Primary cilia and lipid raft dynamics

Primary cilia, antenna-like structures of the plasma membrane, detect various extracellular cues and transduce signals into the cell to regulate a wide range of functions. Lipid rafts, plasma membrane microdomains enriched in cholesterol, sphingolipids and specific proteins, are also signalling hubs involved in a myriad of physiological functions. Although impairment of primary cilia and lipid rafts is associated with various diseases, the relationship between primary cilia and lipid rafts is poorly understood. Here, we review a newly discovered interaction between primary cilia and lipid raft dynamics that occurs during Akt signalling in adipogenesis. We also discuss the relationship between primary cilia and lipid raft-mediated Akt signalling in cancer biology. This review provides a novel perspective on primary cilia in the regulation of lipid raft dynamics.

Although both primary cilia and lipid rafts act as signalling hubs, their relationship has been poorly understood. In this review, we explain a novel role of primary cilia on certain types of lipid raft dynamics related to IR-Akt signalling upon adipogenic stimuli in mouse adipose progenitor cells (APs) and mouse mesenchymal progenitor C3H10T1/2 cells [27]. We also discuss potential therapeutic approaches for obesity and cancer targeting lipid raft dynamics and primary cilia.

Lipid rafts in the regulation of RTKs signalling
RTKs, such as IR, IGF1R, epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR) and plateletderived growth factor receptor (PDGFR), are transmembrane proteins that are activated by ligand binding, which usually causes dimerization and/or oligomerization of the receptors followed by trans-autophosphorylation of multiple tyrosine residues [28]. The phosphorylated RTKs activate various downstream signalling pathways, such as the PI3K-Akt cascade, the mitogen-activated protein kinase cascade and the Janus kinasesignal transducer and activator of transcription cascade [28]. Some of the RTKs signallings events can be modulated by lipid rafts [29][30][31]. The mechanisms include positive or negative regulation of RTK trans-phosphorylation by clustering within the lipid rafts and concentration or exclusion of signalling molecules downstream of RTKs within lipid rafts [29,30,32]. Lipid rafts can be grouped based on the main components [30,31,[33][34][35]. Caveolins and flotillins are representative scaffold proteins in lipid rafts [31,33]. Caveolin 1 (CAV1) inserts into the inner leaflet of the membrane via a hairpin loop with the N-and C-termini exposed to the cytoplasm [36]. The N-terminal domain facilitates the formation of homooligomers [37] and interaction with other proteins that have a binding motif to CAV1 (e.g. IR) [36,38]. In the presence of Caveolae-associated protein 1, CAV1 can make caveola, structurally a unique lipid raft that is characterized by flaskshaped invagination of the plasma membrane [39]. CAV1 accelerated IR-Akt signalling in the differentiation of preadipocytes and the energy metabolism of adipocytes [27,40]. By contrast, CAV1 inhibited the autophosphorylation of EGFR and PDGFR in epidermoid carcinoma and fibroblasts, respectively [41,42]. Flotillins are two homologous proteins, flotillin 1 and flotillin 2 [43]. Flotillin 1 positively regulated the phosphorylation of EGFR and a substate of FGFR in HeLa cells [44,45]. The correlation between the composition and the function of lipid rafts, however, remains elusive [17].

Primary cilia regulate lipid raft dynamics related to IR-Akt signalling in adipogenesis
Adipogenesis plays an important and complex role in metabolic health [46]. Differentiation from preadipocytes to adipocytes is an adaptive response to nutritional overload [47]. Of note, adipogenesis can be detrimental depending on the context. For example, adipogenesis in the skeletal muscle induced by over-nutrition impairs wound healing [48]. Adipogenesis in bone marrow caused by ageing is associated with an increased risk of bone fracture [49]. Primary cilia are involved in this physiological and pathological adipogenesis. Differentiation from preadipocytes to adipocytes is stimulated by adipogenic signalling through activation of IR/IGF1R located at the ciliary base [27,50]. Upon adipogenic stimulation, IR/IGF1R observed at the region surrounding the ciliary base stimulate the PI3K-Akt pathway [27,51,52]. The activated Akt increases the expression of PPARγ, the master regulator of adipogenesis, by inhibiting the translocation of FOXO1, a key transcriptional suppressor in adipogenesis, to the nucleus of preadipocytes [53,54]. Impairment of IR signalling was associated with the metabolic dysfunction in Bardet-Biedl syndrome, a rare autosomal recessive ciliopathy [55][56][57]. Primary cilia also regulate adipogenesis through omega-3 fatty acidactivated FFAR4/GPR120 and desert hedgehog-activated smoothened [58,59]. However, the relationship between primary cilia and lipid rafts in the regulation of Akt signalling remains largely unknown [26,60].
We have shown that trichoplein (TCHP), a centriolar protein originally identified as a keratin-binding protein, is a key activator of aurora A kinase (AURKA) at centrioles and that knockdown (KD) of TCHP causes elongation of primary cilia in human retinal pigmental epithelium cells by suppressing the inhibitory effect of AURKA in ciliogenesis [7,[61][62][63][64][65][66]. We recently found that Tchp knockout (KO) mice show elongated cilia in APs, reduced body fat and smaller adipocytes than wild-type mice under a high-fat diet [27]. In APs from wildtype mice, CAV1-or ganglioside GM3-positive lipid rafts moved to the ciliary base upon adipogenic stimulation [27] (figure 1a), while GM1 and flotillin-2, other markers of lipid rafts, did not accumulate around the primary cilia [27]. In APs from Tchp KO mice, all four types of lipid rafts did not appear near the ciliary base upon adipogenic stimulation.
We also found that lipid raft dynamics were impaired in C3H10T1/2 cells with Tchp KD, in which the primary cilia were elongated, but not in C3H10T1/2 cells with double KD of Tchp and Ift88, in which the elongation of primary cilia caused by Tchp KD was suppressed by concomitant KD of Ift88, resulting in primary cilia being of similar length to that of control C3H10T1/2 cells [27] (figure 1b). The phosphorylation of Akt in mouse APs upon adipogenesis and the differentiation into adipocytes were also suppressed by Tchp KD but not by double Tchp and Ift88 KD [27]. The correlation between the impaired accumulation of CAV1-positive lipid rafts and the decreased phosphorylation of Akt at the ciliary base where IRs are localized is consistent with previous studies demonstrating that CAV1 positively modulates IR/IGF1R-Akt signalling in adipogenesis [67,68].
The finding that primary cilia regulate Akt signalling in adipogenesis by modulating lipid raft dynamics provides a novel perspective on the crosstalk between primary cilia and lipid rafts [27]. Co-localization of actin filaments with primary cilia was significantly lower in C3H10T1/2 cells with Tchp KD than in control C3H10T1/2 cells or C3H10T1/2 cells with double Tchp and Ift88 KD (figure 1c). CAV1 clusters are tethered to cortical actin filaments through interaction with filamin, an actin-binding protein [69]. These results indicate that the elongated cilia in C3H10T1/2 cells caused by Tchp KD may affect lipid raft dynamics through impairment of the actinbased transport system. The mechanisms by which primary cilia regulate lipid raft dynamics warrant further examination.

