Role Of Primary Cilia Research Articles

The Roles of Primary Cilia in Cardiovascular Diseases.

Primary cilia are microtubule-based organelles found in most mammalian cell types. Cilia act as sensory organelles that transmit extracellular clues into intracellular signals for molecular and cellular responses. Biochemical and molecular defects in primary cilia are associated with a wide range of diseases, termed ciliopathies, with phenotypes ranging from polycystic kidney disease, liver disorders, mental retardation, and obesity to cardiovascular diseases. Primary cilia in vascular endothelia protrude into the lumen of blood vessels and function as molecular switches for calcium (Ca2+) and nitric oxide (NO) signaling. As mechanosensory organelles, endothelial cilia are involved in blood flow sensing. Dysfunction in endothelial cilia contributes to aberrant fluid-sensing and thus results in vascular disorders, including hypertension, aneurysm, and atherosclerosis. This review focuses on the most recent findings on the roles of endothelial primary cilia within vascular biology and alludes to the possibility of primary cilium as a therapeutic target for cardiovascular disorders.

Primary Cilia as Signaling Hubs in Health and Disease.

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.

Abstract 575: Primary Cilia of Cardiac Neural Crest Cells Orchestrate Multiple Aspects of Cardiovascular Development

Cardiac neural crest cells (CNCC) are a specialized population of migratory neural crest cells that contribute to the developing cardiac outflow tract (OFT) and endocardial cushions (ECC). Like many eukaryotic cells, CNCC also possess primary cilia, which function as membrane-bound sensory organelles involved in intra- and extra-cellular communications in developing tissues. Previous work in our laboratory has shown that primary cilia of cardiac progenitor populations are critical orchestrators of both the septation and alignment of the developing OFT and interventricular septum. Our current in vivo models have refined the elimination of primary cilia to CNCC of the embryonic mouse heart using multiple neural crest-specific Cre drivers ( Wnt1 :Cre-Danielian et al,1998; Wnt1 :Cre2-Soriano et al, 2013) crossed with the Ift88 flox/flox mouse (Haycraft et al, 2007), resulting in elimination of primary cilia from CNCC during early embryonic heart development. Additionally, we have utilized a Td-Tomato (Tdt) reporter mouse line to track the migration of CNCC during various embryonic timepoints. Use of this reporter line revealed transient Tdt-positive CNCC contributions to the ventricular portion of the developing myocardium, in addition to the expected CNCC contributions to in the OFT and ECC regions. The resultant mutant phenotypes included a high incidence of outflow tract defects, impaired septation of the membranous interventricular septum, non-compaction of the developing ventricular myocardium, and perinatal lethality. Our data combine various immunohistochemical approaches to document, characterize, and quantify the novel presence of CNCC in the developing embryonic ventricular myocardium. These data suggest a potential modulatory role for primary cilia of CNCC in the development and maturation of the ventricular myocardium. Primary cilia-mediated signal transduction events that could account for the descried phenotypes are currently being elucidated.

Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells

The role of primary cilia in mechanosensation is essential in endothelial cell (EC) shear responsiveness. Here, we find that venous, capillary, and progenitor ECs respond to shear stress invitro in a cilia-dependent manner. We then demonstrate thatprimary cilia assembly in human induced pluripotent stem cell (hiPSC)-derived ECs varies between different cell lines with marginal influence of differentiation protocol. hiPSC-derived ECs lacking cilia do not align to shear stress, lack stress fiber assembly, have uncoordinated migration during wound closure invitro, and have aberrant calcium influx upon shear exposure. Transcriptional analysis reveals variation in regulatory genes involved in ciliogenesis among different hiPSC-derived ECs. Moreover, inhibition of histone deacetylase 6 (HDAC6) activity in hiPSC-ECs lacking cilia rescues cilia formation and restores mechanical sensing. Taken together, these results show the importance of primary cilia in hiPSC-ECmechano-responsiveness and its modulation through HDAC6 activity varies among hiPSC-ECs.

