J Biol Chem. 2006 Jan 19;

Smoothened regulates activator and repressor functions of Hedgehog signaling via two distinct mechanisms.

Ogden SK, Casso DJ, Ascano M Jr, Yore MM, Kornberg TB, Robbins DJ.

Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755-7650.

The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through activation of the seven transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos), the protein kinase Fused (Fu) and the Drosophila Gli homolog Cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion, but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci.

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Cancer Res. 2006 Jan 15;66(2):839-45.

Protein Kinase C-{delta} and Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase-1 Control GLI Activation in Hedgehog Signaling.

Riobo NA, Haines GM, Emerson CP Jr.

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania and Boston Biomedical Research Institute, Watertown, Massachusetts.

One third of all lethal cancers are associated with excessive activation of the Hedgehog (HH) pathway by mutations of its signaling components or by increased responsiveness of cells to the HH ligand. HH signaling through the GLI transcription factors leads to increased cell proliferation by up-regulation of the extracellular regulated kinase (ERK) pathway and by expression of S phase cyclins. In this study, we have tested the hypothesis that the HH pathway can integrate ERK signaling to modulate the activity of GLI. Using NIH 3T3 cells, we show that phorbol esters, acting through protein kinase C-delta (PKCdelta) and mitogen-activated protein/extracellular signal-regulated kinase-1 (MEK-1), fully stimulate the transcriptional activity of endogenous and overexpressed GLI proteins, as assessed by GLI-luciferase reporter assays, and induce the expression of endogenous GLI1 and PTCH-1 target genes, as assessed by reverse transcription-PCR. Moreover, activation of GLI elicited by Sonic Hedgehog also requires PKCdelta and MEK-1 function. Remarkably, coexpression of activated MEK-1 and GLI1 or GLI2 induced a 10-fold synergistic increase in GLI-luciferase activity that was totally blocked by PD98059. The NH(2)-terminal region of GLI1 (amino acids 1-130) is required for sensing the ERK pathway, as deletion of this domain produces active GLI1 protein with greatly reduced response to activation by MEK-1. Basic fibroblast growth factor activation of the ERK pathway also stimulated GLI1 activity through its NH(2)-terminal domain. Our results identify PKCdelta and MEK-1 as essential, positive regulators of GLI-mediated HH signaling. Furthermore, our findings suggest that tumors with deregulated HH and ERK synergize to stimulate cell proliferation pathways. (Cancer Res 2006; 66(2): 839-45).

Treatment of Down Syndrome in Mice Restores Nerve Growth in Cerebellum

BALTIMORE, Jan. 24 (AScribe Newswire) -- Researchers at Johns Hopkins restored the normal growth of specific nerve cells in the cerebellum of mouse models of Down syndrome (DS) that were stunted by this genetic condition. The cerebellum is the rear, lower part of the brain that controls signals from the muscles to coordinate balance and motor learning.

The finding is important, investigators say, because the cells rescued by this treatment represent potential targets for future therapy in human babies with DS. And it suggests that similar success for other DS-related disruptions of brain growth, such as occurs in the hippocampus, could lead to additional treatments -- perhaps prenatally -- that restore memory and the ability to orient oneself in space.

DS is caused by an extra chromosome 21, a condition called trisomy -- a third copy of a chromosome in addition to the normal two copies. Children with DS have a variety of abnormalities, such as slowed growth, abnormal facial features and mental retardation. The brain is always small and has a greatly reduced number of neurons.

A report on the Hopkins work with trisomic mice, led by Roger H. Reeves, Ph.D., professor in the Department of Physiology and the McKusick-Nathans Institute for Genetic Medicine at Hopkins, appears in the January 24 issue of the Proceedings of the National Academy of Sciences (PNAS).

Reeves and his team used an animal model of DS called the Ts65Dn trisomic mouse to show that pre-nerve cells called granule cell precursors (GCP) fail to grow correctly in response to stimulation by a natural growth-triggering protein. This protein, called Sonic hedgehog (Shh), normally activates the so-called Hedgehog pathway of signals in these cells. These signals stimulate mitosis (cell division) and multiplication of the cells in the growing, newborn brain, according to the researchers.

The GCP originate near the surface of the cerebellum and migrate deeper into the brain to form the internal granule layer (IGL), the researchers note. Therefore, the team studied the growth of the cerebellum in Ts65Dn trisomic mice at seven time points -- beginning at birth -- to determine when GCP abnormalities first occurred. The IGL was similar in both normal and Ts65Dn mice at birth, but was significantly reduced in the trisomic mice by day six after birth.

