Disruption of the Hedgehog signaling pathway in inflammatory bowel disease fosters chronic intestinal inflammation.

Hedgehog (Hh) signaling is essential for intestinal homeostasis and has been associated with inflammation and tissue repair. We hypothesized that Hh signaling could affect the inflammatory process in inflammatory bowel disease (IBD). For this purpose, colon specimens from the inflamed and non-inflamed mucosa of 15 patients with Crohn's disease (CD), 15 with ulcerative colitis, and 15 controls were analyzed by immunohistochemistry and real-time PCR. The production and modulation of cytokines were measured by ELISA from culture explants. Apoptosis was assessed by TUNEL and caspase-3 activity assays. Chemotaxis was evaluated using a transwell system. Primary human intestinal and skin fibroblasts were used for analyzing migration and BrdU incorporation. Hh proteins were generally expressed at the superficial epithelium, and a marked reduction was observed in CD. In the lamina propria, Gli-1 predominantly co-localized with vimentin- and alpha-smooth muscle actin-positive cells, with lower levels observed in CD. In colon explants, Hh stimulation resulted in reduction, while blockade increased, TNF α, IL-17, and TGF ß levels. Apoptotic rates were higher in inflamed samples, and they increased after Hh blockade. Levels of Gli-1 mRNA were negatively correlated with caspase-3 activity. Hh blockade increased chemoattraction of monocytes. Primary fibroblasts incorporated more BrdU, but migrated less after Hh blockade. These results suggest that Hh signaling provides a negative feedback to the lamina propria, down-regulating inflammatory cytokines, and inhibiting leukocyte migration and fibroblast proliferation, while favoring fibroblast migration. Therefore, Hh signaling is strongly implicated in the pathogenesis of intestinal inflammation, and it may represent a novel therapeutic target for IBD.

Hedgehog pathway signaling regulates human colon carcinoma HT-29 epithelial cell line apoptosis and cytokine secretion.

The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted ß-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.

A via de sinalização hedgehog regula a apoptose e a secreção de citocinas na linhagem de células epiteliais de carcinoma de cólon humano HT-29

Introdução: A via hedgehog (Hh) está envolvida na embriogênese e em processos fisiológicos incluindo sobrevivência e proliferação celular. Objetivos: células de carcinoma de cólon humano HT-29 foram utilizadas parainvestigar a via de sinalização Hh e as funções biológicas nas células epiteliais colônicas. Métodos: Foram realizadas culturas de células HT-29 sob diferentes condições e exposição a vários estímulos. A expressão dos componentes da via de sinalização Hh, proteínas e genes relacionados, foram avaliados por munofluorescência e por reação de cadeia de polimerase (PCR) em tempo real. Viabilidade, proliferação celular e apoptose foram avaliados, pelo método de 3-(4,5-Dimetiltiazol-2-yl)- ,5-difeniltetrazolium bromida, tetrazol (MTT), por incorporação de Brom odeoxiuridina (BrdU) e pela reação de Annexina - V/7- AAD, respectivamente. A produção de quimiocinas no sobrenadante de culturas de células HT -29 foi avaliada por reação imunoenzimática (ELISA). Resultados: Níveis de RNA mensageiro de Indian e Sonic Hh e o s fatores de transcrição Gli-1 e Gli-2 aumentaram após tratamento com agonistas Hh e butirato,masdiminuiramapós exposição a iclopamina. A expressão de proteínas orfogenéticas 4 e 7 (BMP4 e BMP7)diminui após o bloqueio da via Hh com ciclopamina. A expressão da proteína Gli-1 aumenta após exposição com agonistas e diminui após ciclopamina. A exposição a agonistas Hh promove a redução da β-catenina e causa sua redistribuição subcelular. Níveis de interleucina 8 (IL-8) e proteína quimiotática de monócitos (MCP-1) diminuem após exposição a agonistas Hh quando comparados a ciclopamina, lipopolissacarídeos (LPS), interferon (IFN-γ) ou fator de crescimento epitelial (EGF). A proliferação e a viabilidade celular diminuem após o bloqueio da via Hh. Agonistas Hh revogaram a apoptose induzida pelo anticorpo anti-CD95. Conclusão: A via Hh é um controlador fundamental das células epiteliais colônicas...

Background & Aims: The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. Methods: The human colon carcinoma HT-29 cell line was used to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components, related proteins and genes were assessed by immunofluorescence and protein chain reaction real -time (PCR). Viability, cell proliferation and apoptosis were measured by the 3-(4,5 Dimethylthiazol -2-yl)-,5 diphenyltetrazolium bromide, tetrazole (MTT) assay, Bromodeoxiuridine (BrdU) uptake and Annexin-V/7-AAD staining, respectively. Chemokines production was measured by enzyme-linked immunosorbent assay (ELISA)in culture supernatants. ResultsIndian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine. Bone morphogen 4 and 7 (BMP4 and BMP7) expression decreased after Hh blockade with cyclopamine. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted β-catenin reduction and subcellular redistribution. Levels of interleukin 8 (IL-8) and monocyte chemotactic protein (MCP-1) decreased upon exposure to Hh agonists compared to cyclopamine, lipopolyssacharide (LPS), interferon(IFN-γ) or epidermal growth factor (EGF). Cellular proliferation and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Conclusions: Hh pathway is a key controller of colonic epithelial cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response...