A Novel Role of Primary Cilia in Breast Cancer Recurrence

Recurrent tumors are the predominant cause of breast cancer‐related deaths, therefore there is an urgent need for more effective therapeutic strategies. However, a critical barrier to their development is a lack of understanding of the molecular mechanisms involved in breast tumor recurrence. Recently, primary cilia, microtubule‐based organelles that project from the surface of vertebrate cells, have emerged into the spotlight for their link to numerous human diseases, including cancer. However, the relationship between primary cilia and breast cancer recurrence remains unknown. Here, using a genetically engineered mouse model of breast cancer, which closely recapitulates the clinical phenomenon of breast cancer recurrence, we have uncovered that primary cilia are more highly enriched in recurrent mammary tumors than in both primary mammary tumors and asymptomatic/contralateral mouse mammary tissues. Congruently, we found that the percentage of ciliated cells was substantially elevated in recurrent breast cancer cells compared to primary breast cancer cells. Moreover, RNA‐sequence analysis also revealed that GLI2 and GLI3, which are major downstream effectors of the morphogenic Sonic Hedgehog signaling pathway and are also well‐known to localize to primary cilia, were upregulated in recurrent breast cancer cells compared to primary breast cancer cells. Collectively, these data suggest that primary cilia may have a role in breast cancer recurrence. Our study also indicates that, at least within the recurrent stages of the disease, the primary cilium may serve as a specialized oncogenic signaling organelle.Support or Funding InformationTo Sarah C. Goetz: R00 HD076444 (from the National Institute of Child Health and Human Development).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased Primary Cilia in Idiopathic Pulmonary Fibrosis.

Primary cilia are solitary, non-motile, axonemal microtubule-based antenna-like organelles that project from the plasma membrane of most mammalian cells and are implicated in transducing hedgehog signals during development. It was recently proposed that aberrant SHH signaling may be implicated in the progression of idiopathic pulmonary fibrosis (IPF). However, the distribution and role of primary cilia in IPF remains unclear. Here, we clearly observed the primary cilia in alveolar epithelial cells, fibroblasts, and endothelial cells of human normal lung tissue. Then, we investigated the distribution of primary cilia in human IPF tissue samples using immunofluorescence. Tissues from six IPF cases showed an increase in the number of primary cilia in alveolar cells and fibroblasts. In addition, we observed an increase in ciliogenesis related genes such as IFT20 and IFT88 in IPF. Since major components of the SHH signaling pathway are known to be localized in primary cilia, we quantified the mRNA expression of the SHH signaling components using qRT-PCR in both IPF and control lung. mRNA levels of SHH, the coreceptor SMO, and the transcription factors GLI1 and GLI2 were upregulated in IPF compared with control. Furthermore, the nuclear localization of GLI1 was observed mainly in alveolar epithelia and fibroblasts. In addition, we showed that defective KIF3A-mediated ciliary loss in human type II alveolar epithelial cell lines leads to disruption of SHH signaling. These results indicate that a significant increase in the number of primary cilia in IPF contributes to the upregulation of SHH signals.

The studies of roles and mechanism of primary cilium in the progressing of multiple osteochondromas

Objective To explore the pathological role of primary cilium in multiple osteochondromas by comparing the difference of primary cilium in the chondrocytes between multiple osteochondromas cartilage cap (MOCC)and normal epiphyseal growth plate cartilage (NEGPC). Methods Chondrocytes lines from MOCC and NEGPC were collected and cultured in vitro. The morphology and incidence of primary cilla in the MOCC and NEGPC were studied by laser confocal scanning microscope, Toluidine blue staining and Sulforhodamine B (SRB) was applied to display different cell morphology and growth rates between MOCC and NEGPC, and alcian blue and alkaline phosphatase (ALP) in MOCC and NEGPC were measured by histochemical staining. Results MOCC and NEGPC cultured in vitro were polygonal or fusiform and were stained with purple particles by toluidine blue staining. The incidence of primary cilla in MOCC [(25.265±1.766)%] was lower than in NEGPC [(53.943±3.631)%], the difference was significant (F=232.692, P=0.000). A values in MOCC were significantly lower than in NEGPC, while both of them were tended to increase with time. Alcian blue staining in MOCC were decreased compared to NEGPC. The area of ALP staining (7.387±2.045) mm2 in MOCC were decreased obviously compared with NEGPC (14.768±2.072) mm2, the difference was significant (F=16.835, P=0.020). Conclusion The primary cilium in chondrocyte was closely related to MO. When the expression of primary cilium in chondrocytes was suppressed, the proliferation, maturation and synthetic function of chondrocytes were delayed in epiphyseal growth plate cartilage. Key words: Multiple osteochondromas; Chondrocytes; Primary cilium; Cell culture

