Cilia-Mediated Insulin/Akt and ST2/JNK Signaling Pathways Regulate the Recovery of Muscle Injury.

Following injury, skeletal muscle regenerates but fatty tissue accumulation is seen in aged muscle or muscular dystrophies. Fibro/adipogenic progenitors (FAPs) are key players in these events; however, the effect of primary cilia on FAPs remains unclear. Here, it is reported that genetic ablation of trichoplein (TCHP), a ciliary regulator, induces ciliary elongation on FAPs after injury, which promotes muscle regeneration while inhibiting adipogenesis. The defective adipogenic differentiation of FAPs is attributed to dysfunction of cilia-dependent lipid raft dynamics, which is critical for insulin/Akt signaling. It is also found that interleukin (IL) 13 is substantially produced by intramuscular FAPs, which are upregulated by ciliary elongation and contribute to regeneration. Mechanistically, upon injury, long cilia excessively activate the IL33/ST2/JNK axis to enhance IL13 production, facilitating myoblast proliferation and M2 macrophage polarization. The results indicate that FAPs organize the regenerative responses to skeletal muscle injury via cilia-mediated insulin/Akt and ST2/JNK signaling pathways.

Primary ciliary signaling: links with the cell cycle.

Primary cilia are solitary, microtubule-based structures emanating from the surface of most vertebrate cells. Although it is understood that ciliary assembly and disassembly both depend upon and impact cell cycle progression, critical mechanistic details of these links remain unresolved. Accumulating evidence shows that the signaling pathways downstream of receptor tyrosine kinases and lysophosphatidic acid receptors control the dynamics of primary cilia. It has also become clear that primary cilia not only serve as signaling hubs but also regulate the composition of the surrounding membrane, which is likely to affect the response to growth factors. Here, we overview recent advances in understanding the interplay between primary cilia and the cell cycle, with a focus on growth factor signaling pathways.

Primary cilia-dependent lipid raft/caveolin dynamics regulate adipogenesis.

Primary cilia play a pivotal role in signal transduction and development and are known to serve as signaling hubs. Recent studies have shown that primary cilium dysfunction influences adipogenesis, but the mechanisms are unclear. Here, we show that mesenchymal progenitors C3H10T1/2 depleted of trichoplein, a key regulator of cilium formation, have significantly longer cilia than control cells and fail to differentiate into adipocytes. Mechanistically, the elongated cilia prevent caveolin-1- and/or GM3-positive lipid rafts from being assembled around the ciliary base where insulin receptor proteins accumulate, thereby inhibiting the insulin-Akt signaling. We further generate trichoplein knockout mice, in which adipogenic progenitors display elongated cilia and impair the lipid raft dynamics. The knockout mice on an extended high-fat diet exhibit reduced body fat and smaller adipocytes than wild-type (WT) mice. Overall, our results suggest a role for primary cilia in regulating adipogenic signal transduction via control of the lipid raft dynamics around cilia.

Targeting E3 Ubiquitin Ligases and Deubiquitinases in Ciliopathy and Cancer.

Cilia are antenna-like structures present in many vertebrate cells. These organelles detect extracellular cues, transduce signals into the cell, and play an essential role in ensuring correct cell proliferation, migration, and differentiation in a spatiotemporal manner. Not surprisingly, dysregulation of cilia can cause various diseases, including cancer and ciliopathies, which are complex disorders caused by mutations in genes regulating ciliary function. The structure and function of cilia are dynamically regulated through various mechanisms, among which E3 ubiquitin ligases and deubiquitinases play crucial roles. These enzymes regulate the degradation and stabilization of ciliary proteins through the ubiquitin-proteasome system. In this review, we briefly highlight the role of cilia in ciliopathy and cancer; describe the roles of E3 ubiquitin ligases and deubiquitinases in ciliogenesis, ciliopathy, and cancer; and highlight some of the E3 ubiquitin ligases and deubiquitinases that are potential therapeutic targets for these disorders.

A novel CDK-independent function of p27Kip1 in preciliary vesicle trafficking during ciliogenesis.

p27Kip1, a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27Kip1 is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27Kip1 is associated with ciliogenesis. Here, we have generated p27Kip1-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27Kip1-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27Kip1 and, surprisingly, its 86-140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27Kip1 abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehd1-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27Kip1 in primary cilia formation.

Intermediate filaments and IF-associated proteins: from cell architecture to cell proliferation.

Intermediate filaments (IFs), in coordination with microfilaments and microtubules, form the structural framework of the cytoskeleton and nucleus, thereby providing mechanical support against cellular stresses and anchoring intracellular organelles in place. The assembly and disassembly of IFs are mainly regulated by the phosphorylation of IF proteins. These phosphorylation states can be tracked using antibodies raised against phosphopeptides in the target proteins. IFs exert their functions through interactions with not only structural proteins, but also non-structural proteins involved in cell signaling, such as stress responses, apoptosis, and cell proliferation. This review highlights findings related to how IFs regulate cell division through phosphorylation cascades and how trichoplein, a centriolar protein originally identified as a keratin-associated protein, regulates the cell cycle through primary cilium formation.

