Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

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Primary cilia formation does not rely on WNT/β-catenin signaling

10.1101/2020.10.30.361642 ◽

2020 ◽

Author(s):

Ondrej Bernatik ◽

Petra Paclikova ◽

Anna Kotrbova ◽

Vitezslav Bryja ◽

Lukas Cajanek

Keyword(s):

Primary Cilia ◽

Wnt Pathway ◽

Inhibitory Effect ◽

Positive Role ◽

Knock Out ◽

Wnt Ligands ◽

Cilia Formation ◽

And Function ◽

Cilium Length

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However gaps exist in our understanding of how cilia formation and function is regulated.Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin.To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6 or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis.These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

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Primary Cilia in Glial Cells: An Oasis in the Journey to Overcoming Neurodegenerative Diseases

Frontiers in Neuroscience ◽

10.3389/fnins.2021.736888 ◽

2021 ◽

Vol 15 ◽

Author(s):

Soo Mi Ki ◽

Hui Su Jeong ◽

Ji Eun Lee

Keyword(s):

Neurodegenerative Diseases ◽

Glial Cells ◽

Primary Cilia ◽

Regulatory Mechanisms ◽

Close Attention ◽

Cellular Processes ◽

Cilia Formation ◽

And Function ◽

Cellular Organelles

Many neurodegenerative diseases have been associated with defects in primary cilia, which are cellular organelles involved in diverse cellular processes and homeostasis. Several types of glial cells in both the central and peripheral nervous systems not only support the development and function of neurons but also play significant roles in the mechanisms of neurological disease. Nevertheless, most studies have focused on investigating the role of primary cilia in neurons. Accordingly, the interest of recent studies has expanded to elucidate the role of primary cilia in glial cells. Correspondingly, several reports have added to the growing evidence that most glial cells have primary cilia and that impairment of cilia leads to neurodegenerative diseases. In this review, we aimed to understand the regulatory mechanisms of cilia formation and the disease-related functions of cilia, which are common or specific to each glial cell. Moreover, we have paid close attention to the signal transduction and pathological mechanisms mediated by glia cilia in representative neurodegenerative diseases. Finally, we expect that this field of research will clarify the mechanisms involved in the formation and function of glial cilia to provide novel insights and ideas for the treatment of neurodegenerative diseases in the future.

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Partial loss of colonic primary cilia promotes inflammation and carcinogenesis

10.1101/2019.12.20.871772 ◽

2019 ◽

Author(s):

Ruizhi Tang ◽

Conception Paul ◽

Rossano Lattanzio ◽

Thibaut Eguether ◽

Hulya Tulari ◽

...

Keyword(s):

Normal Tissue ◽

Primary Cilia ◽

Colon Carcinogenesis ◽

Partial Loss ◽

Colonic Epithelial Cells ◽

Knock Out ◽

Chemically Induced ◽

Colonic Primary ◽

Increased Susceptibility

AbstractPrimary cilia (PC) are important signaling hubs in cells and their deregulation has been associated with various diseases including cancer. Here we explored the role of PC in colorectal cancer (CRC) and colitis. In the colon we found PC to be mostly present on different subtypes of fibroblasts. Colons of mice exposed to either chemically induced colitis-associated colon carcinogenesis (CAC) or dextran sodium sulfate (DSS)-induced colitis had decreased numbers of PC. We employed conditional knock-out strains for the PC essential genes, Kif3A and Ift88, to generate mice with reduced numbers of PC on colonic fibroblasts. These mice showed an increased susceptibility in the CAC model as well as in DSS-induced colitis. Colons from DSS-treated mice with PC-deficiency on fibroblasts displayed an elevated production of the pro-inflammatory cytokine IL-6 and colonic epithelial cells had diminished levels of HES-1, a key transcription factor of Notch signaling. Notably, an analysis of PC presence on biopsies of patients with ulcerative colitis as well as CRC patients revealed decreased numbers of PC on colonic fibroblasts in pathological versus surrounding normal tissue. Taken together, we provide evidence that a decrease in colonic PC numbers promotes colitis and CRC.Graphical Abstract

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NRF2-dependent gene expression promotes ciliogenesis and Hedgehog signaling

Scientific Reports ◽

10.1038/s41598-019-50356-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 6

Author(s):

Ana Martin-Hurtado ◽

Raquel Martin-Morales ◽

Natalia Robledinos-Antón ◽

Ruth Blanco ◽

Ines Palacios-Blanco ◽

...