Repeated restraint stress promotes hippocampal neuronal cell ciliogenesis and proliferation in mice

Stress severely disturbs physiological and mental homeostasis which includes adult neurogenesis in hippocampus. Neurogenesis in hippocampus is a key feature to adapt to environmental changes and highly regulated by multiple cellular signaling pathways. The primary cilium is a cellular organelle, which acts as a signaling center during development and neurogenesis in adult mice. However, it is not clear how the primary cilia are involved in the process of restraint (RST) stress response. Using a mouse model, we examined the role of primary cilia in repeated and acute RST stress response. Interestingly, RST stress increased the number of ciliated cells in the adult hippocampal dentate gyrus (DG). In our RST model, cell proliferation in the DG also increased in a time-dependent manner. Moreover, the analysis of ciliated cells in the hippocampal DG with cell type markers indicated that cells that were ciliated in response to acute RST stress are neurons. Taken together, these findings suggest that RST stress response is closely associated with an increase in the number of ciliated neurons and leads to an increase in cell proliferation.

The primary cilium - once a "rudimentary" organelle that is now a ubiquitous sensory cellular structure involved in many pathological disorders.

This article looks mostly at the steps that have led to the primary cilium finding its place in our understanding of cell biology, developmental biology, and medical syndromes due to its aberrations. It is a personal account that stresses, if nothing else, the value of the adage "stick to your guns". My obsession with this organelle, following on from fascination with the centriole, has led to a whole career devoted to determining the nature and role of primary cilia in basic cell biology, which has proved much more important than had been appreciated for almost a century. They are heavily involved in very many aspects of cell physiology that have much wider implications with regard to human biology and probably throughout the animal kingdom. That aberrations, to the surprise of many researchers in their structure or functioning has led to their being implicated or perhaps deeply involved in an extraordinary range of medical conditions. This invitation allows me to raise crucial questions that need answers regarding the regulation of their genesis, their cache of both intracellular and extracellular signal, and their association with a multitude of development processes from embryo to adult status.

Triple Vectors Expand AAV Transfer Capacity in the Retina.

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

A role for primary cilia in aortic valve development and disease.

Bicuspid aortic valve (BAV) disease is the most common congenital heart defect, affecting 0.5-1.2% of the population and causing significant morbidity and mortality. Only a few genes have been identified in pedigrees, and no single gene model explains BAV inheritance, thus supporting a complex genetic network of interacting genes. However, patients with rare syndromic diseases that stem from alterations in the structure and function of primary cilia ("ciliopathies") exhibit BAV as a frequent cardiovascular finding, suggesting primary cilia may factor broadly in disease etiology. Our data are the first to demonstrate that primary cilia are expressed on aortic valve mesenchymal cells during embryonic development and are lost as these cells differentiate into collagen-secreting fibroblastic-like cells. The function of primary cilia was tested by genetically ablating the critical ciliogenic gene Ift88. Loss of Ift88 resulted in abrogation of primary cilia and increased fibrogenic extracellular matrix (ECM) production. Consequentially, stratification of ECM boundaries normally present in the aortic valve were lost and a highly penetrant BAV phenotype was evident at birth. Our data support cilia as a novel cellular mechanism for restraining ECM production during aortic valve development and broadly implicate these structures in the etiology of BAV disease in humans. Developmental Dynamics 246:625-634, 2017. © 2017 Wiley Periodicals, Inc.

Simulation of intra-ciliary diffusion suggests a novel role of primary cilia as a cell-signaling enhancer.