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.

Tetraploidy in cancer and its possible link to aging.

Tetraploidy, a condition in which a cell has four homologous sets of chromosomes, is often seen as a natural physiological condition but is also frequently seen in pathophysiological conditions such as cancer. Tetraploidy facilitates chromosomal instability (CIN), which is an elevated level of chromosomal loss and gain that can cause production of a wide variety of aneuploid cells that carry structural and numerical aberrations of chromosomes. The resultant genomic heterogeneity supposedly expedites karyotypic evolution that confers oncogenic potential in spite of the reduced cellular fitness caused by aneuploidy. Recent studies suggest that tetraploidy might also be associated with aging; mice with mutations in an intermediate filament protein have revealed that these tetraploidy-prone mice exhibit tissue disorders associated with aging. Cellular senescence and its accompanying senescence-associated secretory phenotype have now emerged as critical factors that link tetraploidy and tetraploidy-induced CIN with cancer, and possibly with aging. Here, we review recent findings about how tetraploidy is related to cancer and possibly to aging, and discuss underlying mechanisms of the relationship, as well as how we can exploit the properties of cells exhibiting tetraploidy-induced CIN to control these pathological conditions.

Regulation of keratin 5/14 intermediate filaments by CDK1, Aurora-B, and Rho-kinase.

We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.

EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase.

Ciliogenesis is generally inhibited in dividing cells, however, it has been unclear which signaling cascades regulate the phenomenon. Here, we report that epidermal growth factor receptor (EGFR) kinase suppresses ciliogenesis by directly phosphorylating the deubiquitinase USP8 on Tyr-717 and Tyr-810 in RPE1 cells. These phosphorylations elevate the deubiquitinase activity, which then stabilizes the trichoplein-Aurora A pathway, an inhibitory mechanism of ciliogenesis. EGFR knockdown and serum starvation result in ciliogenesis through downregulation of the USP8-trichoplein-Aurora A signal. Moreover, primary cilia abrogation, which is induced upon IFT20 or Cep164 depletion, ameliorates the cell cycle arrest of EGFR knockdown cells. The present data reveal that the EGFR-USP8-trichoplein-Aurora A axis is a critical signaling cascade that restricts ciliogenesis in dividing cells, and functions to facilitate cell proliferation. We further show that usp8 knockout zebrafish develops ciliopathy-related phenotypes including cystic kidney, suggesting that USP8 is a regulator of ciliogenesis in vertebrates.

Sphingosine 1-phosphate signaling through its receptor S1P5 promotes chromosome segregation and mitotic progression.

Sphingosine kinase 1 (SphK1) promotes cell proliferation and survival, and its abundance is often increased in tumors. SphK1 produces the signaling lipid sphingosine 1-phosphate (S1P), which activates signaling cascades downstream five G protein-coupled receptors (S1P1-5) to modulate vascular and immune system function and promote proliferation. We identified a new function of the SphK1-S1P pathway specifically in the control of mitosis. SphK1 depletion in HeLa cells caused prometaphase arrest, whereas its overexpression or activation accelerated mitosis. Increasing the abundance of S1P promoted mitotic progression, overrode the spindle assembly checkpoint (SAC), and led to chromosome segregation defects. S1P was secreted through the transporter SPNS2 and stimulated mitosis by binding to and activating S1P5 on the extracellular side, which then activated the intracellular phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Knockdown of S1P5 prevented the S1P-induced spindle defect phenotype. RNA interference assays revealed that the mitotic kinase Polo-like kinase 1 (PLK1) was an important effector of S1P-S1P5 signaling-induced mitosis in HeLa cells. Our findings identify an extracellular signal and the downstream pathway that promotes mitotic progression and may indicate potential therapeutic targets to inhibit the proliferation of cancer cells.

Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues.

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.

Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein-Aurora A pathway.

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly.

Phospho-Specific Antibody Probes of Intermediate Filament Proteins.

Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.

Vimentin Phosphorylation Underlies Myofibroblast Sensitivity to Withaferin A In Vitro and during Corneal Fibrosis.

Vimentin is a newly recognized target for corneal fibrosis. Using primary rabbit corneal fibroblasts and myofibroblasts we show that myofibroblasts, unlike fibroblasts, display impaired cell spreading and cell polarization, which is associated with increased levels of soluble serine-38 phosphorylated vimentin (pSer38Vim). This pSer38Vim isoform is inefficiently incorporated into growing vimentin intermediate filaments (IFs) of myofibroblasts during cell spreading, and as a result, myofibroblasts maintain higher soluble pSer38Vim levels compared to fibroblasts. Moreover, the soluble vimentin-targeting small molecule and fibrotic inhibitor withaferin A (WFA) causes a potent blockade of cell spreading selectively in myofibroblasts by targeting soluble pSer38Vim for hyperphosphorylation. WFA treatment does not induce vimentin hyperphosphorylation in fibroblasts. This hyperphosphorylated pSer38Vim species in WFA-treated myofibroblasts becomes complexed with adaptor protein filamin A (FlnA), and these complexes appear as short squiggles when displaced from focal adhesions. The extracellular-signal regulated kinase (ERK) is also phosphorylated (pERK) in response to WFA, but surprisingly, pERK does not enter the nucleus but remains bound to pSer38Vim in cytoplasmic complexes. Using a model of corneal alkali injury, we show that fibrotic corneas of wild type mice possess high levels of pERK, whereas injured corneas of vimentin-deficient (Vim KO) mice that heal with reduced fibrosis have highly reduced pERK expression. Finally, WFA treatment causes a decrease in pERK and pSer38Vim expression in healing corneas of wild type mice. Taken together, these findings identify a hereto-unappreciated role for pSer38Vim as an important determinant of myofibroblast sensitivity to WFA.

