Prostaglandin signaling in ciliogenesis during development

Prostaglandins regulate a wide variety of physiological and pathological processes, including inflammation, reproduction, cardiovascular homeostasis, and cancer progression. Cyclooxygenase (COX) catalyzes the rate-limiting step in the production of prostaglandins from arachidonic acid (Fig. 1). In 2 reaction steps, arachidonic acid is firstly converted to PGH₂, subsequently metabolized to form structurally related prostanoids in various tissues, including PGE₂, PGD₂, PGF2α, PGI₂ and Thromboxane A₂ (TxA₂).¹ Until now, most of the studies evaluating prostaglandin signaling have come from mammalian models. We propose that zebrafish provides a useful model system to elucidate the roles of prostaglandin signaling in embryogenesis and organ development.

Figure 1. Prostaglandin signaling regulates ciliogenesis by modulating intraflagellar transport. PGE₂ is synthesized by COX1 and COX2 and then exported by LKT/ABCC4 transporter. Released PGE₂ binds to EP4 receptor on the cilium, which activates adenylate cyclase (AC) to promote accumulation of intracellular cAMP. The cAMP-dependent PKA phosphorylates as-yet unknown substrates, leading to increase anterograde IFT and promote ciliogenesis.

Through analyzing a zebrafish mutant leakytail (lkt), we demonstrate that cilia formation and elongation require prostaglandin signaling during development.² Our findings indicate that Lkt/ABCC4-mediated PGE₂ signaling affects cAMP level and promotes ciliogenesis. A model is proposed where PGE₂ is exported from cells via Lkt/ABCC4 transporter on the plasma cell membrane and signals through the G-protein coupled receptor EP4 on the cilium or at the base of the cilium, thereby activating adenylate cyclase (AC) and cAMP signaling to promote the anterograde intraflagellar transport (IFT)² (Fig. 1). In support of this model, we found that PGE₂ treatment causes an increase of intracellular cAMP but not Ca²⁺ in inner medullary collecting duct 3 (IMCD3) cells during ciliogenesis.² PGE₂ treatment increases anterograde velocities of IFT particles but has no obvious effects on retrograde velocities² (Fig. 1). We further observed that deficiency in Lkt/Abcc4 or Ep4 activities failed to alter expression of the ciliary transcriptional factor Foxj1,² suggesting that PGE₂ signaling regulates ciliogenesis through facilitating the axonemal assembly rather than cilia biosynthesis. These findings linking PGE₂ signaling to the second messenger represent a key regulatory step in the control of the anterograde IFT during ciliogenesis.

Cilia formation and elongation require coordinate regulation of bidirectional traffic of IFT particles, including frequency and speed. One plausible mechanism in control of IFT speed during ciliogenesis can be inferred from studies of the nematode cilium, where 2 motors, kinesin-II and OSM3, act in a concerted fashion and produce an intermediate speed in lengthening cilia³ (Fig. 1). Deletion of the fast motor OSM3 causes IFT particles to move at the slow velocity and ablation of the slow motor Kinesin-II results in IFT particles to move at the fast velocity.³ A mechanism to enzymatically modulates motor or IFT proteins could be used to regulate particle velocity. It is unknown whether the motors can be served as substrates of PKA in response to PGE₂ (Fig. 1). During the anterograde axonal transport of vesicle in the leg giant axon, several axoplasmic proteins including kinesin can be phosphorylated by cAMP/PKA activation.⁴ Alternatively, kinesin or cytoplasmic dynein proteins form complexes with PKA subunits in response to PGE₂ during IFT. In melanophores, cAMP/PKA regulates intracellular organelle transport during aggregation and dispersion of pigment granules. Motor proteins dynein and kinesin II form 2 separate complexes with PKA regulatory subunits. Removal of PKA from granules causes disruption of dynein-dependent pigment aggregation, leading to kinesin II-dependent pigment dispension.⁵ This direct contact model appears to be the efficient way to switch between various kinds of intracellular transport.

The roles of PGE₂ signaling in ciliary development were not apparent from previous murine genetic studies. This is likely due to the maternal contribution of prostaglandins in the placenta allowing PGE₂ deficient mouse embryos to develop normally. We circumvented the maternal interference by using externally developing zebrafish embryos to reveal the roles of Lkt/ABCC4-mediated PGE₂ signaling in regulating ciliogenensis and organ laterality. In agreement with animal model studies, cultured mammalian cells deficient in PGE₂ signaling display defective ciliogenesis.² Zebrafish are an attractive model system to reveal the roles of prostaglandins in embryogenesis, stem cell formation and organ development.¹ PGE₂ signaling has been identified to regulate morphogenetic movements of convergence and extension and is essential for gastrulation movements in zebrafish embryos.¹ Importantly, activation of PGE₂ signaling expands haematopoietic stem cells in zebrafish and mouse, thereby providing potential therapy for human patients with depleted haematopoietic stem cells. Recent studies indicate that PGE₂ activity has a critical role in the specification and outgrowth of liver and pancreas, and can determine the fate of liver versus pancreas progenitor cells.⁶ These findings place prostaglandin signaling among the canonical protein signaling pathways in regulating embryo development and organ formation, in addition to its long-standing roles in physiology and pathology.

From an evolution perspective, prostaglandins have been found in all major phyla, and can regulate development of metazoans similar to protein signaling pathways.⁷ Prostaglandins offer unique ancestral advantages compared to protein signaling pathways. Prostaglandins are synthesized de novo from membrane-released arachidonic acid when cells are activated, and they do not need the energy and storage requirements of proteins. The important properties of prostaglandins are their stereochemical precision in recognition, their potency in the nanomolar range and their evanescent half-life. This enables them to have more quick and accurate response in spatial and temporal manners in the setting of development and physiology. Prostaglandin molecules are truly a conundrum. New insight into their roles in development and disease, and the potential therapeutics based on the novel findings will undoubtedly arise in the near future.