Besides the role to generate a fluid flow in the surrounding medium, eukaryotic cilia have a crucial function in sensing external signals such as chemical or mechanical stimuli. A large body of work has shown that cilia are frequently found in various types of sensory cells and are closely related to many regulatory mechanisms in differentiation and development. However, we do not yet have a definitive answer to the fundamental question, "why cilia?" It has been a long-standing mystery why cells use cilia for sensing external signals. To shed light on this, we sought to describe the kinetics of signaling with theoretical approaches. Based on the results, here we propose a new role of cilia as a cell-signaling enhancer. The enhancing effect comes from restricted volume for the free intra-ciliary diffusion of molecules due to the cylindrical shape of cilia, which can facilitate quick accumulation of intracellular signaling molecules. Our simulations demonstrate that both the rate and amplitude of response in signal transduction depend on where the membrane receptors or channels are located along the ciliary shaft. In addition, the calculated transfer function of cilia regarded as a transmitter of external signals also suggests the properties of cilia as a signal enhancer. Since such unique composition of receptors and channels in cilia is found in various types of eukaryotic cells, signal enhancing is presumably one of the most essential and conserved roles of cilia.

A novel role for primary cilia in airway remodeling

Primary cilia (PC) are solitary cellular organelles that play critical roles in development, homeostasis, and disease pathogenesis by modulating key signaling pathways such as Sonic Hedgehog and calcium flux. The antenna-like shape of PC enables them also to facilitate sensing of extracellular and mechanical stimuli into the cell, and a critical role for PC has been described for mesenchymal cells such as chondrocytes. However, nothing is known about the role of PC in airway smooth muscle cells (ASMCs) in the context of airway remodeling. We hypothesized that PC on ASMCs mediate cell contraction and are thus integral in the remodeling process. We found that PC are expressed on ASMCs in asthmatic lungs. Using pharmacological and genetic methods, we demonstrated that PC are necessary for ASMC contraction in a collagen gel three-dimensional model both in the absence of external stimulus and in response to the extracellular component hyaluronan. Mechanistically, we demonstrate that the effect of PC on ASMC contraction is, to a small extent, due to their effect on Sonic Hedgehog signaling and, to a larger extent, due to their effect on calcium influx and membrane depolarization. In conclusion, PC are necessary for the development of airway remodeling by mediating calcium flux and Sonic Hedgehog signaling.

Role of Primary Cilia in Cell Migration

Cellular migration is a common activity for many different types of cells. If cellular migration is disrupted, cells may be unable to repair damaged tissue (wound repair), migrate appropriately as in cancer, or correctly signal their cellular state. In several situations, cilia have been implicated in helping to determine the direction of cellular migration. As primary cilia express a number of distinct receptors for particular hormones, these organelles have the required machinery to influence cellular behavior. We are using ARPE‐19 cells, a human retinal pigment epithelial cell line capable of forming cilia in culture, as an assay for cellular motility. Using a 3D model of cellular migration (matrigel invasion chambers) and a 2D scratch assay for cellular motility, we are testing various reagents for their ability to alter RPE cell motility. Preliminary results suggest that a number of agents which alter ciliary function, particularly protein kinase A modulators, are able to inhibit the migratory ability of ARPE cells. These results suggest that protein kinase A may be a key link between ciliary signaling and the direction of cellular migration.Support or Funding InformationUniversity of Michigan Graduate Student Research Assistantship; University Research and Creative Activities grant

Primary cilia: a link between hormone signalling and endocrine-related cancers?

Primary cilia are sensory organelles that play a role as signalling hubs. Disruption of primary cilia structure and function is increasingly recognised in a range of cancers, with a growing body of evidence suggesting that ciliary disruption contributes to tumourigenesis. This review considers the role of primary cilia in the pathogenesis of endocrine-related cancers.

The role of primary cilia in the molecular pathogenesis of phaeochromocytoma

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The role of primary cilia in obesity and diabetes.

One in 12 people worldwide suffers from diabetes and more than 90% of affected adult individuals are diagnosed with type 2 diabetes mellitus (T2DM). Obesity adds to the personal risk to develop T2DM, and both metabolic diseases are rampantly increasing worldwide. Over recent years, primary cilia have moved into the focus of basic and clinical research, after several human diseases have been identified as ciliopathies (i.e., they are linked to ciliary dysfunction). A subset of ciliopathies presents with obesity and diabetes, either as core symptoms or major complications. Several studies have shown a role for ciliary signaling in the satiety signaling centers of the hypothalamus and in other metabolically active tissues, such as pancreatic islets. Here, we discuss recent advances and perspectives in ciliary metabolic research.

