Arl6 controls ciliogenesis and Hh signaling from the basal body to the primary cilium
Arl6 is a small GTPase critical for ciliary transport of BBSome [15, 20]. To determine the ciliary localization of Arl6, we ectopically expressed mouse Arl6 tagged with RFP (Arl6-RFP) in MEF cells, then serum-starved those MEF cells with 0.5% FBS for 24 h to induce ciliogenesis. The Arl6-RFP predominantly distributed in cytoplasm but not nucleus. With the anti-acetylated tubulin staining of primary cilia, we also found accumulation of Arl6-RFP at the base of cilia (Fig. 1a). However we didn’t find localization of Arl6 in primary cilia as it reported before [15]. The failed modification of ectopically expressed Arl6 is one possible reason for its failed transport into cilia. To access whether the GTP binding is necessary for localization of Arl6 at ciliary base, we ectopically expressed Arl6 mutants T31R and Q73L in MEFs as well. T31R is a dominant-negative mutant appears exclusively bound to GDP, while Q73L is a constitutively active mutant appears strictly bound to GTP. Under confocal microscope, we found that Q73L mutant accumulated at the base of cilia as WT did, while T31R didn’t, although they expressed at similar levels (Fig. 1a, b). It suggested that GTP-bound Arl6 functions at the base of primary cilia.
It was reported that Arl6 functions at or near the ciliary gate to modulate mammalian ciliary assembly or disassembly and ciliary signaling, for example Wnt Signaling [17]. To understand the function of Arl6 at the base, we immortalized Arl6−/− MEFs derived from E13.5 Arl6 knockout mice [18]. Our immortalized Arl6−/− MEFs showed reduced numbers and length of primary cilia (Fig. 1c, d), although ciliary staining was normal in tissue sections from kidney, eye, and pancreas of Arl6−/− mice [18]. Vertebrate Hh signaling absolutely requires IFT [4]. To assess the role of Arl6 in Hh signaling, we quantified the transcriptional responses of endogenous Gli1 by RT-PCR and Q-PCR in Arl6−/− MEFs, and found that lack of Arl6 led to marked curtailment of Gli1 activation (Fig. 1e, f; Additional file 1: Figure S1). Knockdown of Arl6 leads to a 42.5% decrease of Hh pathway activation. This result is consistent with the disruption of ciliogenesis in Arl6−/− MEFs. Taken together, the above results show that Arl6 is required for ciliogenesis and vertebrate Hh signaling.
Arl6 is expressed dynamically during muscle differentiation
To address the function of Arl6, we first examined the expression pattern in adult mouse tissues by Q-PCR. High expression of Arl6 was found in brain, kidney and lung, which have plenty of ciliated cells (Fig. 2a). In contrast, little expression was found in heart, skeletal muscle and colon, which are muscle tissues without cilia (Fig. 2a). Arl6 expression pattern in mouse is quite similar as that in human according to records from The Human Protein Atlas database (http://www.proteinatlas.org/ENSG00000113966-ARL6/tissue). It suggested that Arl6 functions in ciliogenesis or ciliary transport in mammals.
Although primary cilia are missing in mature muscle, they grow in myoblasts. Recent study reported that assembly and disassembly of primary cilia are controlled during the differentiation of myoblasts [9]. To understand whether Arl6 contributes to ciliogenesis during muscle differentiation, we induced the differentiation of C2C12 myoblasts with 2% HS for 3 days, and analyzed ciliary formation and Arl6 expression at different time points. The increased expression of differentiation marker Myogenin over time indicates C2C12 cells differentiated well as we expected (Additional file 2: Figure S2B). We found that primary cilia were assembled during the early stage of myogenic differentiation (0–24 h after HS treatment), subsequently disappeared during the later period (24–72 h after HS treatment) through immunofluorescent staining of acetylated tubulin (Additional file 2: Figure S2A). The numbers and length of cilia reached the peak at 24 h time point (Fig. 2b, c). This result is consistent with that of primary mouse myoblasts [9]. Interestingly, the transcription of Arl6 was also dynamically regulated during the differentiation of C2C12 cells. The mRNA level of Arl6 was also at the peak at 24 h time point according to Q-PCR analysis (Fig. 2d). So the regulation of Arl6 expression synchronized with that of ciliogenesis. These results suggested that Arl6 participates ciliogenesis during muscle differentiation.
Up-regulation of Arl6 is related to RMS
It is reported that ciliogenesis is deregulated in RMS, which is derived from myogenic precursors [9]. We found that primary cilia were observed only in RH30 cells but not RD cells after 48 h treatment with 2% HS (Fig. 3a), which is consistent with the reports from other groups [7, 9]. Interestingly, mRNA level of Arl6 was increased dramatically in RH30 cells compared with SKM and RD cells (Fig. 3b left). Meanwhile our Q-PCR also showed dramatic increase of Gli1 in RH30 cells as well (Fig. 3b right). It is well known that aberrant Hh activity initiates or promotes RMS tumorigenesis [21, 22]. Previous studies have shown that RMS formation in the patients with Ptch1 mutation and Ptch1+/− mice as well [23]. So we are curious whether Arl6 is involved in Hh-associated RMS in vivo. To answer this question, we separated RMS tumor tissues and normal gastrocnemius tissues from same Ptch1+/− mouse for IHC staining. The IHC results showed a robust Arl6 and Gli1 expression in RMS tissues (Fig. 3d, f). In contrast, in the gastrocnemius control, Arl6 and Gli1 expression was barely detectable (Fig. 3c, e). The typical primary cilia were seen in RMS sections but not in normal skeletal muscle control (Fig. 3g, h). These in vitro and in vivo evidences suggested that up-regulation of Arl6 caused abnormal assembly of primary cilia and aberrant activation of Hh signaling in RMS.
