Transcription profiling of mouse E18.5 intestinal mesenchyme treated with Sonic or Indian hedgehog ligand to identify targets regulated by Hedgehog signaling
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ABSTRACT: Epithelial Hedgehog (Hh) ligands regulate several aspects of fetal intestinal organogenesis and emerging data implicate the Hh pathway in inflammatory signaling in adult colon. We investigated the effects of chronic Hh inhibition in vivo and profiled molecular pathways acutely modulated by Hh signaling in the intestinal mesenchyme. Experiment Overall Design: E18.5 intestinal mesenchyme was isolated and cultured. Mesenchyme was treated with Sonic (Shh) or Indian (Ihh) hedgehog ligand or Vehicle (control) acutely to identify targets regulated by Hh signaling in intestinal mesenchyme.
Project description:Epithelial Hedgehog (Hh) ligands regulate several aspects of fetal intestinal organogenesis and emerging data implicate the Hh pathway in inflammatory signaling in adult colon. We investigated the effects of chronic Hh inhibition in vivo and profiled molecular pathways acutely modulated by Hh signaling in the intestinal mesenchyme.
Project description:Canonical Hedgehog (Hh) signaling regulates the expression of genes that are critical to the patterning and development of a variety of organ systems. In adult, both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligand, activation occurs within the stromal microenvironment (Yauch et al., 2009). In situ hybridization of the pathway target gene, Ptch1, shows that signaling is located at stromal perivascular fibroblast-like cells in xenograft tumor sections derived from Hh-expressing colorectal cancer cell lines. To study the downstream genes regulated by Hh signaling, we treated a primary human colon myofibroblast, CCD-18Co, with SHH (1 ug/ml) or no treatment (control) in serum-free medium supplemented with 0.1% BSA for 72 hrs and performed microarray analysis (Affymetrix U133P) on these samples. Three biological replicates of SHH stimulated and three replicates of unstimulated primary colon myofibroblast cells CCD-18Co were used in the experiment to analyze their gene expression.
Project description:Canonical Hedgehog (Hh) signaling regulates the expression of genes that are critical to the patterning and development of a variety of organ systems. In adult, both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligand, activation occurs within the stromal microenvironment (Yauch et al., 2009). In situ hybridization of the pathway target gene, Ptch1, shows that signaling is located at stromal perivascular fibroblast-like cells in xenograft tumor sections derived from Hh-expressing colorectal cancer cell lines. To study the downstream genes regulated by Hh signaling, we treated a primary human colon myofibroblast, CCD-18Co, with SHH (1 ug/ml) or no treatment (control) in serum-free medium supplemented with 0.1% BSA for 72 hrs and performed microarray analysis (Affymetrix U133P) on these samples.
Project description:Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1+/- mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for treatment of medulloblastoma and BCC. Results clearly demonstrate a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1+/- mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidate the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in the tumor cells, showing the maximum inhibitory effect on Gli1 activity. MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways , were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity. Gene expression data was generated (in replicates) from Medulloblastoma allografts collected at various time points and following low (40mpk) or high (80 mpk) or vehicle single dose (QD) or mutiple dose (BID) treatment with a SHH pathway inhibitor.
Project description:Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1+/- mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for treatment of medulloblastoma and BCC. Results clearly demonstrate a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1+/- mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidate the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in the tumor cells, showing the maximum inhibitory effect on Gli1 activity. MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways , were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity.
Project description:The morphogen Indian Hedgehog plays a very important role during intestinal embryogenesis, but also maintains homeostasis of the adult gut. Intestinal Indian Hedgehog is expressed by the intestinal epithelium and signals in paracrine manner to fibroblasts in the stromal compartment. Unresolved deletion of Ihh from the intestinal epithelium leads to a severe enterocolitis. We studied the short term changes in the colon upon deletion of Ihh from the epithelial layer.
Project description:In the intestine, Hedgehog (Hh) signalling orchestrates epithelial homeostasis in a bidirectional loop. Differentiated enterocytes secrete the ligand leading to active downstream signaling exclusively in the stroma. In turn, Hh-driven stromal factors contribute to the control of intestinal stem cell numbers and induce epithelial differentiation. To investigate the consequences of stromal Hh activation on the gene expression level, we performed microarray analysis on full-thickness colonic tissue from Col1a2CreER;Ptch1fl/fl mice (C57BL/6 background). Upon Cre-driven recombination, these mice lack the inhibiting Hh receptor Ptch1 specifically in Col1a2 expressing stroma cells, leading to stroma-specific activation of downstream Hh signaling.
Project description:Hedgehog (HH) signaling, transduced at the primary cilium, plays a crucial role in limb longitudinal growth and digit formation, yet its involvement in thumb development has been underestimated. This study identifies Merlin (Nf2) as a novel regulator of limb development, modulating the ciliary trafficking of the HH receptor, Smoothened. Merlin is predominantly expressed in limb buds and growth plates. Conditional knockout of Merlin in limb mesenchyme in mice results in dwarfism, brachydactyly, and thumb hypoplasia, with transcriptomic profiling and molecular analyses revealing disrupted HH signaling. Mechanistically, Merlin interacts with ARF6 to regulate the ciliary transport of Smoothened via RAB11+ vesicles. Importantly, pharmacological enhancement of HH signaling significantly corrected the limb defects caused by Merlin deletion. These findings highlight the essential role of Merlin in regulating longitudinal limb growth and thumb morphogenesis via primary cilia-HH signaling, suggesting potential therapeutic strategies for related limb dysplasias.
Project description:Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes. Phosphorylation, orchestrated by multiple kinases, is pivotal in regulating Gli activity. While kinases in this context have been extensively studied, the role of protein phosphatases, particularly Protein Phosphatase 2A (PP2A), remains less explored. This study unveils a novel role for the B'' gamma subunit of PP2A, PPP2R3C, in Hh signaling regulation. PPP2R3C interacts with Gli proteins, and its disruption reduces Hedgehog pathway activity. Moreover, we establish a connection between PPP2R3C and MEKK1 kinase in Gli protein phosphorylation, underscoring the intricate interplay between kinases and phosphatases in Hh signaling pathway. This study sheds light on the previously understudied role of protein phosphatases in Hh signaling and provides insights into their significance in cellular regulation.
Project description:Postnatal tissue quiescence is generally thought to be a default state in the absence of a proliferative stimulus such as injury. We now demonstrate that in the lung, quiescence in the adult is an actively maintained state and is regulated by paracrine hedgehog signaling. Epithelial-specific deletion of Sonic Hedgehog during normal homeostasis results in a proliferative expansion of the adjacent lung mesenchyme. Injury to the lung epithelium results in decreased hedgehog activation, accompanied by proliferative expansion of the adjacent mesenchyme. Moreover, reconstitution of Hedgehog signaling during epithelial injury attenuated the proliferative expansion of the adjacent mesenchyme. Hedgehog signaling maintains lung quiescence by attenuating PDGF signaling through blocking post-translational processing of PDGF receptor α/β into the mature isoforms. These results indicate that in postnatal tissues, epithelial cells can actively maintain mesenchymal quiescence via paracrine hedgehog activation, and that imbalances in this pathway could lead to aberrant mesenchymal expansion and postnatal disease. Fibroblasts were isolated from mouse lungs and grown in culture in triplicate wells. Samples were treated with vehicle or purmorphamine 5um for 24 hours and RNA was isolated for microarray.