Research Article: Primary Cilia as Signaling Hubs in Health and Disease

Date Published: November 16, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Yuhei Nishimura, Kousuke Kasahara, Takashi Shiromizu, Masatoshi Watanabe, Masaki Inagaki.

http://doi.org/10.1002/advs.201801138

Abstract

Primary cilia detect extracellular cues and transduce these signals into cells to regulate proliferation, migration, and differentiation. Here, the function of primary cilia as signaling hubs of growth factors and morphogens is in focus. First, the molecular mechanisms regulating the assembly and disassembly of primary cilia are described. Then, the role of primary cilia in mediating growth factor and morphogen signaling to maintain human health and the potential mechanisms by which defects in these pathways contribute to human diseases, such as ciliopathy, obesity, and cancer are described. Furthermore, a novel signaling pathway by which certain growth factors stimulate cell proliferation through suppression of ciliogenesis is also described, suggesting novel therapeutic targets in cancer.

Partial Text

Primary cilia are nonmotile, 1–10 µm long antenna‐like structures observed in a variety of vertebrate cells. Primary cilia detect extracellular cues, such as mechanical flow and chemical stimulation, and transduce these signals into the cell.1, 2, 3, 4, 5, 6, 7 Therefore, the dysregulation of primary cilia can cause various diseases, including congenital anomalies, neurodevelopmental disorders, obesity, and cancer.8, 9, 10, 11, 12, 13, 14

Primary cilia have three compartments, the basal body, the transition zone, and the axoneme.1, 2, 3, 4, 5, 6, 7 The basal body is derived from the mother centriole and has distal and subdistal appendages and docks to the apical plasma membrane through the distal appendage. The axoneme consists of nine circularly arranged microtubule doublets and forms a projection extending from the basal body. The transition zone is a short (0.5 µm) area located above the basal body, characterized by Y‐shaped connectors between the microtubule doublets and primary cilia membrane. Although primary cilia are nonmotile, the formation is dynamically regulated. In 1979, Tucker et al. found that primary cilia were assembled when cultured mouse 3T3 fibroblasts exited the cell cycle under serum deprivation (i.e., G0 phase).36, 37 They also reported that when the quiescent fibroblasts were stimulated with serum, primary cilia were disassembled after the serum stimulation,36, 37 which is also the case for RPE1 cells (an immortalized cell line derived from human retinal pigment epithelium).38 Deciliation after serum stimulation corresponded to the G0/G1 transition.38 Subsequent analyses revealed that the progression to S phase after cell cycle reentry was delayed and shortened if primary cilia were longer and shorter in the G0 phase, respectively.39, 40 In contrast, forced ciliation in growing cells resulted in the arrest of cell‐cycle progression.25, 26, 41, 42, 43 These findings suggest that primary cilia themselves can work as negative regulators of the cell cycle. In the following sections, we describe the primary cilia dynamics with an eye on (i) the assembly of primary cilia responding to serum withdrawal, (ii) the disassembly of primary cilia responding to serum stimulation, and (iii) suppression of ciliogenesis in the presence of serum (growth factors).

Two pathways to generate primary cilia, namely, the extracellular and intracellular pathways, have been demonstrated in the assembly of primary cilia responding to serum withdrawal.6, 44, 45, 46 In the extracellular pathway, the mother centriole first docks to the plasma membrane, after which axonemal microtubules are nucleated and the cilia grow directly in the extracellular setting. Centrosomal protein 83 (CEP83), which is localized at the distal appendage of the mother centriole, plays important roles in centriole‐to‐membrane docking47 and the recruitment of intraflagellar transport 20 (IFT20) protein, which is mandatory for axoneme formation,48 to the basal body.49 The extracellular pathway‐dependent ciliogenesis is frequently observed in epithelial cells.45, 46

