Ciliary Proteins Research Articles

Mechanisms of Bone Morphogenetic Protein 2 in Respiratory Diseases.

Bone morphogenetic protein 2 (BMP2) belongs to the transforming growth factor-β (TGF-β) superfamily and plays an important role in regulating embryonic development, angiogenesis, osteogenic differentiation, tissue homeostasis, and cancer invasion. Increasing studies suggest BMP2 is involved in several respiratory diseases. This study aimed to review the role and mechanisms of BMP2 in respiratory diseases. BMP2 signaling pathway includes the canonical and non-canonical signaling pathway. The canonical signaling pathway is the BMP2-SMAD pathway, and the non-canonical signaling pathway includes mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. The BMP2 is related to pulmonary hypertension (PH), lung cancer, pulmonary fibrosis (PF), asthma, and chronic obstructive pulmonary disease (COPD). BMP2 inhibits the proliferation of pulmonary artery smooth muscle cells (PASMCs), promotes the apoptosis of PASMCs to reduce pulmonary vascular remodeling in PH, which is closely related to the canonical and non-canonical pathway. In addition, BMP2 stimulates the proliferation and migration of cells to promote the occurrence, colonization, and metastasis of lung cancer through the canonical and the non-canonical pathway. Meanwhile, BMP2 exert anti-fibrotic function in PF through canonical signaling pathway. Moreover, BMP2 inhibits airway inflammation to maintain airway homeostasis in asthma. However, the signaling pathways involved in asthma are poorly understood. BMP2 inhibits the expression of ciliary protein and promotes squamous metaplasia of airway epithelial cells to accelerate the development of COPD. In conclusion, BMP2 may be a therapeutic target for several respiratory diseases.

New functions of B9D2 in tight junctions and epithelial polarity

Ciliopathies are a diverse group of disorders resulting from abnormalities in the development or function of multiple organs. While significant research has clarified the role of the primary cilium in transducing numerous signalling pathways, elucidating causes of neuronal and skeletal development disorders, the origins of other ciliopathy-related conditions, such as hepatic fibrocystic diseases, remain elusive. Additionally, attempts to correlate specific ciliary proteins with distinct phenotypes have been largely unsuccessful due to the variable and overlapping symptoms of ciliopathies. This study aims to elucidate the extraciliary roles of the protein B9D2 in the development of biliary dysgenesis, a condition present in Meckel-Gruber and Joubert syndromes caused by mutations in this protein. Traditionally, B9D2 is known for its role at the transition zone of the primary cilium in the transduction of signalling pathways notably Wingless and Hedgehog. Our work demonstrates that before ciliogenesis occurs, B9D2 is crucial for the maturation and maintenance of tight junctions ensuring epithelial barrier tightness and appropriate biliary lumen formation. This study provides new insights into the mechanisms underlying biliary dysgenesis in hepatic ciliopathies, suggesting that further exploration of the non-ciliary functions of proteins involved in ciliopathies could lead to a better understanding and treatment of these complex disorders.

Primary cilia shape postnatal astrocyte development through Sonic Hedgehog signaling.

Primary cilia function as specialized signaling centers that regulate many cellular processes including neuron and glia development. Astrocytes possess cilia, but the function of cilia in astrocyte development remains largely unexplored. Critically, dysfunction of either astrocytes or cilia contributes to molecular changes observed in neurodevelopmental disorders. Here, we show that a sub-population of developing astrocytes in the prefrontal cortex are ciliated. This population corresponds to proliferating astrocytes and largely expresses the ciliary protein ARL13B. Genetic ablation of astrocyte cilia in vivo at two distinct stages of astrocyte development results in changes to Sonic Hedgehog (Shh) transcriptional targets. We show that Shh activity is decreased in immature and mature astrocytes upon loss of cilia. Furthermore, loss of cilia in immature astrocytes results in decreased astrocyte proliferation and loss of cilia in mature astrocytes causes enlarged astrocyte morphology. Together, these results indicate that astrocytes require cilia for Shh signaling throughout development and uncover functions for astrocyte cilia in regulating astrocyte proliferation and maturation. This expands our fundamental knowledge of astrocyte development and cilia function to advance our understanding of neurodevelopmental disorders.