Keyword(s):

Hedgehog Signaling ◽

Primary Cilia ◽

Embryonic Stem ◽

Mtor Inhibitors ◽

Gene Promoters ◽

Protein Levels ◽

Functional Connections ◽

Differentiated Cells ◽

Cilia Formation ◽

And Function

Abstract The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implications.

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Phosphoinositide lipids in primary cilia biology

Biochemical Journal ◽

10.1042/bcj20200277 ◽

2020 ◽

Vol 477 (18) ◽

pp. 3541-3565

Author(s):

Sarah E. Conduit ◽

Bart Vanhaesebroeck

Keyword(s):

Primary Cilia ◽

Critical Role ◽

Phospholipid Composition ◽

Cell Types ◽

Phosphoinositide Metabolism ◽

Lipid Phosphatases ◽

Regulatory Enzymes ◽

Lipid Species ◽

Cilia Formation ◽

And Function

Primary cilia are solitary signalling organelles projecting from the surface of most cell types. Although the ciliary membrane is continuous with the plasma membrane it exhibits a unique phospholipid composition, a feature essential for normal cilia formation and function. Recent studies have illustrated that distinct phosphoinositide lipid species localise to specific cilia subdomains, and have begun to build a ‘phosphoinositide map’ of the cilium. The abundance and localisation of phosphoinositides are tightly regulated by the opposing actions of lipid kinases and lipid phosphatases that have also been recently discovered at cilia. The critical role of phosphoinositides in cilia biology is highlighted by the devastating consequences of genetic defects in cilia-associated phosphoinositide regulatory enzymes leading to ciliopathy phenotypes in humans and experimental mouse and zebrafish models. Here we provide a general introduction to primary cilia and the roles phosphoinositides play in cilia biology. In addition to increasing our understanding of fundamental cilia biology, this rapidly expanding field may inform novel approaches to treat ciliopathy syndromes caused by deregulated phosphoinositide metabolism.

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Involvement of NFAT Transcription Factors in NK Cell Reactivity

Blood ◽

10.1182/blood.v128.22.1344.1344 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1344-1344 ◽

Cited By ~ 2

Author(s):

Kathrin Rothfelder ◽

Melanie Märklin ◽

Julia Wild ◽

Daniela Dörfel ◽

Lothar Kanz ◽

...

Keyword(s):