When quiescent mouse 3T3 fibroblasts or human RPE1 cells are stimulated with serum, primary cilia disassemble after serum stimulation.36, 37, 38 Deciliation after serum stimulation corresponds to the G0/G1 transition.38 AurA has important roles in the deciliation.69 AurA is activated by serum stimulation through Ca2+/calmodulin signaling, the noncanonical wingless (WNT) pathway, and phosphatidyl inositol signaling.12, 70, 71, 72 Calmodulin activated by Ca2+ binds to multiple motifs on AurA and activates it.73 Neural precursor cells express developmentally downregulated protein 9 (NEDD9), which is activated via the noncanonical WNT pathway and binds to and activates AurA.74 Inositol polyphosphate‐5‐phosphatase E stimulates autophosphorylation and activates AurA, probably through producing phosphatidylinositol‐(3,4)‐diphosphate (PtdIns(3,4)P2).72 The activated AurA phosphorylates itself and targets proteins during G1, which stimulate the disassembly of primary cilia.12 One important substrate of AurA is histone deacetylase 6 (HDAC6). Phosphorylated HDAC6 deacetylates α‐tubulin and reduces the stability of axoneme microtubules.38 Several studies support the role of HDAC6 in the disassembly of primary cilia.75, 76 However, mice lacking HDAC6 develop normally,77 suggesting that there may be other important pathways regulated by AurA in the disassembly of primary cilia.

We have recently found that knockdown of trichoplein, a centriolar protein originally identified as a keratin‐binding protein (and now as an activator protein of AurA),78, 79 caused ciliogenesis through the inactivation of AurA and cell cycle arrest in RPE1 cells in the presence of serum.41 This experimental setting was quite different from that of ciliogenesis induced by serum starvation. These effects were prevented by simultaneous knockdown of IFT20 or CEP164, suggesting that the ciliogenesis is required for the cell cycle arrest induced by knockdown of trichoplein.41 This was the first report indicating that ciliogenesis could inhibit the cell cycle and that the trichoplein‐AurA pathway could be an important player in ciliogenesis‐induced cell cycle arrest. Recently, we analyzed the molecular mechanisms of the regulation of the trichoplein‐AurA pathway (Figure2).

Primary cilia and RTKs, especially PDGFRα, play critical roles in cell migration.15, 18, 22 When tissue is wounded, PDGF‐AA, a dimeric glycoprotein composed of two A subunits of PDGF, is secreted mainly from platelets. The PDGF‐AA binds to PDGFRα located in primary cilia in dermal fibroblasts, which causes the autophosphorylation of PDGFRα, resulting in the activation of MEK1/2‐ERK1/2‐p90 ribosomal S6 kinase (p90RSK)‐Na+/H+ exchanger (NHE1) and PI3K‐AKT‐ NHE1 pathways. If primary cilia are defective, these signaling modules are not activated, suggesting that localization of PDGFRα in primary cilia is critical in response to PDGF‐AA to regulate cell migration. The activated NHE1 translocates to the leading and mobile edge of the cell (called the lamellipodium) and organizes the actin cytoskeleton with Ezrin/Radixin/Moesin proteins, which enable directional cell migration. The localization of PDGFRα is regulated by Cbl E3 ubiquitin ligases.24 Both c‐Cbl and Cbl‐b interact with IFT20 and ubiquitinate PDGFRα in response to PDGF‐AA stimulation, resulting in the internalization of PDGFRα for negative regulation. If primary cilia are impaired by the depletion of IFT20, both c‐Cbl and Cbl‐b are ubiquitinated and degraded, resulting in the overactivation of PDGFRα localized in the plasma membrane in response to the ligand activation.24 PDGF signaling in primary cilia has been excellently reviewed.15, 21, 22