A multistage Plasmodium CRL4WIG1 ubiquitin ligase is critical for the formation of functional microtubule organization centers in microgametocytes.

Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium. Here, we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to CRL4 in other eukaryotes. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1, and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include WD-repeat protein important for gametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both the schizont and gametocyte stages. WIG1 disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired the formation of the microtubule organization centers that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the formation of microgametes by regulating proteostasis of ciliary and DNA replication proteins.IMPORTANCEPlasmodium parasites undergo fascinating lifecycles with multiple developmental steps, converting into morphologically distinct forms in both their mammalian and mosquito hosts. Protein ubiquitination by ubiquitin ligases emerges as an important post-translational modification required to control multiple developmental stages in Plasmodium. Here, we identify a cullin RING E3 ubiquitin ligase (CRL) complex expressed in the replicating asexual blood stages and in the gametocyte stages that mediate transmission to the mosquito. WIG1, a putative substrate recognition protein of this ligase complex, is essential for the maturation of microgametocytes into microgametes upon ingestion by a mosquito. More specifically, WIG1 is required for proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. This requirement is linked to DNA replication and microtubule organization center formation, both critical to the development of flagellated microgametes.

Cell-cell interaction determines cell fate of mesoderm-derived cell in tongue development through Hh signaling.

Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma, and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested tongue swelling formation. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations to disturb cellular processes which were disrupted in mutant mice. Our results provide hints for possible future treatment in ciliopathies.

Astrogliosis and neuroinflammation underlie scoliosis upon cilia dysfunction.

Cilia defects lead to scoliosis in zebrafish, but the underlying pathogenic mechanisms are poorly understood and may diverge depending on the mutated gene. Here, we dissected the mechanisms of scoliosis onset in a zebrafish mutant for the rpgrip1l gene encoding a ciliary transition zone protein. rpgrip1l mutant fish developed scoliosis with near-total penetrance but asynchronous onset in juveniles. Taking advantage of this asynchrony, we found that curvature onset was preceded by ventricle dilations and was concomitant to the perturbation of Reissner fiber polymerization and to the loss of multiciliated tufts around the subcommissural organ. Rescue experiments showed that Rpgrip1l was exclusively required in foxj1a-expressing cells to prevent axis curvature. Genetic interactions investigations ruled out Urp1/2 levels as a main driver of scoliosis in rpgrip1 mutants. Transcriptomic and proteomic studies identified neuroinflammation associated with increased Annexin levels as a potential mechanism of scoliosis development in rpgrip1l juveniles. Investigating the cell types associated with annexin2 over-expression, we uncovered astrogliosis, arising in glial cells surrounding the diencephalic and rhombencephalic ventricles just before scoliosis onset and increasing with time in severity. Anti-inflammatory drug treatment reduced scoliosis penetrance and severity and this correlated with reduced astrogliosis and macrophage/microglia enrichment around the diencephalic ventricle. Mutation of the cep290 gene encoding another transition zone protein also associated astrogliosis with scoliosis. Thus, we propose astrogliosis induced by perturbed ventricular homeostasis and associated with immune cell activation as a novel pathogenic mechanism of zebrafish scoliosis caused by cilia dysfunction.

Lipid droplets sequester palmitic acid to disrupt endothelial ciliation and exacerbate atherosclerosis in male mice

Disruption of ciliary homeostasis in vascular endothelial cells has been implicated in the development of atherosclerosis. However, the molecular basis for the regulation of endothelial cilia during atherosclerosis remains poorly understood. Herein, we provide evidence in male mice that the accumulation of lipid droplets in vascular endothelial cells induces ciliary loss and contributes to atherosclerosis. Triglyceride accumulation in vascular endothelial cells differentially affects the abundance of free fatty acid species in the cytosol, leading to stimulated lipid droplet formation and suppressed protein S-palmitoylation. Reduced S-palmitoylation of ciliary proteins, including ADP ribosylation factor like GTPase 13B, results in the loss of cilia. Restoring palmitic acid availability, either through pharmacological inhibition of stearoyl-CoA desaturase 1 or a palmitic acid-enriched diet, significantly restores endothelial cilia and mitigates the progression of atherosclerosis. These findings thus uncover a previously unrecognized role of lipid droplets in regulating ciliary homeostasis and provide a feasible intervention strategy for preventing and treating atherosclerosis.