Polycystins and intercellular mechanotransduction: A precise dosage of polycystin 2 is necessary for alpha-actinin reinforcement of junctions upon mechanical stimulation

Polycystins 1 and 2, which are mutated in Autosomal Polycystic Kidney Disease, are involved in mechanotransduction through various mechanisms. In renal cells, polycystins not only have an important mechanotransductive role in primary cilia but are also present in intercellular contacts but their role there remains unclear. Here, we address the hypothesis that polycystins are involved in mechanotransduction via intercellular junctions, which would be expected to have consequences on tissue organization. We focused on the role of polycystin 2, which could be involved in mechanical organization at junctions either by its channel activity or by the direct recruitment of cytoskeleton components such as the F-actin cross-linker α-actinin. After mechanical stimulation of intercellular junctions in MDCK renal epithelial cells, α-actinin is rapidly recruited but this is inhibited upon overexpression of PC2 or the D509V mutant that lacks channel activity, and is also decreased upon PC2 silencing. This suggests that a precise dosage of PC2 is necessary for an adequate mechanosensitive α-actinin recruitment at junctions. At the multicellular level, a change in PC2 expression was associated with changes in velocity in confluent epithelia and during wound healing together with a loss of orientation. This study suggests that the mechanosensitive regulation of cytoskeleton by polycystins in intercellular contacts may be important in the context of ADPKD.

A finite elements study on the role of primary cilia in sensing mechanical stimuli to cells by calculating their response to the fluid flow

The primary cilium which is an organelle in nearly every cell in the vertebrate body extends out of the cell surface like an antenna and is known as cell sensor of mechanical and chemical stimuli. In previous numerical simulations, researchers modeled this organelle as a cantilevered beam attached to the cell surface. In the present study, however, we present a novel model that accommodates for both pivoting and bending of primary cilium in response to the fluid flow. In this model, primary cilium is attached to the cell using a thin elastic layer. This layer, which comprises the bottom boundary of the cilium, provides the possibility of pivoting of the cilium around its base so that we are able to analyze the other part of ciliary response to the fluid flow. In this study, we have used finite element method and fluid-structure interaction techniques to simulate the problem. Domains of solid cilium and the surrounding fluid were meshed using triangular elements. The governing equations in this problem were fully coupled and were solved by the direct method. Graphs of maximum stress in the cilium base versus elastic constant of the thin layer depict two different trends. By scrutinizing colored plots of stress distribution in the cilium base it is construed that these trends represent two different mechanisms in ciliary deformation due to the flow of the fluid. In case of low elasticity constants, for which attachment of the cilium to the cell is soft, pivoting mechanism is essential, while in case of harder attachments, bending mechanism dominates.

P-005 Prognostic significance of the frequency of primary cilia in cells of small bowel and colorectal adenocarcinoma

Purpose: The primary cilium is a solitary, sensory, immotile microtubule-based structure that arises from the centrosome and is projected from the surface of most human cell types. It has been hypothesized that primary cilia could serve as a tumor suppressor organelle. The objective of this pilot study was to investigate the presence and frequency of primary cilia in cells of small bowel and colorectal adenocarcinoma and to evaluate the prognostic significance of their frequency. Methods: The presence of primary cilia in cells in samples of small bowel (8 patients) and colorectal adenocarcinoma (32 patiens) was evaluated. The primary cilia of cells were immunofluorescently labeled using primary monoclonal anti-acetylated alpha-tubulin antibody and cell nuclei were labeled using DAPI. Results: Primary cilia were identified in all examined specimens. The median frequency of primary cilia was 0.49% in cells of small bowel cancer and 0.22% in cells in colorectal cancer. Overall survival according to frequency of primary cilia in all intestinal adenocarcinomas was significantly longer in patients with higher frequency (>= 0.187) than in patients with lower frequency of primary cilia ( = 0.187) than in patients with lower frequency of primary cilia (< 0.187) (p=0.028). Conclusions: The present pilot study provides the first evidence of the prognostic significance of the frequency of primary cilia in small bowel and colorectal adenocarcinoma. Because of significantly higher median frequency of primary cilia in the rare small bowel adenocarcinoma than in the frequent colorectal adenocarcinoma (p<0.001), the results of this study support a potential role for primary cilia as a biomarker in these types of cancer.