EGF signaling regulates not only cell proliferation but also the development of various tissues and the maintenance of body homeostasis. In the kidney, EGFR is localized in the primary cilia of epithelial cells of the thick ascending loop of Henle and the distal convoluted tube within the vicinity of polycystin 2 (PKD2).83, 84, 85 PKD2 belongs to the transient receptor potential superfamily of channel proteins and conveys extracellular stimuli to ion, mainly Ca2+, currents.14, 86, 87 EGFR in kidney epithelial cells is activated by various EGF ligands during tubulogenesis.88, 89, 90, 91, 92 The activation of EGFR causes a decreased local concentration of phosphatidylinositol (4,5)‐biphosphate (PtdIns(4,5)P2) partly through the phosphorylation of PIP2 into phosphatidylinositol (3,4,5)‐triphosphate (PtdIns(3,4,5)P3) by PI3K and the hydrolysis of PIP2 by PLCγ85, 93 (Figure3). The function of PKD2 is suppressed by PtdIns(4,5)P2. When EGF signaling is activated, the local concentration of PtdIns(4,5)P2 is decreased, which causes activation of PKD2, resulting in increased Ca2+ influx.85, 93 Therefore, EGF signaling can sensitize the primary cilium‐based mechanosensation by reducing the threshold of PKD2 for activation by mechanical stimulation.87 Targeted disruption of genes involved in EGF signaling, including EGFR,94, 95 PKD2,96 and USP8,26 causes cystic kidney in animal models. The localization of EGFR and PKD2 in primary cilia is also observed in human airway smooth muscle cells97 and mouse odontoblasts.98 The interaction between EGFR and PKD2 may be involved in the mechanosensation that is necessary for directed migration of these cells.

Although the human body is externally symmetrical, the visceral organs are arranged asymmetrically in a stereotyped manner.99 For example, the heart, spleen, and pancreas are located on the left side of the body, whereas the liver and gall bladder are located on the right side. The left‐right (L‐R) asymmetry is generated through the three steps: (i) the initial breakage of the L‐R symmetry at the ventral node of the mammalian embryo or an equivalent structure of other vertebrates, such as Kupffer’s vesicle in zebrafish; (ii) transfer of the L‐R biased signal from the node to the lateral plate mesoderm (LPM), which also has primary cilia,100 resulting in the expression of transforming growth factor β (TGFβ)‐related proteins, such as Nodal and Lefty2 on the left side of the LPM; and (iii) L‐R asymmetric morphogenesis of the visceral organs induced by these signaling proteins.101 Nodal protein is expressed bilaterally in perinodal cells located at the periphery of the node (crown cells).102 When the nonmotile cilia of crown cells sense the leftward flow generated by the rotational movement of motile cilia of pit cells, which are located in the node as an epithelial sheet of a few hundred monociliated cells, the mRNA for Cerberus‐like 2 (Cerl2), an antagonist of Nodal, is degraded crown cells103, 104, 105 located at the left side (Figure4). Cerl2 usually binds to Nodal and prevents the formation of a heterodimer composed of Nodal and growth differentiation factor‐1 (Gdf1).103 Gdf1, another TGFβ‐related factor, is expressed in crown cells and is essential for Nodal expression in LPM.106 If the Cerl2 protein is reduced in the left side crown cell, Nodal forms a heterodimer with Gdf1 and travels to left side of the LPM, where the heterodimer binds to type I and type II TGFβ receptors.107 Next, the transcription factor forkhead box protein 1 (FoxH1), interacting with SMAD2/3, is activated and binds to the Nodal‐responsive enhancer region in promoters of FoxH1 target genes, including Nodal itself, Lefty2, and Pitx2.108 The promoter of Pitx2 also has a conserved binding sequence for Nhx2, enabling asymmetric expression of Pitx2 longer than those of Nodal and Lefty2.109 LPM‐derived cells expressing Pitx2 develop left‐side morphogenesis.110 These findings suggest that physiological L‐R asymmetry is disturbed if the function of cilia in pit cells and/or crown cells are impaired. Consistent with this, situs inversus, a type of laterality disorder in which all internal organs are reversed, is observed in ciliopathy, a genetic disorder linked to ciliary dysfunction.111, 112 It remains unknown whether the primary cilia of crown cells and LPM possess receptors for growth factors, such as EGF and TGFβ.