Evolutionary trajectory for nuclear functions of ciliary transport complex proteins.

SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.

Expansion microscopy of axonemal dyneins in islet primary cilia.

Primary cilia are sensory organelles that mediate critical cellular functions yet are difficult to visualize because of their small size and low abundance. Expansion microscopy (ExM) is an imaging method that physically expands biological specimens using a swellable hydrogel, facilitating downstream imaging of small cellular structures. In this study, we apply ExM with confocal imaging to probe axonemal dynein expression in murine and human pancreatic islets. Innovations include an updated ExM workflow adapted from 2D cultured cells to 3D tissue optimized for intact pancreatic islets, and demonstration of axonemal dynein protein expression in islet primary cilia and centrioles. Four dynein subunits were consistently detected in both expanded and nonexpanded samples across species, including DNAI1, DNAH5, DNAH11, and DNALI1, where DNAI1 expression was the most readily detectable and seen to exhibit a distinct ring-like symmetry on super-resolution imaging. We discuss technical issues contributing to ExM success, including antibody performance across ExM and conventional sample preparation and imaging protocols, and the need to validate findings using gene knockdowns and controls. We conclude that ExM is a useful approach for identifying ciliary proteins in pancreatic islet tissue and may be broadly applicable to studying protein composition of other organelles.

Contribution of intraflagellar transport to compartmentalization and maintenance of the photoreceptor cell.

The first steps of vision take place in the ciliary outer segment compartment of photoreceptor cells. The protein composition of outer segments is uniquely suited to perform this function. The most abundant among these proteins is the visual pigment, rhodopsin, whose outer segment trafficking involves intraflagellar transport (IFT). Here, we report three major findings from the analysis of mice in which ciliary transport was acutely impaired by conditional knockouts of IFT-B subunits. First, we demonstrate the existence of a sorting mechanism whereby mislocalized rhodopsin is recruited to and concentrated in extracellular vesicles prior to their release, presumably to protect the cell from adverse effects of protein mislocalization. Second, reducing rhodopsin expression significantly delays photoreceptor degeneration caused by IFT disruption, suggesting that controlling rhodopsin levels may be an effective therapy for some cases of retinal degenerative disease. Last, the loss of IFT-B subunits does not recapitulate a phenotype observed in mutants of the BBSome (another ciliary transport protein complex relying on IFT) in which non-ciliary proteins accumulate in the outer segment. Whereas it is widely thought that the role of the BBSome is to primarily participate in ciliary transport, our data suggest that the BBSome has another major function independent of IFT and possibly related to maintaining the diffusion barrier of the ciliary transition zone.

Central regulation of feeding and body weight by ciliary GPR75.

Variants of the G protein-coupled receptor 75 (GPR75) are associated with a lower BMI in large-scale human exome-sequencing studies. However, how GPR75 regulates body weight remains poorly understood. Using random germline mutagenesis in mice, we identified a missense allele (Thinner) of Gpr75 that resulted in a lean phenotype and verified the decreased body weight and fat weight in Gpr75-knockout (Gpr75-/-) mice. Gpr75-/- mice displayed reduced food intake under high-fat diet (HFD) feeding, and pair-feeding normalized their body weight. The endogenous GPR75 protein was exclusively expressed in the brains of 3xFlag-tagged Gpr75-knockin (3xFlag-Gpr75) mice, with consistent expression across different brain regions. GPR75 interacted with Gαq to activate various signaling pathways after HFD feeding. Additionally, GPR75 was localized in the primary cilia of hypothalamic cells, whereas the Thinner mutation (L144P) and human GPR75 variants in individuals with a lower BMI failed to localize in the cilia. Loss of GPR75 selectively inhibited weight gain in HFD-fed mice but failed to suppress the development of obesity in leptin ob-mutant (Lepob-mutant) mice and adenylate cyclase 3-mutant (Adcy3-mutant) mice on a chow diet. Our data reveal that GPR75 is a ciliary protein expressed in the brain and plays an important role in regulating food intake.

