The Sonic hedgehog / Patched / Gli Signal Transduction Pathway

Developmental pathways are networks of genes that act coordinately to establish the body plan. Disruptions of these genes, which can be associated with environmental exposures, can result in serious dysmorphogenesis or cancer in both children and adults. The ligand Sonic hedgehog, the receptors Patched and Smoothened, and the GLI family of transcription factors represent one such pathway critical to the normal development of many organs due to their regulatory functions at the nexus of mesenchymal differentiation. The Sonic hedgehog-Patched-Gli pathway is a highly conserved signal transduction pathway. Remarkable similarity in gene sequence and function exists from the round worm (C. elegans) to human. A very significant human disease burden is associated with disruption of the pathway and a number of environmental agents (including alcohol, phytoalkloids, bacterial metabolites and sunlight) are known or suspected to disrupt gene function in the pathway. Although some gene targets of the pathway are known from work in Drosophila, key downstream targets and upstream regulators are being elucidated in mammals and the roles of these molecules established in normal development in order to better understand their role in dysmorphogenesis and neoplasia. For example, basal cell carcinoma (BCC) is the most common cancer in man and mutations in Patched or overexpression of GLI are both strongly associated with BCC. Prostatic cancer is a serious problem in the US and there is an association of GLI expression with prostatic cancer in humans.
    The Sonic hedgehog signal in vertebrates is mediated by three C2H2 zinc finger transcription factors, GLI1, GLI2 and GLI3. Near identity of gene sequence exists between mouse and human GLI1. We established that GLI1 protein regulates a set of genes that coordinately control proliferation and may in part explain malignant transformation by mis-expression of GLI1. Our research is on the regulation of GLI promoters and microarray studies to identify gene targets that involve transformation and oncogenesis particularly of rhabdomyosarcoma and medulloblastoma.
    The 150 kD GLI protein localizes predominantly to the nucleus and binds DNA in a sequence specific fashion. Three GLI DNA binding sequences have been identified by DNAse footprinting all of which share the 9 base pair sequence GACCACCCA. Crystallographic data indicate that GLI zinc fingers 2 through 5 mediate DNA binding to GACCACCCA. We have shown that GLI functions as a transcription factor and that a critical domain in the COOH end of the protein is a transactivator with VP16-like structure. Other members of the GLI-Kruppel gene family including YY1 and Kruppel have been shown by transfection assays in tissue culture to function as transcription regulators of reporter chloramphenicol acetyltransferase (CAT) constructs. Transcriptional regulatory properties of these proteins have been shown to be affected by protein-protein interactions. Physical interactions between YY1 and the transcription factor SP1 have been shown to result in an apparent functional cooperation, whereas physical interactions between YY1 and the c-myc protein have been shown to result in functional inhibition of both the repressor and activator functions of YY1. The interaction of GLI with other proteins and any effect such interactions might have on transcriptional regulation is not well understood, this is an active area of investigation in our lab.

	Figure 1 Autoradiographs of hybridization of frozen sections of day 13 mouse embryos. A and C sense controls, B and D anti-sense with non-zinc finger gli probe. High levels of message are apparent in the digits (B arrow), the basis occipitus (D bo) and cervical vertebrae (D cv). D arrow is Meckel’s cartilage. Bar = 0.5 mm
	Figure 2 Autoradiographs of hybridization of frozen sections of day 13 mouse embryo. B and D sense controls, A and C anti-sense gli probe. Arrow in A proliferative zone of the spinal cord, arrow in C the mesenchymal layer of the stomach. e is the epithelial layer of the stomach, lv is the liver which does not have true signal but is visible because it is refractile. bar = 0.5 mm in B, 0.25 mm in D
	Autoradiograph of hindlimb bud of a day 14 mouse embryo. gli expression is present in phalanges but not presumptive joint space (top arrow). Expression is also seen in the long bone (e.g. bottom arrow) and spinal cord of tail (t)

Regulation of GLI1 transcription

GLI1 has a complex transcriptional regulatory domain. The function of the human GLI1 promoter is conserved in transgenic mice.