Arl6 promotes RH30 cell growth and inhibits ciliogenesis in RH30 cells
Having found the correlation between Arl6 expression, ciliogenesis and Hh signaling, we sought to address the physiological relevance of Arl6 regulation of ciliogenesis in RMS tumorigenesis. For this purpose, we generated RD and RH30 stable cell lines expressing shArl6 or shCon, and measured their proliferation. Relative to those expressing shCon, RH30 cells expressing shArl6 grew much slower and had reduced rate of metabolism as evident by quantification using MTT (Fig. 4a). Companying with the decrease of Arl6 expression, the mRNA level of Gli1 was significantly decreased in RH30 cells expressing shArl6 (Fig. 4c, d). However, knockdown of Arl6 didn’t reduce the rate of metabolism and Gli1 expression in RD cells (Fig. 4b, e, f). The mRNA levels of Hh target genes Ptch1, Hhip, and Gli2 were significantly decreased in RH30 but not RD cells knockdown of Arl6 (Fig. 4g–l). The decreased expression of Dhh and increased Ihh was found in RH30 and RD cells knockdown of Arl6 (Fig. 4m–p). Shh was undetectable. This result suggested that knockdown of Arl6 suppress Hh pathway activity through retarding the signaling transduction not ligands expression in RH30 cells. We then examined the role of Arl6 in sustaining the proliferation of RD and RH30 cells. Consistent with the decrease of rate of metabolism, the proliferation of RH30 cells was blocked by knockdown of Arl6 (Fig. 4q, r) according to the EdU incorporation results. Taken together, these results indicate that knockdown of Arl6 inhibits the growth and Hh activity of RH30 cells.
To understand the mechanisms underlying the growth suppression effect of Arl6 knockdown, the cell cycle distribution of RH30 cells was analyzed. Knockdown of Arl6 induced G2/M phase arrest in RH30 cells. The fraction of RH30 cells in the G2/M phase increased from around 11.6% in the shCon to around 15.4% in the shArl6 treatment groups (Fig. 5a, b). The G2/M arrest was largely at the expense of the G0/G1 phase.
To further characterize the tumor suppression properties of Arl6 knockdown, we examined the ability of RH30 cells to undergo apoptosis when stably expressing shArl6. Apoptosis was induced by maintaining the cells in DMEM supplemented with 0.2% FBS for 24 h and quantified by using Annexin V and propidium iodide double staining. Flow cytometry analyses indicated that only around 1.5% of RH30 cells expressing shCon became Annexin V positive, but this percentage increased to 11.6% in the cells expressing shArl6 after serum depletion (Fig. 5c, d). This result indicated that knockdown of Arl6 promotes the apoptosis of RH30 cells, and suggested that cell cycle arrest at G2/M phase is involved in apoptosis induction.
Since knockdown of Arl6 only obstructed the growth of RH30 cells but not RD cells, we expected the effect of shArl6 is related to the interruption of ciliogenesis in RH30 cells. So we stained the primary cilia in those RH30 stable cells, and found that RH30 cells stably expressing shArl6 could not form cilia even after 48 h treatment with 2% HS (Fig. 6a). As shown in Fig. 6b, around 26.1% RH30 cells stably expressing shCon showed typical structures of primary cilia, while only 7.5% RH30 cells stably expressing shArl6 had primary cilia after the induction of ciliogenesis with HS. Those data suggested that knockdown of Arl6 suppressed RH30 cell proliferation through interrupted ciliogenesis.
To identify the role of cilia assembly in RMS cell proliferation, we tried to knockdown IFT88 in RH30 cells (Additional file 3: Figure S3A). IFT88 is a well-known central component of the IFT complex B, essential for cilia assembly. Knockdown of IFT88 blocked cilia assembly, retarded Hh signaling, and suppressed cell proliferation (Additional file 3: Figure S3). The siIFT88 data identified that blocking cilia assembly suppresses Hh signaling and cell proliferation in RH30 cells.
To further investigate the functions of Arl6 in ciliogenesis and cell proliferation of RH30 cells, we used the CRISPR/Cas9 genome editing technology to knockout Arl6. Western blotting showed that the expression of Arl6 was significantly decreased in RH30 cells edited by Arl6 CRISPR, indicated Arl6 was knockout in most cells of the selected individual clone (Additional file 4: Figure S4A). The ciliogenesis and cell proliferation was also suppressed in edited RH30 cells when compared with that in untransfected cells (Additional file 4: Figure S4B–D). The data again indicated that Arl6 promotes cell growth and ciliogenesis in RH30 cells.
Hh signal induces the transcription of Arl6
Until now, we understand that Arl6 is involved in ciliogenesis and Hh signaling in myoblast differentiation and RMS tumorigenesis. However, the up-regulation of Arl6 in RMS tissues from Ptch1+/− mouse is still not explained. To answer this question, we incubated wild type MEFs with Shh conditioned medium. As shown in Fig. 7, the Shh ligand induced Arl6 transcription in MEFs, indicating that Arl6 may be the direct or indirect target gene of Hh pathway. It suggested a positive feedback loop between Hh signal and Arl6.