Absence of Aquaporin-4 (AQP4) Prolongs the Presence of a CD11c+ Microglial Population during Postnatal Corpus Callosum Development.

Aquaporin-4 (AQP4) expression is associated with the development of congenital hydrocephalus due to its structural role in the ependymal membrane. Gene expression analysis of periaqueductal tissue in AQP4-knockout (KO) mice at 11 days of age (P11) showed a modification in ependymal cell adhesion and ciliary protein expression that could alter cerebrospinal fluid homeostasis. A microglial subpopulation of CD11c+ cells was overexpressed in the periaqueductal tissue of mice that did not develop hydrocephalus, suggesting a possible protective effect. Here, we verified the location of this CD11c+ expression in the corpus callosum (CC) and cerebellum of AQP4-KO mice and analysed its time course. Immunofluorescence labelling of the CD11c protein in the CC and cerebellum of WT and KO animals at P3, P5, P7 and P11 confirmed an expanded presence of these cells in both tissues of the KO animal; CD11c+ cells appeared at P3 and reached a peak at P11, whereas in the WT animal, they appeared at P5, reached their peak at P7 and were undetectable by P11. The gene expression analysis in the CC samples at P11 confirmed the presence of CD11c+ microglial cells in this tissue. Among the more than 4000 overexpressed genes, Spp1 stood out with the highest differential gene expression (≅600), with other genes, such as Gpnmb, Itgax, Cd68 and Atp6v0d2, also identified as overexpressed. Therefore, CD11c+ cells appear to be necessary for normal corpus callosum development during postnatal life, and the absence of AQP4 prolonged its expression in this tissue.

Role of endosomal pathway in the ciliary transport and the membrane organization of outer segment disc membrane in photoreceptors

A photoreceptor is a specialized neuron that is responsible for the conversion of light into an electrical signal. Photoreceptors are classified into rods and cones, and both photoreceptors possess light-sensing ciliary organelles called outer segments (OSs), anchored in the cells by a microtubule-based axoneme. The OS consists of a stack of disc membranes, which are abundant for the retinal phototransduction proteins such as rhodopsin. Recently, modern protein synchronization techniques using in vivo transfection in rodents revealed that rhodopsin transits through Rab11-positive recycling endosomes, preferentially entering the OS in the dark. Moreover, Peripherin-2 (PRPH2, also called retinal degeneration slow, RDS), a photoreceptor-specific tetraspanin protein essential for the morphogenesis of disc membranes, is delivered to the OS following complementary to that of rhodopsin. Various PRPH2 disease-causing mutations have been found in humans, and most of the mutations in the cytosolic C-terminus of PRPH2 are linked to cone-dominant macular dystrophies. It has been shown that the late endosome is the waystation that sorts newly synthesized PRPH2 into the cilium. The multiple C-terminal motifs of PRPH2 regulate its late endosome and ciliary targeting through ubiquitination and binding to an Endosomal Sorting Complexes Required for Transport (ESCRT) component, Hrs. These findings suggest that the late endosomes play an important role in the biosynthetic pathway of ciliary proteins and can be a new therapeutic target for the diseases caused by ciliary defects.

The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling.