Transcription Factors ◽

Nk Cells ◽

Nk Cell ◽

Inhibitory Effect ◽

Target Cells ◽

Cell Reactivity ◽

Tumor Immunosurveillance ◽

Metastatic Burden ◽

And Function

Abstract NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), as highlighted by its important role in T and B cell development and function. With regard to NK cells, available data indicate that NFAT is dispensable for development. However, several lines of evidence including the observation that the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, influence NK reactivity implicate a role of this family of transcription factors in NK cell reactivity and function. Here we employed different genetic mouse models on the C57BL/6 background to directly study the functional role of NFAT in NK cells. We found that except for NFAT3 mRNA and protein of all family members (NFAT 1, 2, 4 and 5) was expressed in resting NK cells of wild type (WT) mice with NFAT1, 2 and 4 being most abundantly detectable. When we employed NK cells with knockout (KO) of NFAT 1, 2, and 4 in comparative in vitro analyses, we surprisingly found that lack of NFAT resulted in enhanced NK cell activation, degranulation and release of immunomodulatory cytokines like IFN-γ after co-culture with YAC-1 target cells as well as increased production of granzyme B and perforin after cytokine activation. The inhibitory effect of NFAT on NK cell effector function was further confirmed in vivo by employing WT and germ line NFAT KO animals in the syngeneic B16 melanoma model, which revealed a significantly reduced metastatic burden in NFAT KO mice. Depletion of NK cells in this model system in turn resulted in increased metastasis, however, with WT animals displaying significantly higher metastatic burden compared to NFAT KO mice. As this pointed to the fact that NFAT influences metastasis via both NK-dependent and independent mechanisms, we further generated mice with a NK cell-specific (conditional Ncr1-Cre dependent) NFAT2 KO. When these animals were employed again in analyses of B16 lung metastasis, comparative analyses with WT animals confirmed the inhibitory effect of NFAT on NK tumor immunosurveillance. Taken together, these results provide the first direct evidence for the functional involvement of NFAT in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. Disclosures No relevant conflicts of interest to declare.

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Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

10.1101/251694 ◽

2018 ◽

Cited By ~ 1

Author(s):

Laura Vuolo ◽

Nicola L. Stevenson ◽

Kate J. Heesom ◽

David J. Stephens

Keyword(s):

Transition Zone ◽

Cell Lines ◽

Primary Cilia ◽

Point Mutations ◽

Intraflagellar Transport ◽

Jeune Syndrome ◽

Cilia Formation ◽

Microtubule Motor ◽

And Function ◽

Intermediate Chains

AbstractThe dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.SUMMARYHere, Vuolo and colleagues use engineered knockout human cell lines to define roles for dynein-2 intermediate chains. WDR34 is required for axoneme extension, while WDR60 is not. Both subunits are required for cilia transition zone organization and bidirectional intraflagellar transport.

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Aurora A and AKT Kinase Signaling Associated with Primary Cilia

Cells ◽

10.3390/cells10123602 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3602

Author(s):

Yuhei Nishimura ◽

Daishi Yamakawa ◽

Takashi Shiromizu ◽

Masaki Inagaki

Keyword(s):

Primary Cilia ◽

Tissue Type ◽

Aurora A ◽

Kinase Signaling ◽

Cellular Processes ◽

Pathological Conditions ◽

Cell Tissue ◽

Selective Targeting ◽

Cilia Formation ◽

And Function

Dysregulation of kinase signaling is associated with various pathological conditions, including cancer, inflammation, and autoimmunity; consequently, the kinases involved have become major therapeutic targets. While kinase signaling pathways play crucial roles in multiple cellular processes, the precise manner in which their dysregulation contributes to disease is dependent on the context; for example, the cell/tissue type or subcellular localization of the kinase or substrate. Thus, context-selective targeting of dysregulated kinases may serve to increase the therapeutic specificity while reducing off-target adverse effects. Primary cilia are antenna-like structures that extend from the plasma membrane and function by detecting extracellular cues and transducing signals into the cell. Cilia formation and signaling are dynamically regulated through context-dependent mechanisms; as such, dysregulation of primary cilia contributes to disease in a variety of ways. Here, we review the involvement of primary cilia-associated signaling through aurora A and AKT kinases with respect to cancer, obesity, and other ciliopathies.

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MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2

Blood ◽

10.1182/blood-2009-02-203943 ◽

2009 ◽

Vol 113 (26) ◽

pp. 6576-6583 ◽

Cited By ~ 416

Author(s):

Grazia Maria Spaggiari ◽

Heba Abdelrazik ◽

Flavio Becchetti ◽

Lorenzo Moretta

Keyword(s):