Therapeutic approaches targeting lipid raft dynamics through primary cilia
Lipid rafts have attracted attention as a novel target in cancer therapy [23,71,73,103,104]. For example, T0901317, a liver X receptor agonist, suppressed lipid raft-mediated Akt signalling affecting the localization and expression of flotillin-2 in a prostate cancer cell line, LNCaP, and a breast cancer cell line, MCF-7 [105,106]. Rh2, a major bioactive constituent of Panax ginseng, a traditional medicine, inhibited lipid raft-mediated Akt signalling, which affected the dynamics of CAV1 and GM1 in an epidermoid carcinoma cell line, A431 [107]. Edelfosin and perifosine, single-chain alkylphospholipids, suppressed the phosphorylation of Akt and the growth of patient-derived xenografts of mantle cell lymphoma [108]. royalsocietypublishing.org/journal/rsob Open Biol. 11: 210130 Strategies for improving the efficacy and safety of these therapeutic drugs have been actively developed [109][110][111]. Statins can also be classified as anti-cancer agents that inhibit lipid raft-mediated Akt signalling [71,73,103,104].
The possibility that primary cilia may take part in lipid raft dynamics [27] provides a novel approach to modulate lipid raft-mediated Akt signalling through targeting primary cilia. Genome-wide screens have identified various ciliary proteins that directly regulate ciliogenesis and the associated proteins that regulate the expression and function of these ciliary proteins [64,66,[112][113][114][115][116][117][118][119]. Multi-omics approaches, including lipidomics and glycomics, can also be used to identify novel components involved in the formation of primary cilia and lipid raft-mediated signalling. For example, O-linked β-N-acetylglycosaminylation (O-GlcNAcylation) is related to lipid raft-mediated Akt signalling [120,121] and ciliogenesis [122,123], and is associated with obesity, diabetes and cancer [124][125][126]. Modulation of O-GlcNAcylation may be a novel therapeutic approach to regulate lipid raft dynamics and primary cilia.
AURKA inhibitors, as well as Akt inhibitors, have been developed and evaluated in cancer and obesity [102,[127][128][129][130][131]. In mouse preadipocytes, KD of Aurka, a downstream target of Tchp [63], stimulated elongation of primary cilia and inhibited IR-Akt signalling and adipogenesis [27]. This suggests that forced elongation of primary cilia caused by AURKA inhibition may rectify the dysregulation of cell proliferation and/or differentiation caused by impairment of ciliogenesis in cancer and obesity.
Identification of novel factors involved in ciliogenesis and lipid raft dynamics in specific cell types may provide critical information to understand the pathophysiology of diseases associated with the dysregulation of these signalling pathways, which could lead to the development of novel therapeutic drugs targeting lipid raft dynamics and primary cilia. Competing interests. We declare we have no competing interests. Funding. This work was supported by the Japan Society for the Pro-  Figure 2. Loss of primary cilia and increase of phosphorylated Akt in PTEN-negative and well-differentiated prostate cancer. Immunohistological analysis of basal cell, primary cilia and nuclei (a), or basal cell, phosphorylated Akt and nuclei (b), in normal prostate and PTEN-negative well-differentiated prostate cancer. Basal cells were stained using an anti-CK34βE12 antibody to detect cytokeratin (CK) 1/5/10/14 (a) or an anti-CK5 antibody (b). The primary cilia and nuclei were stained using an anti-Arl13b antibody and DAPI, respectively. Phosphorylated Akt was stained using an anti-phosphorylated Akt antibody.