The primary cilium, an antenna-like sensory organelle that protrudes from the surface of most eukaryotic cell types, has become a signaling hub of growing interest given that defects in its structure and/or function are associated with human diseases and syndromes, known as ciliopathies. With the continuously expanding role of primary cilia in health and diseases, identifying new players in ciliogenesis will lead to a better understanding of the function of this organelle. It has been shown that the primary cilium shares similarities with the immune synapse, a highly organized structure at the interface between an antigen-presenting or target cell and a lymphocyte. Studies have demonstrated a role for known cilia regulators in immune synapse formation. However, whether immune synapse regulators modulate ciliogenesis remains elusive. Here, we find that programmed death ligand 1 (PD-L1), an immune checkpoint protein and regulator of immune synapse formation, plays a role in the regulation of ciliogenesis. We found that PD-L1 is enriched at the centrosome/basal body and Golgi apparatus of ciliated cells and depleting PD-L1 enhanced ciliogenesis and increased the accumulation of ciliary membrane trafficking proteins Rab8a, BBS5, and sensory receptor protein PC-2. Moreover, PD-L1 formed a complex with BBS5 and PC-2. In addition, we found that depletion of PD-L1 resulted in the ciliary accumulation of Gli3 and the downregulation of Gli1. Our results suggest that PD-L1 is a new player in ciliogenesis, contributing to PC-2-mediated sensory signaling and the Hh signaling cascade.

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

CEP41, a ciliopathy-linked centrosomal protein, regulates microtubule assembly and cell proliferation.

Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies. Understanding the multifaceted roles of centrosomal proteins is vital to comprehend their involvement in disease development. Here, we report novel cellular functions of CEP41, a centrosomal and ciliary protein implicated in Joubert syndrome. We show that CEP41 is an essential microtubule-associated protein with microtubule-stabilizing activity. Purified CEP41 binds to preformed microtubules, promotes microtubule nucleation and suppresses microtubule disassembly. When overexpressed in cultured cells, CEP41 localizes to microtubules and promotes microtubule bundling. Conversely, shRNA-mediated knockdown of CEP41 disrupts the interphase microtubule network and delays microtubule reassembly, emphasizing its role in microtubule organization. Further, we demonstrate that the association of CEP41 with microtubules relies on its conserved rhodanese homology domain (RHOD) and the N-terminal region. Interestingly, a disease-causing mutation in the RHOD domain impairs CEP41-microtubule interaction. Moreover, depletion of CEP41 inhibits cell proliferation and disrupts cell cycle progression, suggesting its potential involvement in cell cycle regulation. These insights into the cellular functions of CEP41 hold promise for unraveling the impact of its mutations in ciliopathies.

Immuno-scanning electron microscopy of islet primary cilia.

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

#2810 Exploring non-renal vasculature: therapeutic implications of proton pump inhibitor and anthelmintic treatment in PCK rat model

Abstract Background and Aims Cystic kidney diseases represent ciliopathies that can cause vascular damage through renal hypertension. Additionally, there is growing experimental evidence of endothelial cell abnormalities associated with ciliary protein deficiencies. PCK rats, a strain derived from Sprague-Dawley (SD) rats that develops both polycystic kidney disease (PKD) and liver disease, serve as a model for recessive PKD. The PCK rat model also presents abnormalities in non-renal vascular function. Another study from our group suggests a potential therapeutic benefit of repurposed therapy with a proton pump inhibitor (PPi) and an anthelmintic agent in attenuating the progression of PKD in a PCK rat model. Here, we investigated whether these drugs can also improve endothelial function. Method The compounds were administered to 3-week-old male and female PCK rats (n = 5-6) for a total of 10 weeks. Untreated PCK rats and age-matched SD rats (n = 5-6) served as controls. After perfusion fixation, the aorta was harvested and its thoracic rings were used to evaluate the contraction/relaxation responses ex vivo by organ bath experiments. Additional rings were preserved for histological and molecular biological analyses. Results Male PCK rats exhibited impaired endothelial function compared to healthy controls. Endothelium-dependent relaxation to acetylcholine was improved by 10% after treatment with PPi, while the anthelmintic administration had no protective effect. PKD also impaired endothelial function in female PCK rats compared to healthy SD controls. There was an improvement in endothelium-dependent relaxation to acetylcholine after treatment with PPi by 5.5%, while administration of the anthelmintic agent further impaired endothelial function. Conclusion 13-week-old PCK rats show endothelial dysfunction in non-renal vasculature. Treatment with a proton pump inhibitor improved endothelial function in male PCK rats to the level of healthy SD controls. By contrast, in a parallel study, the anthelmintic had a positive effect on renal function but a detrimental impact on endothelial function. These intricate relationships underscore the need for further exploration to understand the underlying mechanisms and broader implications of our findings, illustrating the challenges and potential avenues for the development of effective treatments for PKD.