The formation of the cerebral cortex begins with the transition from neuroepithelial to radial glial cells, the proliferating progenitors of the developing neocortex, in the ventricular zone113 (Figure5A). The precise coordination between lateral expansion of radial glial cells and differentiation from radial glial to intermediate progenitor cells is critical to form the cerebral cortex. The apical domains of radial glial cells face lateral ventricles filled with cerebrospinal fluid into which the primary cilia of radial glial cells protrude. IGF2 is secreted into the cerebrospinal fluid from the choroid plexus, a highly vascularized tissue located in each ventricle of the brain, and binds to IGF1R located on the primary cilia of radial glial cells.114 The binding of IGF2 to IGF1R causes the proliferation of radial glial cells.115 The level of IGF2 is highest in late neocortical development, suggesting that IGF2 may preferentially regulate upper‐layer corticogenesis.115 IGF1 in cerebrospinal fluid also contributes to the proliferation of radial glial cells116 (Figure 5B). The binding of IGF1 to IGF1R on the primary cilia of radial glial cells activates and phosphorylates IGF1R. Although IGF1R is known as a RTK, it also has noncanonical G protein‐coupled receptor activity.117, 118 The activated IGF1R binds to Gα, releasing Gβγ. The free Gβγ triggers the release of Tctex‐1 from the dynein complex, enabling subsequent phosphorylation and recruiting of the phosphorylated Tctex‐1 to the transition zone of the primary cilia of radial glial cells at the ventricular zone.116 The phosphorylated Tctex‐1 stimulates the resorption of primary cilia and subsequent S phase progression.119 Shortening G1 increases the proliferation of radial glial cells, whereas lengthening G1 stimulates differentiation of radial glial cells into neurons.120, 121 These findings suggest that impairment of these IGF signaling events may cause abnormal corticogenesis and neurodevelopmental diseases. In fact, microcephaly and mental retardation have been observed in the patients suffering from mutations in IGF1 or IGF1R.122, 123 The mutation of ADP‐ribosylation factor‐like protein 13B, a causative gene of Joubert syndrome, which is accompanied by autism,124, 125 disrupts the localization of IGF1R in primary cilia of radial glial cells and impairs the migration and placement of interneurons in the developing cerebral cortex.126, 127

Ciliopathies, such as Bardet‐Biedl syndrome (BBS) and Alström syndrome (ALMS), often accompany obesity, suggesting that the impairment of primary cilia may be involved in the pathogenesis of obesity.10, 11, 128 Obesity results from excessive calorie intake relative to the energy expenditure. The arcuate nucleus of the hypothalamus is the center that regulates calorie intake and energy expenditure. The arcuate nucleus is composed of different types of ciliated neurons, including anorexigenic neurons expressing pro‐opiomelanocortin (POMC) and orexigenic neurons expressing Agouti‐related peptide (AgRP) (Figure6). POMC is cleaved by proteases to generate α‐melanocyte‐stimulating hormone (αMSH), which is the anorexigenic neuropeptide. These neurons express the leptin receptor in primary cilia.129 If leptin, a hormone secreted from adipocytes in response to food intake, binds to its receptor, the transcription of POMC and AgRP is increased and decreased, respectively, through the JAK‐STAT3 pathway.130, 131 The negative‐feedback system of appetite does not work if primary cilia are ablated by knockout of IFT88 or KIF3A, suggesting the fundamental role of primary cilia in appetite regulation by leptin.10, 132 Genes associated with BBS and ALMS encode ciliary proteins, and the mutated proteins impair the function of primary cilia in hypothalamic neurons, which is the potential cause of obesity in these disorders.133

