Kevin C. Corbit

Vertebrate Smoothened functions at the primary cilium

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.

Kif3a constrains β-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms

Primary cilia are microtubule-based organelles involved in signal transduction and project from the surface of most vertebrate cells. Proteins that can localize to the cilium, for example, Inversin and Bardet-Biedl syndrome (BBS) proteins, are implicated in both β-catenin-dependent and -independent Wnt signalling. Given that Inversin and BBS proteins are found both at the cilium and elsewhere in the cell, the role of the cilium itself in Wnt signalling is not clear. Using three separate mutations that disrupt ciliogenesis (affecting Kif3a, Ift88 and Ofd1), we show in this study that the primary cilium restricts the activity of the canonical Wnt pathway in mouse embryos, primary fibroblasts, and embryonic stem cells. Interestingly, unciliated cells activate transcription only in response to Wnt stimulation, but do so much more robustly than ciliated cells. Loss of Kif3a, but not other ciliogenic genes, causes constitutive phosphorylation of Dishevelled (Dvl). Blocking the activity of casein kinase I (CKI) reverses this constitutive Dvl phosphorylation and abrogates pathway hyper-responsiveness. These results suggest that Kif3a restrains canonical Wnt signalling both by restricting the CKI-dependent phosphorylation of Dvl and through a separate ciliary mechanism. More generally, these findings reveal that, in contrast to its role in promoting Hedgehog (Hh) signalling, the cilium restrains canonical Wnt signalling.

Disruption of a ciliary B9 protein complex causes Meckel syndrome.

Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.

The Extracellular Domain of Smoothened Regulates Ciliary Localization and Is Required for High-Level Hh Signaling

Members of the Hedgehog (Hh) family of secreted proteins function as morphogens to pattern developing tissues and control cell proliferation. The seven-transmembrane domain (7TM) protein Smoothened (Smo) is essential for the activation of all levels of Hh signaling. However, the mechanisms by which Smo differentially activates low- or high-level Hh signaling are not known. Here we show that a newly identified mutation in the extracellular domain (ECD) of zebrafish Smo attenuates Smo signaling. The Smo agonist purmorphamine induces the stabilization, ciliary translocation, and high-level signaling of wild-type Smo. In contrast, purmorphamine induces the stabilization but not the ciliary translocation or high-level signaling of the Smo ECD mutant protein. Surprisingly, a truncated form of Smo that lacks the cysteine-rich domain of the ECD localizes to the cilium but is unable to activate high-level Hh signaling. We also present evidence that cilia may be required for Hh signaling in early zebrafish embryos. These data indicate that the ECD, previously thought to be dispensable for vertebrate Smo function, both regulates Smo ciliary localization and is essential for high-level Hh signaling.

Adipocyte JAK2 mediates growth hormone–induced hepatic insulin resistance

For nearly 100 years, growth hormone (GH) has been known to affect insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here, we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Adipocyte GH signaling directly impinged on both adipose and hepatic insulin signal transduction. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extrahepatic, adipose tissue-dependent mechanism.

Adipocyte JAK2 Regulates Hepatic Insulin Sensitivity Independently of Body Composition, Liver Lipid Content, and Hepatic Insulin Signaling

Disruption of hepatocyte growth hormone (GH) signaling through disruption of Jak2 (JAK2L) leads to fatty liver. Previously, we demonstrated that development of fatty liver depends on adipocyte GH signaling. We sought to determine the individual roles of hepatocyte and adipocyte Jak2 on whole-body and tissue insulin sensitivity and liver metabolism. On chow, JAK2L mice had hepatic steatosis and severe whole-body and hepatic insulin resistance. However, concomitant deletion of Jak2 in hepatocytes and adipocytes (JAK2LA) completely normalized insulin sensitivity while reducing liver lipid content. On high-fat diet, JAK2L mice had hepatic steatosis and insulin resistance despite protection from diet-induced obesity. JAK2LA mice had higher liver lipid content and no protection from obesity but retained exquisite hepatic insulin sensitivity. AKT activity was selectively attenuated in JAK2L adipose tissue, whereas hepatic insulin signaling remained intact despite profound hepatic insulin resistance. Therefore, JAK2 in adipose tissue is epistatic to liver with regard to insulin sensitivity and responsiveness, despite fatty liver and obesity. However, hepatocyte autonomous JAK2 signaling regulates liver lipid deposition under conditions of excess dietary fat. This work demonstrates how various tissues integrate JAK2 signals to regulate insulin/glucose and lipid metabolism.

