In this report we present findings concerning the cellular function of the SYT proto-oncoprotein. The studies follow the initial observation that SYT forms a nuclear, G1-specific complex with p300, a histone acetyl transferase and a transcription co-activator, and this SYT/p300 complex is actively involved in the regulation of cell adhesion. While p300 and other chromatin remodeling complexes interact with the N-terminal regions of SYT including the SNH domain, cell adhesion control appeared to be mediated by its C-terminal domains [13
]. With this in mind, we decided to extend the analysis of SYT function to 3D-systems that are physiologically closer to tissue morphogenesis. In the present study we used the MDCK-collagen system as a 3D model to study the role of SYT in maintaining tissue integrity. In a collagen gel matrix, single MDCK cells proliferate and differentiate to form multicellular, highly polarized, fluid-filled cysts. The architecture of these cysts recapitulates that of epithelial cell-lined organs and their intricate signaling [29
]. Epithelial morphogenesis is a tightly regulated phenomenon that involves cell-cell adhesion, cell-matrix interactions and cell polarization.
In the present study, we found that the proto-oncoprotein SYT regulates MDCK cyst formation through its distinct domains (SNH and QPGY). SYT lacking the QPGY domain (SYT 1–164NLS) allowed a few rudimentary cysts to form at best while SYTdl 1–40 which has lost half of the SNH domain formed very large cysts in significantly high numbers. These results indicate that the SYT-QPGY domain is important for the formation of 3D structures and the N-terminal region of SYT exerts a regulatory effect on epithelial cyst morphogenesis.
Intriguingly, the defective phenotype of SYT 1–164NLS did not translate into any apparent abnormal growth or adhesion when the cells were grown in 2D monolayers. This indicates that this particular function of SYT requires and involves the signaling network integrated in the growth of 3D structures. When tested in monolayers, we detected no change in the levels of key polarity markers such as ZO-1, cadherin, β-catenin, or GM-130 in the cells expressing SYT 1–164NLS or SYT dl 1–40. Moreover, confocal microscopy of cysts derived from the various mutants, including the rudimentary structures without lumen of SYT 1–164NLS, demonstrated a normal distribution of the same markers on polarized cells. Confocal imaging did reveal however what appeared to be an enlarged volume of accumulated fluid in the lumen and a flattened shape of the surrounding polarized monolayer in the SYTdl 1–40-derived cysts. This phenotype prompted the thinking that the N-terminal region of SYT may regulate the signal (s) for fluid accumulation within the cyst.
Our next quest was to begin to understand the biochemical mechanism by which SYT stimulates MDCK cystogenesis. P2Y receptors are GPCRs mainly activated by ATP, ADP and UTP agonists [30
]. They belong to a family of eight members and induce divergent signaling pathways (phospholipase C, Rac, adenylate cyclase, RhoA), depending on the heterotrimeric G proteins they activate [31
]. In addition to their immediate effectors, the P2Y receptors interact with and regulate a variety of signaling molecules such as ion channels, integrins and growth factor receptors, thereby integrating an intricate signaling network and several biological processes in the polarized cell. MDCK cells express several P2Y receptor subtypes and use multiple signaling pathways for both direct and feedback regulation. Such pathways confer on released nucleotides, in particular ATP, a key role in establishing signaling systems in MDCK cells as well as in other cell types [32
]. Once activated by the purine nucleotide, P2Y receptor signaling may lead to phosphoinositide hydrolysis, MAP kinase activation, cAMP production and ERK stimulation via interacting receptor tyrosine kinases. Altogether, these potent signals will exert a mitogenic effect on the epithelial cells and allow them to proliferate and organize into a polarized layer with their apical surface facing a fluid-filled lumen [15
]. Several of these P2Y pathways can increase ion transport as well and lead to fluid accumulation, by osmosis, in the cyst lumen. Moreover, MDCK cells possess a Cl−
ion secretory mechanism driven by ATP [33
The definite increase in number and lumen size WT SYT confers on MDCK cysts indicates that the proto-oncoprotein induces positive modulators of the cystogenesis signaling cascade. Deletion of the N-terminal region of SYT eliminated a regulatory domain and led to, if not enhanced proliferation of polarized cells, an increase in the volume of the lumen that resulted in a flattened surrounding monolayer. Increase in luminal fluid by secreted anions is considered the end process of an active signaling network initiated by ATP stimulation of the P2Y purinergic receptors [31
]. Inhibition of several members of this network abrogated SYT-promoted cystogenesis (). Moreover, inhibition of WT-SYT- and SYTdl 1–40- cysts by PPADS, a potent functional inhibitor of P2 receptors and Reactive Blue 2 (RB2), a potent P2Y receptor blocker [23
], suggested that SYT-enhanced cystogenesis likely relies on P2Y signaling. Finally, The drastic decrease in ATP release from the surface of SYT1-164NLS cells (with absent QPGY domain) that are incapable of forming cysts provided a functional link between activation of the ATP-driven cystogenesis cascade and an intact SYT-QPGY domain. The enhanced ATP release by polarized SYT 1-164 cells may therefore explain the increased fluid accumulation and expansion of their cyst lumen.
The role of SYT on ATP release by polarized MDCK cells was further supported by the RNA interference experiments where depletion of SYT resulted in a significant reduction in apical and basolateral ATP release. So far we have been unsuccessful in depleting SYT from MDCK cell lines in a stable fashion. In the transient siRNA assays, SYT levels reverted to normal on the fourth day and we were therefore unable to study SYT-depleted MDCK cells in the 3-D cultures that needed 7–10 days for cyst formation. Combined with the embryonic lethality of SYT-null mice, our data on the effect of SYT on cellular ATP indicate that SYT is a vital protein and the cells are unable to survive without it. This may justify our failure in generating cells with permanent SYT-loss of function.
Altogether the above data clearly indicate a significant role of SYT in MDCK cyst formation. They also suggest that SYT regulates MDCK cyst by controlling the release of ATP from the polarized cells and subsequent activation of the P2Y receptors.
The mechanism of ATP regulation by SYT remains to be discovered. However, given the prevalent function of SYT, it could be surmised that this control occurs at the level of gene expression (), and that it is mediated by the QPGY domain. Conversely, inhibition of ATP release and cyst formation by SYT 1–164 NLS indicates that this mutant exerts a dominant-negative effect on WT SYT. This likely occurs as a consequence of SYT 1–164 sequestering away necessary factors required for QPGY-mediated transcription.
Nucleotides like ATP are ubiquitous extracellular signaling molecules that induce a wide spectrum of biological effects. In recent years considerable efforts have been made in the area of nucleotide signaling to understand their release mechanism in the extracellular fluids, their degradation by ectoenzymes, and the receptors mediating their cellular effects [34
]. Altered ATP release and signaling has also been shown to be detrimental to the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). Elevated ATP release in the lumen of the kidney cysts elicits an autocrine and/or paracrine activation of P2 receptors that ultimately leads to Cl−
and fluid accumulation and volume expansion in ADPKD cysts [24
The exciting and novel observation of SYT involvement in the regulation of a fundamental metabolic molecule such as ATP has significant ramifications that will help understand essential metabolic processes. It also provides one likely mechanism for failure of proper organ formation during embryogenesis in SYT knock-out mice. SYT is essentially a gene regulator involved in chromatin remodeling and transcription. We can surmise that its apparent effect on ATP release is the consequence of a positive feedback modulator whose expression is induced by nuclear SYT and either directly or indirectly stimulates ATP release machineries (). Extensive molecular characterization is needed to dissect this event and identify the intermediate effectors of this pathway. Such characterization promises to uncover key events in cellular metabolism.