Although primary cilia are lost in a wide range of cancer types, as described in the section below, primary cilia can also promote tumor progression in different types of cancer, including medulloblastoma, basal cell skin cancer, and basal‐like breast cancer.2, 147, 148, 149 Medulloblastoma comprises four major subgroups: sonic hedgehog (SHH), WNT, group 3, and group 4.150, 151, 152 The SHH subgroup accounts ≈30% of all cases. In the SHH groups, somatic mutations and amplifications of genes involved in hedgehog pathway have been identified, including Patched (PTCH1), SMO, suppressor of fused (SUFU), and Gli transcription factor 2 (GLI2).153 SHH is a secretory protein that binds to the receptor PTCH1. In the absence of SHH, PTCH1 is located in primary cilia and keeps SMO, a seven‐pass transmembrane protein, outside primary cilia. When SHH binds to PTCH1, it disappears from primary cilia, allowing accumulation of SMO in primary cilia (Figure7A). SUFU is also accumulated in primary cilia in the presence of SHH. There are three Gli transcription factors: GLI1, GLI2, and GLI3. GLI2 and GLI3 can be converted to transcriptional activators or repressors, depending on their proteolytic processing. GLI1 is a transcriptional target of GLI2 and GLI3 and acts as a transcriptional activator. In the absence of SHH, GLI2 and GLI3 (predominant) are converted to transcriptional repressors (GLI2‐R and GLI3‐R) in primary cilia and translocate to the nucleus, resulting in the suppression of hedgehog signaling (Figure 7B). In the presence of SHH, both GLI2 and GLI3 are converted to transcriptional activators (GLI2‐A and GLI3‐A) in primary cilia and translocate to the nucleus, where they mediate hedgehog signaling at the level of transcription (Figure 7A). SMO and SUFU accumulated in primary cilia are positively and negatively involved in the conversion and translocation of GLI2/3‐A, respectively.147, 148, 154, 155 Loss‐of‐function mutation in PTCH1 or SUFU and gain‐of‐function mutation in SMO or GLI2 can enhance hedgehog signaling, resulting in tumorigenesis. Because PTCH1 and SMO work in primary cilia to generate GLI2‐A and GLI3‐A, primary cilia promote the progression of SHH medulloblastoma caused by mutations of PTCH1 and SMO. In the case of mutation or amplification of GLI2, the amount of GLI2‐A can increase without the help of PTCH1 and SMO (e.g., cilia‐independent) (Figure 7B). In this case, GLI3‐R is predominantly generated in primary cilia in the absence of SHH and antagonizes GLI2‐A. Therefore, primary cilia suppress the progression of SHH medulloblastoma caused by the mutation/amplification of GLI2.156 The dual and opposing roles of primary cilia are also observed in basal cell carcinoma (BCC), a skin cancer caused by dysregulated hedgehog signaling.157, 158, 159 Primary cilia are expressed in BCC caused by activated form of SMO and ablation of primary cilia inhibits the progression of BCC. In contrast, ablation of primary cilia expressed in BCC caused by activated GLI2 accelerates the progression of BCC.157 These findings suggest that primary cilia can promote and suppress tumorigenesis depending on the context.

Primary cilia are lost in a wide range of cancer types, including clear‐cell renal‐cell carcinoma,191, 192, 193 epithelial ovarian cancer,16 cholangiocarcinoma,194 pancreatic ductal adenocarcinoma (PDAC),195 astrocytoma/glioblastoma,196 luminally derived breast cancer,197 melanoma,198 chondrosarcoma,199 and prostate cancer.200 These findings and the negative role of primary cilia in cell cycle progression suggest the hypothesis that ciliopathies may predispose tissue to the development of cancer. In fact, renal cancers can be comorbid in two ciliopathies, Birt‐Hogg‐Dube syndrome and VHL syndrome.201, 202 However, except for these ciliopathies, cancer incidence is not increased in human ciliopathies.203 Thus, the relationship between primary cilia and cancer remains to be fully elucidated.

Growth factor and morphogen signaling through primary cilia are undoubtedly important for both health and disease. Although many mechanisms underlying these signaling events have been elucidated, many questions still exist. The clinical phenotypes observed in ciliopathies are highly heterogeneous, even in the same syndrome, depending on the organ system in which primary cilia are impaired14 and the mutation state of the individual patient.208 Growth factor and morphogen signaling are highly context dependent.209, 210, 211 In addition, complex crosstalk between various growth factor and morphogen signaling pathways through primary cilia occurs.15, 19, 22 A primary cilium can express various receptors for growth factors and morphogens. Several molecules have been identified as hubs of signaling pathways activated by different growth factors and morphogens at the primary cilium. For example, the expression of Gli2, one of the most important targets of hedgehog signaling, is also increased by TGFβ signaling through a SMAD2/3 and tuberous sclerosis complex protein 1‐dependent pathway in the primary cilia of mouse embryonic fibroblasts.212 USP8 is also activated by EGF, PDGF, and FGF signaling.26 The complete mechanism by which each growth factor and morphogen signaling pathway contributes to the activation of these hub molecules and how the integrated activity of the hubs is involved in health and disease remain to be elucidated.

The authors declare no conflict of interest.

 

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http://doi.org/10.1002/advs.201801138

 

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