Disruption of Hepatocyte Jak2 leads to Spontaneous NASH in Aged Mice and Uncouples Metabolic Liver Disease from Insulin Resistance

Growth Hormone (GH) is a master regulator of metabolic homeostasis and longevity. Whole body GH insensitivity (GHI) augments insulin sensitivity, age-related disease resistance, adiposity, and occurrence of NAFLD. Conversely, acromegalic patients are prone to diabetes and increased mortality due to constitutive high levels of circulating GH. However, which tissues control the various metabolic aspects of GH physiology are unknown. Therefore, we determined the role of GH in age-related metabolic dysfunction by inducing hepatocyte- (JAK2L) or adipocyte-specific (JAK2A) GHI individually or combinatorially (JAK2LA) via deletion of Jak2, an obligate transducer of GH signaling. Aged JAK2L mice were insulin resistant but lean and had significant NASH, hepatic inflammation, and fibrosis. In contrast, JAK2A animals had increased adiposity and were completely resistant to age-associated hepatic steatosis, NASH, and insulin resistance. Interestingly, while JAK2LA mice retained enhanced whole-body insulin sensitivity, they still developed NASH to an almost identical degree as JAK2L mice but with a substantial reduction in the degree of microvesicular steatosis. Collectively, loss of adipocyte Jak2 conferred whole body insulin sensitivity even in the face of obesity and NASH. Deletion of hepatocyte Jak2 promoted NASH in aged mice without any dietary or drugs perturbations. The effect appears to be liver autonomous and cannot be overcome by the insulin sensitizing effect of adipocyte Jak2 deletion. Here, we describe the first model of spontaneous NASH that is coupled to augmented insulin sensitivity. Further, there was an inverse correlation between insulin sensitivity and the degree of microvesicular steatosis. Therefore, GH signaling independently mediates insulin/glucose and lipid homeostasis and directly regulates the development of NASH in aged mice. Financial Support: This study was supported by National Institutes of Health (NIH) Grants 1R01DK091276 (to E.J.W.). We also acknowledge the support of the University of California, San Francisco (UCSF) Cardiovascular Research Institute, the UCSF Diabetes Center (P30 DK063720), the UCSF Liver Center (P30 DK026743, and the James Peter Read Foundation. Abbreviations NASH non-alcoholic steato-hepatitis NAFLD non-alcoholic fatty liver disease GH growth hormone JAK2 Janus kinase 2 CON CON mice JAK2L hepatocyte-specific deletion of JAK2 JAK2A adipocyte-specific deletion of JAK2 JAK2LA hepatocyte and adipocyte JAK2 knockout TG triglyceride AST aspartate aminotransferase ALT alanine transaminase Stat5 signal transducer and activator of transcription 5 qRT-PCR quantitative reverse-transcription polymerase chain reaction Mcp1 monocyte chemoattractant protein-1 Cd11b cluster of differentiation molecule 11b F4/80 EGF-like module-containing mucin-like hormone receptor-like 1 FcgR1 high affinity immunoglobulin gamma Fc receptor I L-Fabp liver fatty acid binding protein PPARγ peroxisome proliferator-activated receptor gamma FATP fatty acid transport protein CD36/FAT Fatty Acid Translocase ITT insulin tolerance test. Lpl lipoprotein lipase IL- interleukin- FcgR1 Fc receptor IgG Tnfα tumor necrosis factor alpha Tgfβ1 transforming growth factor beta 1 αSMA, alpha 2 smooth muscle actin IGF-1 insulin-like growth factor 1.

Adipocyte JAK2 mediates hepatic insulin sensitivity and the diabetogenic action of Growth Hormone

For nearly 100 years, Growth Hormone (GH) has been known to impact insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Adipocyte GH signaling directly impinged on both adipose and hepatic insulin signal transduction. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extra-hepatic, adipose tissue-dependent mechanism.For nearly 100 years, Growth Hormone (GH) has been known to regulate insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extra-hepatic, adipose tissue-dependent mechanism.

A transition zone complex of ciliopathy proteins regulates ciliary composition

We have identified a complex of proteins that form part of the transition zone, a region at the base of the cilium. This complex includes the three members of the Tectonic family, extracytosolic glycoproteins that interact with transmembrane components of the transition zone such as Tmem67, Tmem216, and Tmem231. These transmembrane proteins connect to an intracellular transition zone complex comprised of many known Joubert- and Meckel-associated proteins including Cc2d2a, B9d1, B9d2, Mks1. Loss of components of this transition zone complex in mice compromise ciliogenesis in some tissues, and deregulate ciliary protein composition in others. In particular, the ciliary localization of Smoothened (Smo), a central component of the Hedgehog pathway, depends on this complex. As Smo functions at the cilium, many mouse transition zone mutants show deregulation of Hh signaling, resulting in ventralization of the neural tube and polydactyly. Defining the components of the transition zone has led to the identification of additional genes underlying Joubert and Meckel syndromes including Tctn1, Tctn2 and B9d2. We hypothesize that Joubert and Meckel syndromes are caused by transition zone dysfunction that disrupts intercellular signaling, leading to developmental defects.

Regulation of ciliogenesis in mammalian development and disease

Heightened cortical plasticity during postnatal critical periods wanes with age to consolidate neural circuits and behavior. Such rigidity in turn limits recovery from injury or developmental disorders. Identifying these mechanisms carries a broad impact for therapeutic approaches, but is often hampered by complex etiologies and distributed networks. An approachable model is the permanent loss of visual acuity (amblyopia) following sensory deprivation within primary visual cortex, which does not readily recover later in life. Here, we identify two classes of “molecular brakes” on (1) structural growth (acute myelin-related Nogo receptor signaling), and (2) neuromodulation (Lynx1 suppression of nicotinic cholinergic receptor signaling) that actively limit plasticity in adulthood. Removal of these brakes notably restores visual acuity to normal levels simply upon reopening the eye rendered amblyopic earlier in life. Given the widespread distribution of these factors, they may offer a more general model for understanding cognitive development and for treating disorders of similar origin in early postnatal life.