF gene expression. EndoMT was detected in inflamed microvessels of human IBD mucosa and TNBS-induced colonic fibrosis of Tie2-GFP reporter mice. Genomic analysis uncovered Sp1 as the dominant transcriptional regulator of EndoMT. Finally, ChIP analysis revealed increased methylation of lysines 9 and 27 in histone H3 associated with repression of vWF gene expression, while increased acetylation of histone H4 accompanied activation of Col1α2 gene expression. CONCLUSIONS: Inflammatory stimuli induce trans-differentiation of mucosal microvascular cells into mesenchymal cells In Vitro and In Vivo, supporting the notion that the microvasculature undergoes EndoMT in IBD and contributes to fibrogenesis. These events involve epigenetic changes such as histone modifications crucial to regulation of EndoMT, and define unique pro-fibrotic epigenotypes that could become targets of selective anti-fibrotic therapies in IBD.