Novel insights into Chk1 regulation by phosphorylation.

Checkpoint kinase 1 (Chk1) is a conserved protein kinase central to the cell-cycle checkpoint during DNA damage response (DDR). Until recently, ATR, a protein kinase activated in response to DNA damage or stalled replication, has been considered as the sole regulator of Chk1. Recent progress, however, has led to the identification of additional protein kinases involved in Chk1 phosphorylation, affecting the subcellular localization and binding partners of Chk1. In fact, spatio-temporal regulation of Chk1 is of critical importance not only in the DDR but also in normal cell-cycle progression. In due course, many potent inhibitors targeted to Chk1 have been developed as anticancer agents and some of these inhibitors are currently in clinical trials. In this review, we summarize the current knowledge of Chk1 regulation by phosphorylation.

Current topics of functional links between primary cilia and cell cycle.

Primary cilia, microtubule-based sensory structures, orchestrate various critical signals during development and tissue homeostasis. In view of the rising interest into the reciprocal link between ciliogenesis and cell cycle, we discuss here several recent advances to understand the molecular link between the individual step of ciliogenesis and cell cycle control. At the onset of ciliogenesis (the transition from centrosome to basal body), distal appendage proteins have been established as components indispensable for the docking of vesicles at the mother centriole. In the initial step of axonemal extension, CP110, Ofd1, and trichoplein, key negative regulators of ciliogenesis, are found to be removed by a kinase-dependent mechanism, autophagy, and ubiquitin-proteasome system, respectively. Of note, their disposal functions as a restriction point to decide that the axonemal nucleation and extension begin. In the elongation step, Nde1, a negative regulator of ciliary length, is revealed to be ubiquitylated and degraded by CDK5-SCF(Fbw7) in a cell cycle-dependent manner. With regard to ciliary length control, it has been uncovered in flagellar shortening of Chlamydomonas that cilia itself transmit a ciliary length signal to cytoplasm. At the ciliary resorption step upon cell cycle re-entry, cilia are found to be disassembled not only by Aurora A-HDAC6 pathway but also by Nek2-Kif24 and Plk1-Kif2A pathways through their microtubule-depolymerizing activity. On the other hand, it is becoming evident that the presence of primary cilia itself functions as a structural checkpoint for cell cycle re-entry. These data suggest that ciliogenesis and cell cycle intimately link each other, and further elucidation of these mechanisms will contribute to understanding the pathology of cilia-related disease including cancer and discovering targets of therapeutic interventions.

Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension.

Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.

PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3γ and is required for metaphase-anaphase transition.

Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1-Thr210 phosphorylation. Plk1-Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1-Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1-Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1-Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1-Ser99 phosphorylation downstream of the PI3K-Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.

P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation.

The ataxia telangiectasia mutated- and rad3-related kinase (ATR)/Chk1 pathway is a sentinel of cell cycle progression. On the other hand, the Ras/mitogen-activated protein kinase/90-kDa ribosomal S6 kinase (p90 RSK) pathway is a central node in cell signaling downstream of growth factors. These pathways are closely correlated in cell proliferation, but their interaction is largely unknown. Here we show that Chk1 is phosphorylated predominantly at Ser-280 and translocated from cytoplasm to nucleus in response to serum stimulation. Nonphosphorylated Chk1-Ser-280 mutation attenuates nuclear Chk1 accumulation, whereas the phosphomimic mutation has a reverse effect on the localization. Treatment with p90 RSK inhibitor impairs Chk1 phosphorylation at Ser-280 and accumulation at the nucleus after serum stimulation, whereas these two phenomena are induced by the expression of the constitutively active mutant of p90 RSK in serum-starved cells. In vitro analyses indicate that p90 RSK stoichiometrically phosphorylates Ser-280 on Chk1. Together with Chk1 phosphorylation at Ser-345 by ATR and its autophosphorylation at Ser-296, which are critical for checkpoint signaling, Chk1-Ser-280 phosphorylation is elevated in a p90 RSK-dependent manner after UV irradiation. In addition, Chk1 phosphorylation at Ser-345 and Ser-296 after UV irradiation is also attenuated by the treatment with p90 RSK inhibitor or by Ser-280 mutation to Ala. These results suggest that p90 RSK facilitates nuclear Chk1 accumulation through Chk1-Ser-280 phosphorylation and that this pathway plays an important role in the preparation for monitoring genetic stability during cell proliferation.