Furthermore, the researchers found that the reduced number of GCP in these mice compared to normal mice was not due to cell death; rather, there were 21 percent fewer GCP undergoing cell division in Ts65Dn mice. This suggested that stimulating these cells might restore normal numbers of GCP, according to Reeves.

The Hopkins team then showed in test-tube experiments that GCP from the brains of Ts65Dn mice had a significantly lower response to increasing concentrations of a potent form of Shh called ShhNp. That is, increasing concentrations of ShhNp triggered increasing rates of mitosis. Despite their lower response, trisomic cells did show a dose response with increasing ShhNp concentrations.

"The fact that trisomic GCP responded to stimulation of their Hedgehog pathway even in a reduced way is significant," says Reeves, the senior author of the PNAS paper. "It suggested that these cells could be stimulated to reach normal levels of cell division by artificially increasing their exposure to Hedgehog growth factor."

Based on this initial discovery, the team injected into newborn Ts65Dn mice a molecule that stimulates the Hedgehog pathway to trigger cell growth. Treatment of the trisomic mice with this molecule, called SAG 1.1, restored both the numbers of GCP and the number of GCP cells undergoing mitosis to levels seen in normal mice by six days after birth.

"The normal mouse cerebellum attains about a third of its adult size in the first week after birth," says Randall J. Roper, Ph.D. "This is the time during which SAG 1.1 treatment of Ts65Dn restored GCP populations and the rate of mitosis of those cells," he adds. "However, further research is needed to determine if it's possible to reverse the effects of trisomy in other parts of the DS mouse." Roper is a postdoctoral fellow in the laboratory of Reeves and a co-first author of the PNAS paper.

The other authors of the Hopkins paper include Drs. Laura L. Baxter, Nidhi G. Saran, Donna K. Klinedinst, and Philip A. Beachy. Baxter is a co-first author of this paper and is currently at the National Human Genome Research Institute of the National Institutes of Health (Bethesda, Md.).

This work was supported in part by the Public Health Service. P.A.B. is a Howard Hughes Medical Institute investigator.

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CONTACT: Gary Stephenson. Johns Hopkins Medicine Office of Corporate Communications, 410-955-5384, gstephenson@jhmi.edu


Proc Natl Acad Sci U S A. 2006 Jan 23;

Defective cerebellar response to mitogenic Hedgehog signaling in Down's syndrome mice.

Roper RJ, Baxter LL, Saran NG, Klinedinst DK, Beachy PA, Reeves RH.

Department of Physiology and McKusick-Nathans Institute for Genetic Medicine, Howard Hughes Medical Institute, and Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Trisomy 21 is the cause of Down's syndrome (DS) which is characterized by a number of phenotypes, including a brain which is small and hypocellular compared to that of euploid individuals. The cerebellum is disproportionately reduced. Ts65Dn mice are trisomic for orthologs of about half of the genes on human chromosome 21 and provide a genetic model for DS. These mice display a number of developmental anomalies analogous to those in DS, including a small cerebellum with a significantly decreased number of both granule and Purkinje cell neurons. Here we trace the origin of the granule cell deficit to precursors in early postnatal development, which show a substantially reduced mitogenic response to Hedgehog protein signaling. Purified cultures of trisomic granule cell precursors show a reduced but dose-dependent response to the Sonic hedgehog protein signal in vitro, demonstrating that this is a cell-autonomous deficit. Systemic treatment of newborn trisomic mice with a small molecule agonist of Hedgehog pathway activity increases mitosis and restores granule cell precursor populations in vivo. These results demonstrate a basis for and a potential therapeutic approach to a fundamental aspect of CNS pathology in DS.

Sci STKE. 2006 Jan 24;2006(319):pe5.

A tale of two signals: Wnt and Hedgehog in dentate neurogenesis.

Pozniak CD, Pleasure SJ.

Department of Neurology, Program in Neuroscience, University of California, San Francisco, CA 94143, USA.

It is now widely accepted that discrete regions of the adult brain contain stem cells that continue to generate new neurons. However, it remains unclear what molecular signals define the neurogenic niche and how such signals act on the heterogeneous cell populations within these regions. Here we discuss two recent studies that demonstrate the role of Wnt and Sonic Hedgehog signaling in neurogenic zones. Wnts act on neuronal precursors that mature and contribute to the dentate gyrus (DG), whereas Sonic Hedgehog affects the bona fide stem cells and transit amplifying cells (the partially committed progeny of stem cells). These studies further define how discrete populations of cells react to specific extracellular signals provided within the neurogenic niche to survive, proliferate, and form functional mature cell types.