The ciliary protein C2cd3 is required for mandibular musculoskeletal tissue patterning

The mandible is composed of several musculoskeletal tissues including bone, cartilage, and tendon that require precise patterning to ensure structural and functional integrity. Interestingly, most of these tissues are derived from one multipotent cell population called cranial neural crest cells (CNCCs). How CNCCs are properly instructed to differentiate into various tissue types remains nebulous. To better understand the mechanisms necessary for the patterning of mandibular musculoskeletal tissues we utilized the avian mutant talpid2 (ta2) which presents with several malformations of the facial skeleton including dysplastic tendons, mispatterned musculature, and bilateral ectopic cartilaginous processes extending off Meckel's cartilage. We found an ectopic epithelial BMP signaling domain in the ta2 mandibular prominence (MNP) that correlated with the subsequent expansion of SOX9+ cartilage precursors. These findings were validated with conditional murine models suggesting an evolutionarily conserved mechanism for CNCC-derived musculoskeletal patterning. Collectively, these data support a model in which cilia are required to define epithelial signal centers essential for proper musculoskeletal patterning of CNCC-derived mesenchyme.

#2804 Mechanisms of cAMP-induced cyst formation observed in Pkhd1-KO cells

Abstract Background and Aims Loss of ciliary protein function leads to altered epithelial properties in hereditary polycystic kidney diseases. To address molecular aspects of defective epithelial homeostasis, monolayered epithelial spheroids can be used to analyze lack of protein function and consequences of pharmacological intervention. Here, we employed epithelial cell clones deficient for fibrocystin/polyductin (FPC) protein function, the origin of ARPKD, to study the impact of FPC cytoplasmic domain constructs on cAMP-induced cystogenesis. Method We used pl-MDCK, sub-cloned principal-like cell lines, with CRISPR/Cas9-based genetic knockout (KO) of Pkhd1 / FPC, and corresponding controls. Cells were grown in matrigel to allow formation of epithelial spheroids within 3 days. Forskolin (Fsk) treatment was employed to induce cAMP-mediated cyst growth mimicking disease conditions. Measurement of proportional lumen, the ratio of lumen to spheroid area, allowed detection the enhanced water and ion transport across the barrier as characteristic for secretory epithelia. Cystogenic signals were modulated by expression of the FPC C-terminal domain constructs and/or pharmacological inhibitors, and involvement of ion channels was addressed. Results In Pkhd1-KO cell lines, enhanced cAMP levels resulted in massive lumen expansion of epithelial spheroids with no increase in cell number. The rise in proportional lumen was sensitive to inhibition of chloride-channels, CFTR and TMEM16A, and Src kinase. Fsk-mediated cyst induction led to activation and increased Y705 phosphorylation of STAT3. To address the impact of FPC on cystogenic signaling, expression of a membrane-bound FPC C-terminal protein domain was studied, and its processing and intracellular localization determined. Controlled expression of the FPC cytoplasmic domain in Pkhd1-KO cell lines suppressed Fsk-induced cyst formation. In addition, the FPC domain reduced activation of Src and STAT3, leading to lower STAT3-dependent transcription. Conclusion In FPC-deficient pl-MDCK cells, cyst formation stimulated by high cAMP levels is associated with enhanced Src/STAT3 signaling. Expression of the FPC cytoplasmic domain, leading to a gain-of-function, suppresses Src and STAT3 activity in Pkhd1-KO epithelia and limits cyst growth in vitro. Control of cystogenic Src/STAT3 signaling can be considered a physiologic function of the FPC protein.