b-Galactosidase staining of transgenic embryos reporting human GLI promoter activity in tissue. (A) Lateral view of the day 10.5 mouse embryo with staining in the posterior forelimb (fd) buds, in the mandibular arch (ma) (upper arrow) and in the midbrain (m). (B) Dorsal view of day 10.5 mouse embryo with staining in the neural tube (n) and posterior forelimb (fd ) buds. (C) Lateral view of day 12.5 non-transgenic mouse embryo as negative control. (D) Lateral view of day 11.5 embryo. T, telencephalon; M, midbrain; C, cerebellar plate; H, hindbrain; N, neural tube. (E) Dorsal view of day 11.5 embryo. Abbreviations are the same as in (D). (F) Ventral view of day 11.5 embryo. Abbreviations are the same as in (D). (G) Lateral view of day 12.5 embryo. fn, frontonasal mesenchyme; uj, upper jaw; lj, lower jaw; vm, ventral mesencephalon; in, interventricular neuroectoderm; bo, basis occipitus; cv, cervical vertebrae; vb, vertebrae; pfl, proximal forelimb; phl, proximal hindlimb; forelimb digits and hindlimb digits are evident under the aer, apical ectodermal ridge. (H) Dorsal view of day 12.5 embryo. hdb, hindlimb bud. Abbreviations are otherwise the same as in (G). (I ) Ventral view of day 12.5 embryo. gt, genital tubercle; t, tip of tail. Abbreviations are otherwise the same as in (G). (J ) Lateral view of day 13.5 embryo, single arrow indicates staining in the forming phalanges, the double arrow indicates basio-sphenoid and basio-occipital staining. (K) Dorsal view of the cervical region of day 13.5 embryo showing intense staining of the cervical vertebrae. (L) Dorsal view of the day 13.5 embryo showing staining of the presumptive axial skeleton. The Gal-E11 construct was injected into one-cell fertilized mouse eggs in (A) and (B) and the Gal-E2 construct was injected into one-cell fertilized mouse eggs in (D) through (L). (Gal-E11, Gal-E2 are the same as Luc 11, Luc-E2 in Fig. 4 except that the marker gene is b-Galactosidase rather than luciferase.

Translational regulation of GLI1

Post-transcriptional regulation of protein levels of GLI1 occur via the 3’UTR of the human gene and this is anactive area of research in the lab. The 3’UTR contains TGEs (Tra-GLI elements) which bind translational repressors.

Photomicrographs of transgenic C. elegans. Reporter expression is enhanced by deletion of the 3”UTR of both tra-2 (worm gene) and GLI1 (human gene) indicating that translational regulation is occurring through this region that contain the TGEs.
Genes transcriptionally regulated by GLI1

As a transcription factor that controls the expression of other genes and as an oncogene GLI1 must regulate genes important in malignant transformation both onset and maintenance. For cancer to be problematic maintenance of the transformed phenotype is important. We used high-throughput gene expression profiling to establish some of the genes that GLI1 regulates as part of the transformation process.





GLI1 is a human oncogene that functions during development in a molecular pathway specifying morphogenesis of many organ systems including the brain, lung, GI system and prostate. Given the importance of disease consequences of GLI1 mis-expression we need to understand what regulates its expression.

The GLI family of transcription factors plays central roles in the development of both vertebrates and invertebrates. In Drosophila the GLI homologue, Cubitus Interuptus (Ci) mediates the Hedgehog signal to regulate a variety of developmental events, such as wing development, embryonic segmentation, and neuronal development. Similar to flies, the mouse Gli proteins, mGli1, mGli2, and mGli3, are thought to act downstream of the Sonic Hedgehog pathway to regulate development in such important systems as brain, gut , lung, bone, and germ line development. In humans GLI factors are also required for development, and mis-expression of these factors is associated with severe birth defects and cancers including basal cell carcinoma, rhabdomyosarcoma and medulloblastoma. In C. elegans, Tra-1, the homologue of hGLI1 is essential for female development in the soma. It is also required for normal germ line development, but its role in this tissue is less clear. Much work from a number of different organisms indicates that the activity of these different factors must be tightly controlled for proper development. Understanding how this occurs and how the genes themselves are regulated has important implications not only for developmental biology but human health as well particularly since the pathway is disrupted by environmental exposures.
Hedgehog (Hh) proteins are secreted glycoproteins that activate a membrane-receptor complex. This in turn, by means of cytoplamic signal transduction, activates Gli zinc-finger transcription factors. Several lines of evidence suggest that the Shh-Gli pathway plays an important role during normal embryogenesis and tumorigenesis. Loss of Shh-Gli function results in a range of developmental defects, whereas when its inappropriately maintained, or its ectopic function, is associated with tumorigenesis in skin, the cerebullum and skeletal muscle.

