Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications.

BACKGROUND: The Hedgehog (Hh) pathway provides inductive signals critical for developmental patterning of the brain and face. In humans and in animal models interference with this pathway yields birth defects, among the most well-studied of which fall within the holoprosencephaly (HPE) spectrum. METHODS: Timed-pregnant C57Bl/6J mice were treated with the natural Hh signaling antagonist cyclopamine by subcutaneous infusion from gestational day (GD) 8.25 to 9.5, or with a potent cyclopamine analog, AZ75, administered by oral gavage at GD 8.5. Subsequent embryonic morphogenesis and fetal central nervous system (CNS) phenotype were respectively investigated by scanning electron microscopy and high resolution magnetic resonance imaging (MRI). RESULTS: In utero Hh signaling antagonist exposure induced a spectrum of craniofacial and brain malformations. Cyclopamine exposure caused lateral cleft lip and palate (CLP) defects attributable to embryonic deficiency of midline and lower medial nasal prominence tissue. The CLP phenotype was accompanied by olfactory bulb hypoplasia and anterior pituitary aplasia, but otherwise grossly normal brain morphology. AZ75 exposure caused alobar and semilobar HPE with associated median facial deficiencies. An intermediate phenotype of median CLP was produced infrequently by both drug administration regimens. CONCLUSIONS: The results of this study suggest that interference with Hh signaling should be considered in the CLP differential and highlight the occurrence of CNS defects that are expected to be present in a cohort of patients having CLP. This work also illustrates the utility of fetal MRI-based analyses and establishes a novel mouse model for teratogen-induced CLP.

Hedgehog pathway responsiveness correlates with the presence of primary cilia on prostate stromal cells.

BACKGROUND: Hedgehog (Hh) signaling from the urogenital sinus (UGS) epithelium to the surrounding mesenchyme plays a critical role in regulating ductal formation and growth during prostate development. The primary cilium, a feature of most interphase vertebrate cell types, serves as a required localization domain for Hh signaling transducing proteins. RESULTS: Immunostaining revealed the presence of primary cilia in mesenchymal cells of the developing prostate. Cell-based assays of a urongenital sinus mesenchymal cell line (UGSM-2) revealed that proliferation-limiting (serum starvation and/or confluence) growth conditions promoted cilia formation and correlated with pathway activation associated with accumulation of Smoothened in primary cilia. The prostate cancer cell lines PC-3, LNCaP, and 22RV1, previously shown to lack demonstrable autocrine Hh signaling capacity, did not exhibit primary cilia even under proliferation-limiting growth conditions. CONCLUSION: We conclude that paracrine Hedgehog signaling activity in the prostate is associated with the presence of primary cilia on stromal cells but that a role in autocrine Hh signaling remains speculative.

Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines.

BACKGROUND: Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice. RESULTS: Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in Gli1-/-2-/-iMEFs was severely reduced while Gli2-/-3-/-iMEFs were incapable of ligand-induced transcriptional response. However, we found that both Gli1-/-2-/- and Gli2-/-3-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent. CONCLUSION: This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.

Unique and complimentary activities of the Gli transcription factors in Hedgehog signaling.

The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. Aberrations in Hedgehog signaling seriously affect vertebrate development. Postnatally, Hedgehog signaling has been postulated to play a pivotal role in healing and repair processes and inappropriate pathway activation has been implicated in several types of cancers. To better understand both the upstream regulation of the Gli transcription factors, as well as their unique and combinatorial roles in regulating the expression of Hedgehog target genes, we have characterized embryonic fibroblasts (MEFs) from Gli mutant mice. Stimulation of wild-type MEFs by Sonic Hedgehog (Shh) peptide elicited unique profiles of induction of Hedgehog target genes Gli1, Ptc1, and Hip1. Gli2 loss-of-function was associated with diminished Shh-induced target gene expression, while Gli3 loss-of-function was associated with increased basal and Shh-induced target gene expression. The loss of Gli1 alone had no effect on target gene induction but did diminish Shh-induced target gene expression when combined with the loss of Gli2 or Gli3. Additionally, overexpression of Gli1 induced target gene expression in Gli2(-/-)3(-/-) MEFs, while Shh stimulation did not. Using MEFs expressing only Gli2 or Gli3, we found that both cyclopamine and the PKA activator forskolin inhibited target gene induction mediated by Gli2 and Gli3. These results demonstrate that Gli2 and Gli3 share common regulatory mechanisms and modulate Hedgehog target gene expression directly and independently while also regulating Gli1 expression, which in specific contexts, coordinately contributes to target gene activation.

Sonic hedgehog signaling regulates the expression of insulin-like growth factor binding protein-6 during fetal prostate development.

At the onset of ductal morphogenesis in the developing prostate, Shh expression condenses at evaginations of urogenital sinus epithelium and activates Gli transcription factors in the adjacent mesenchyme. Abrogation of Hedgehog signaling disrupts proper prostatic budding, ductal growth, and branching. We now show that Hedgehog signaling regulates the expression of insulin-like growth factor binding protein-6 (Igfbp-6) in the developing mouse prostate. Igfbp-6 is a secreted factor that specifically binds insulin-like growth factor-II (IGF-II), prevents its binding to the IGF-I receptor, and is thought to regulate the activity of IGF-II in growth and differentiation. Igfbp-6 is expressed in both the developing and adult prostate. In the urogenital sinus, Igfbp-6 mRNA colocalized with Ptc1 and Gli1 mRNA in the mesenchyme, while Igfbp-6 protein was found in both the mesenchymal and epithelial layers. Exogenous Shh peptide induced expression of Igfbp-6 in the developing prostate while the chemical inhibitor of Hedgehog signaling, cyclopamine, reduced its expression. These studies show that Igfbp-6 is an actual target of Shh signaling in the urogenital sinus and provide the first evidence for a linkage between the Hedgehog and IGF signaling pathways in prostate development.

H2O2-dependent activation of GCLC-ARE4 reporter occurs by mitogen-activated protein kinase pathways without oxidation of cellular glutathione or thioredoxin-1.

The gp91phox homologue Nox1 produces H2O2, which induces cell growth, transformation, and tumorigenicity. However, it has not been clear whether H2O2 effects are mediated indirectly via a generally oxidizing cellular environment or whether H2O2 more directly targets specific signaling pathways. Here, we investigated signaling by H2O2 induced by Nox1 overexpression using a luciferase reporter regulated by the antioxidant response element ARE4. Surprisingly, Nox1-derived H2O2 activated the reporter gene 15-fold with no effect on the redox state of the major thiol antioxidant substances, glutathione and thioredoxin. H2O2 signaling to ARE4 was mediated by activation of both the c-Jun N-terminal kinase and ERK1/2 pathways modulated by Ras. Thus, "redox signaling" resulting in kinase signaling pathways is distinct from "oxidative stress," and is mediated by discrete, localized redox circuitry.

Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence.

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.