The major function of a cilium is to transduce signals from the cellular milieu into intracellular responses (Praetorius and Leipziger 2013, Pluznick and Caplan 2015, Han, Xiong et al. 2017, Malicki and Johnson 2017, Pala, Alomari et al. 2017). Particularly, the primary cilium contains receptors and potentially controls calcium signalling, Hedgehog, Wnt, PDGFR, Notch, TGF-β, mTOR, OFD1 autophagy, and some other GPCR-associated pathways (Pala, Alomari et al. 2017).
30 Figure 15. A. Cilia-dependent calcium and Wnt signalling. Fluid flow bends the cilium and triggers intracellular Ca2+ to entry through the ciliary channels. Intracellular signalling cascades are activated by the Ca2+ influx. This stimulates gene expression. In another recently proposed model, ciliary bending does not open Ca2+ channels. Ca2+
influx is due to its diffusion from the cell body or caused by a damage in the cilium. B. In the absence of fluid flow Wnt ligand binds to the co-receptors frizzled and DSH is recruited to frizzled but GSK3 is inactivated. β-catenin translocates to the nucleus, where it acts as a transcriptional co-activator in tandem with LEF and TCF family proteins to induce transcription of Wnt target genes. In noncanonical Wnt signalling fluid flow causes intracellular Ca2+ increase and an upregulates inversin expression. Inversin
31 targets cytoplasmic DSH for ubiquitylation and degradation, making it unavailable for canonical Wnt signaling. Figures adapted from Pala, et al. 2017.
1.14.1 Calcium signalling
Primary cilia were thought to regulate calcium signalling via polycystins channels, polycystin1 (PC1) and polycystin 2 (PC2) encoded by PKD1 and PKD2 genes, respectively (Fig.14A). In kidney epithelial cells, these proteins are co-localised and are thought to function as a mechanosensory unit (Nauli, Alenghat et al. 2003, AbouAlaiwi, Takahashi et al. 2009, Raghavan and Weisz 2016). PC2 is activated upon cilium bending by a fluid flow and lead to calcium entry into the cell through the cilium. This calcium signalling would alter gene expression and regulate cellular functions (Nauli, Alenghat et al. 2003, Praetorius and Spring 2003, AbouAlaiwi, Takahashi et al. 2009, Besschetnova, Kolpakova-Hart et al. 2010, Nauli, Jin et al. 2013, Jin, Mohieldin et al. 2014, Praetorius 2015). In opposition to these early experimental results, Delling et al. engineered mice expressing a sensor protein that fluoresces in response to increased Ca2+ influx in primary cilia and measured Ca2+ signals following application of a mechanical force.
Using this model, they found no evidence of mechanical force-driven Ca2+ influx and therefore conclude that the primary cilia are not involved in calcium-based mechanosensation (Delling, Indzhykulian et al. 2016, Norris and Jackson 2016). An alternative theory is that the urinary flow brings metabolic signalling molecules that are recognized by other ciliary receptors to control water and salt reabsorption.
Transient receptor potential (TRP) channels are candidates for this role that was recently liked with calcium signalling (Minke 2006, Hasan and Zhang 2018). The key regulatory mechanism of ciliary signalling in collecting ducts is calcium-dependent but how the signal affects physiology remains to be determined (Norris and Jackson 2016).
1.14.2 Hedgehog Signalling
32 Hedgehog (Hh) signalling pathway regulates homeostasis, tissue patterning and embryonic development in many organisms (Davenport and Yoder 2005, Malicki and Johnson 2017, Pala, Alomari et al. 2017). The primary cilium is thought to be a Hh transduction hub (Goetz, Ocbina et al. 2009). This theory stands on results obtained in experiments with conditional genetic deletion of Ift88 or Kif3a which demonstrated that the primary cilium is essential for Hh signalling responses (Bangs and Anderson 2017).
The Hh signalling comprises serial inhibitory reactions. Under basal condition (Fig15B), in the absence of Hh ligand, the Hh receptor Patched (Ptch) inhibits the activity of smoothened (Smo). Afterwards the suppressor of fused (SUFU) binds to the transcription factor GLI and prevents its activation. After Hh ligand binding to Ptch, Smo migrates to the cilia which results in conversion of full-length GLI/Ci into transcriptional activator form. GLI-activator translocates from the cilium to the nucleus and activates the GLI target gene expression (Goetz, Ocbina et al. 2009, Nachury 2014, Bangs and Anderson 2017). SUFU/KIF7 isolate Gli2 and Gli3 repressors in the ciliary tip where they will be phosphorylated by PKA and Ck1 (Li, Nieuwenhuis et al. 2012). This PTM is recognised by the elements of the ubiquitin proteasome degradation system where Gli2 and, partially, Gli3 are being degraded (Pan, Wang et al. 2009, Snouffer, Brown et al.