Prostaglandin E2 ◽

T Cell Activation ◽

Cell Activation ◽

Molecular Mechanisms ◽

Inhibitory Effect ◽

Interleukin 4 ◽

Myeloid Dendritic Cells ◽

And Function ◽

Dc Maturation

Abstract Various studies analyzed the inhibitory effect exerted by mesenchymal stem cells (MSCs) on cells of the innate or acquired immunity. Myeloid dendritic cells (DCs) are also susceptible to such inhibition. In this study, we show that MSCs strongly inhibit DC generation from peripheral blood monocytes. In the presence of MSCs, monocytes supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) did not acquire the surface phenotype typical of immature (CD14−, CD1a+) or mature (CD80+, CD86+, CD83+) DCs, failed to produce IL-12, and did not induce T-cell activation or proliferation. Analysis of the molecular mechanism(s) responsible for the inhibitory effect revealed a major role of prostaglandin E2 (PGE2). Thus, addition of the PGE2 inhibitor NS-398 restored DC differentiation and function. Moreover, PGE2 directly added to cultures of monocytes blocked their differentiation toward DCs in a manner similar to MSCs. Although IL-6 has been proposed to play a role in MSC-mediated inhibition of DC differentiation, our data indicate that PGE2 and not IL-6 represents the key inhibitory mediator. Indeed, NS-398 inhibited PGE2 production and restored DC differentiation with no effect on IL-6 production. These data emphasize the role of MSCs in inhibiting early DC maturation and identifying the molecular mechanisms responsible for the inhibitory effect.

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Arrestin-2 Differentially Regulates PAR4 and P2Y12 Receptor Signaling in Platelets

Blood ◽

10.1182/blood.v112.11.110.110 ◽

2008 ◽

Vol 112 (11) ◽

pp. 110-110 ◽

Cited By ~ 2

Author(s):

Dongjun Li ◽

Donna Woulfe

Keyword(s):

Ferric Chloride ◽

Phosphatidyl Inositol ◽

Resting Cells ◽

Positive Role ◽

Knock Out ◽

Akt Phosphorylation ◽

Fibrinogen Binding ◽

P2y12 Antagonists

Abstract Arrestins play important roles in the function of G Protein-Coupled Receptors (GPCRs) in many cells, but their roles in platelets remain uncharacterized. While the classical role of arrestins is considered to be the internalization and desensitization of GPCRs, more recent studies suggest that arrestins can serve as molecular scaffolds to recruit phosphatidyl inositol-3 kinases (PI3Ks) to GPCRs and promote PI3K-dependent signaling. Due to the multifunctional role of arrestins, we sought to determine whether arrestins regulate Akt activation in platelets and thrombosis in living animals. Co-immunoprecipitation experiments indicate that arrestin-2 associates with PAR4 in thrombin-treated platelets and P2Y12 in ADP-treated platelets, but neither receptor in resting cells. Interestingly, association of arrestin-2 with PAR4 was also stimulated by ADP and PAR4-induced association of arrestin with PAR4 was inhibited by P2Y12 antagonists or apyrase. To determine the functional role of arrestin-2 in platelets, ADP- and thrombin receptor-stimulated Akt phosphorylation was compared in platelets from arrestin-2 knock-out versus WT mice. Akt phosphorylation stimulated by 0.8 mM AYPGKF PAR4 agonist peptide was reduced by an average of 77% in arrestin-2 knock-out platelets compared to WT controls (significantly different, p=0.007, n=3 in each group), but ADP-stimulated Akt phosphorylation was unaffected (p=.38, n=3 each). PAR4-stimulated fibrinogen binding was also reduced in arrestin2−/− platelets (by 58.5% in 1 mM AYPGKF-stimulated platelets compared to WT controls), whereas ADP-stimulated fibrinogen binding was not. Finally, arrestin2 knock-out mice were less sensitive to ferric chloride-induced thrombosis than WT mice: 55% of WT mice (n-=9) formed occlusive thrombi after 2min15sec exposure of the carotid artery to 10% ferric chloride, whereas only 11% of WT mice (n=9) formed occlusive thrombi under the same conditions. In conclusion, arrestin-2 associates with both PAR4 and P2Y12 receptors, but differentially regulates their signaling to Akt and fibrinogen binding and appears to play a net positive role in regulating thrombosis in vivo.

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