Lab Publications (Comprehensive List):

Yoon, J., Gilbertson, R., Iannaccone, S., Iannaccone, P. and Walterhouse, D. Defining a Role for Hedgehog Pathway Activation in Desmoplastic Meduloblastoma by Identifying GLI1 Target Genes. Int. J. Cancer 124:109-119, (2009).

Min, Y., Gipp, J., Yoon, J.W., Iannaccone, P., Walterhouse, D., and Bushman, W. Sonic Hedgehog-Responsive Genes in the Fetal Prostate. J. Biol. Chem. 284:5620-5629, (2009).
 
Zunich, S., Douglas, T., Valdovinos, M., Chang, T., Bushman, W., Walterhouse, D., Iannaccone, P., and Lamm, M.L.G. Paracrine Sonic Hedgehog Signaling by Prostate Cancer Cells Induces Osteoblast Differentiation. Molecular Cancer 8:12, (2009).
 
Laursen, K.B., Mieke, E., Iannaccone, P., Fuchtbauer, E.M., Mechanism of Transcriptional Activation by the proto-oncogene Twist1, J. Biol. Chem, 282 (48) 34623-33 (2007). (Paper of the week, cover).
 
Lakiza, O., Frater, L., Yoo, Y., Villavicencio, E., Walterhouse, D.O., Goodwin, E.B., and Iannaccone, P.M., STAR Proteins Quaking-6 and GLD-1 Regulate the Translation of Homologues GLI1 and Tra-1 Through a Conserved RNA 3'UTR Based Mechanism. Dev. Biol., 287:98-110. (2005).
 
Walterhouse, D.O., Lamm, M.L.G., Villavicencio, E., and Iannaccone, P., Emerging roles for Hedgehog-Patched- GLI signal transduction in reproduction. Biol. of Reprod. 69:8-14, (2003).
 
Lamm, M.L.G., Catbagan, W.S., Laciak, R.J., Barnett, D.H., Hebner, C.M., Gaffield, W., Walterhouse, D.O., Iannaccone, P., and Bushman, W., Sonic hedgehog activates mesenchymal Gli1 expression during prostate ductal bud formation. Dev. Biol. 249:349-366, (2002).

Villavicencio, E., Yoon, J.W., Frank, D., Füchtbauer, E., Walterhouse, D.O., and Iannaccone, P., Cooperative E-box Regulation of Human GLI1 by TWIST and USF. Genesis: J. of Genetics and Development 32:247-258, (2002).

Jan , E., Yoon, J.W., Walterhouse, D.O., Iannaccone, P., Goodwin, E.B., Conservation of the C.elegans tra-2 3’UTR translational control. EMBO J., 16:6301-6313, (1997).

Yang, J.T., Liu, C.Z., Villavicencio, E., Yoon, J.W., Walterhouse, D.O., and Iannaccone, P., Expression of human GLI in mice results in failure to thrive, early death, and patchy Hirschprung-like gastrointestinal dilatation. Molecular Medicine, 3:826-835, (1997).

Walterhouse, D.O., Ahmed, M., Slusarski, D., Kalamaras, J., Boucher, D., Holmgren, R., and Iannaccone, P., gli, a zinc finger transcription factor and oncogene, is expressed during normal mouse development. Dev. Dynamics 196: 91-102, (1993).