The hedgehog receptor patched is involved in cholesterol transport

Background

Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.

Methodology/Principal Findings

Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.

Conclusion/Significance

Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.     

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.     

The sonic hedgehog receptor patched associates with caveolin-1 in cholesterol-rich microdomains of the plasma membrane

The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.     

宫颈鳞癌演进过程P16INK4a及Hh-Gli信号通路相关蛋白表达及其相关性研究

探讨P16^INK4a及Sonic hedgehog(Hh-Gli)信号通路蛋白在宫颈癌及癌前病变(CIN)中的表达相关性及其意义．采用Western-blot方法检测HPV16阳性及HPV18阳性宫颈癌细胞系P16^INK4a及Hh-Gli信号通路蛋白Smo、Ptch及Gli表达．免疫组化检测组织芯片P16^INK4a、Shh、Smo、Ptch及Gli表达,包括20例正常宫颈、18例癌旁组织、54例CIN及28例宫颈鳞癌组织．分析P16^INK4a与Hh-Gli信号通路蛋白间表达相关性及与临床病理因素的关系．结果显示P16^INK4a、Smo、Ptch及Gli蛋白在HPV16及HPV18阳性宫颈癌细胞系中表达无显著差异(P>0.05)．P16^INK4a、Shh、Smo、Ptch及Gli蛋白在宫颈癌中表达强度显著高于癌旁及正常组织(P<0.05),在CINⅠ与正常组织间差异不显著(P>0.05)．P16^INK4a、Shh、Smo及Gli蛋白,在CINⅠ、CINⅡ与CINⅢ之间均有显著性差异(P<0.05)．相关分析显示,CINⅡ-CINⅢ中P16^INK4a与Shh和Smo蛋白表达正相关,浸润癌中P16^INK4a与Shh、Smo和Gli蛋白正相关．结论认为,P16^INK4a及Hh-Gli信号通路异常激活与宫颈癌发生及演进密切相关,且二者间具有相关性．Hh-Gli信号通路的激活可能是Shh配体增高调控Smo高表达而上调Gli蛋白所致．     

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.     

Dual Function of UNC-51-like Kinase 3 (Ulk3) in the Sonic Hedgehog Signaling Pathway

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.     

Shh signaling within the dental epithelium is necessary for cell proliferation,growth and polarization

Sonic hedgehog (Shh), a member of the mammalian Hedgehog (Hh) family, plays a key role during embryogenesis and organogenesis. Tooth development, odontogenesis, is governed by sequential and reciprocal epithelial-mesenchymal interactions. Genetic removal of Shh activity from the dental epithelium, the sole source of Shh during tooth development, alters tooth growth and cytological organization within both the dental epithelium and mesenchyme of the tooth. In this model it is not clear which aspects of the phenotype are the result of the direct action of Shh on a target tissue and which are indirect effects due to deficiencies in reciprocal signalings between the epithelial and mesenchymal components. To distinguish between these two alternatives and extend our understanding of Shh's actions in odontogenesis, we have used the Cre-loxP system to remove Smoothened (Smo) activity in the dental epithelium. Smo, a seven-pass membrane protein is essential for the transduction of all Hh signals. Hence, removal of Smo activity from the dental epithelium should block Shh signaling within dental epithelial derivatives while preserving normal mesenchymal signaling. Here we show that Shh-dependent interactions occur within the dental epithelium itself. The dental mesenchyme develops normally up until birth. In contrast, dental epithelial derivatives show altered proliferation, growth, differentiation and polarization. Our approach uncovers roles for Shh in controlling epithelial cell size, organelle development and polarization. Furthermore, we provide evidence that Shh signaling between ameloblasts and the overlying stratum intermedium may involve subcellular localization of Patched 2 and Gli1 mRNAs, both of which are targets of Shh signaling in these cells.