2017).
1.14.3 Wnt signalling
Wnt signalling is involved in regulation of cell migration, healing process, neural patterning, planar cell polarity, skeletal development and organogenesis (Gao 2012, Barker, Thomas et al. 2014, Pala, Alomari et al. 2017, Meyer and Leuschner 2018).
Dysregulation of the canonical Wnt signalling (Fig.15A) shown in cancer development is suspected in polycystic kidney disease (Benzing, Simons et al. 2007, Lancaster and Gleeson 2010). Wnt/β-catenin signalling is hyperactive in 70% of the aldosterone producing adenomas (APA) where it controls aldosterone production and cell proliferation acting in cooperation with miRNA-203 (Berthon, Drelon et al. 2014, Peng, Chang et al. 2018).
33 Wnt signalling comprises canonical (β-catenin dependent) and non-canonical pathways;
either of them can be activated by Wnt binding to the membrane receptor frizzled.
When fluid flow is absent, canonical Wnt signalling predominates. Soluble Wnt molecules bind to Frizzled receptors which results in recruitment of the dishevelled (DSH) co-receptors and glycogen synthase kinase-3 (GSK3) becomes inactive. Beta-catenin (β-cat) is no longer degraded and translocates to the nucleus to initiate TCF-dependent transcription of its target genes (Lancaster, Louie et al. 2009). Non-canonical Wnt signalling functions under fluid flow conditions and the generation of calcium signalling upon cilium bending is required. This stimulates Inversin expression and localization in the ciliary base and other cellular locations. Inversin has been proposed to be a switch between canonical and non-canonicat Wnt signalling activity. It targets the cytoplasmic fraction of DSH for ubiquitination and degradation which results in suppression of the β-cat activity (Lienkamp, Ganner et al. 2012).
1.14.4 Notch signalling
Notch signalling is required for various aspects of biogenesis including patterning and differentiation decision of progenitor cells during neurogenesis as well as adult tissue growth and development (Liu, Kiseleva et al. 2018). There are four Notch receptors (Notch1-4) distinguished by a transmembrane domain associated with a calcium ion. It is thought that Notch signalling is related to the primary cilium (Pala, Alomari et al.
2017). Regulation of the left-right asymmetry via cilium length modulation is controlled by notch signalling (Lopes, Lourenço et al. 2010). Notch also facilitates transition of the Shh mediators to the primary cilium subsequently enhancing Hh signalling response (Pala, Alomari et al. 2017)
1.14.5 mTOR signalling
Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, nucleates a major eukaryotic signalling cascade which coordinates cell growth and metabolism, cell cycle as well as organismal survival (Malicki and Johnson 2016, Pala, Alomari et al. 2017).
34 mTOR signalling in kidney is associated with cyst formation, hyper-proliferation of renal cells and hypercalcemia (Huber, Walz et al. 2011, Armour, Carson et al. 2012). Fluid flow bend the cilium which results in mTOR inhibition. This effect is coordinated by LKB1-AMPK-mTOR regulatory network, which is also required for regulation of the cell size by autophagy (Boehlke, Kotsis et al. 2010). Tumor suppressor kinase LKB1 and AMP dependent protein kinase are localized to the primary cilium and its basal body. Upon cilium bending, LKB1 becomes active and translocates to the basal body region and activates AMPK. AMPK in turn phosphorylates tuberin (TSC2) which recruits hamartin (TSC1). TSC1/TSC2 complex stimulates the Pheb GTPase activity which becomes sequestered away from mTOR and therefore cannot activate it (Huber, Walz et al. 2011, Armour, Carson et al. 2012, Pala, Alomari et al. 2017).
1.14.6 Other signalling mechanisms
Platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase receptor, localized to the primary cilium, which regulates cell growth, proliferation and migration. It also plays an important role in embryonic development and tissue growth. When activated, it can induce a cellular response via MEK/ERK signalling cascades (Pala, Alomari et al.
2017). Another receptor protein, localized to the ciliary membrane is transforming growth factor β ( β) receptor. It regulates bone development and metabolism. TGF-β stimulation is transduced into cellular response via SMAD2-3/ERK1-2 signalling (Pala, Alomari et al. 2017).