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Project

Study of osteoarthritis-associated genes using zebrafish and chick developmental models.

Osteoarthritis (OA) is a disorder of the synovial joints and a leading cause of impaired movement and pain, affecting millions of people worldwide. The disease is characterized by a progressive degradation of the articular cartilage and associated defects of the subchondral bone. Over the past decades, genetic and environmental risk factors that predispose to OA have been intensively investigated. Remarkably, many of the OA-associated genes regulate cartilage and bone development during embryogenesis, which led to the hypothesis that a reactivation of embryonic pathways could be involved in the disease process. Nevertheless, the current research did not result in a treatment that arrests or reverses the disease process yet.

The high impact on the quality of life and the increasing costs linked to OA in the aging population, underscores the need for good diagnostic markers and eventually, a curative therapy. Therefore, a pan-European TREAT-OA research consortium was launched to identify novel genes associated with the onset and progression of OA and eventually, develop new treatments or diagnostic markers. Genome-wide association scans (GWAS) performed within the TREAT-OA-project, revealed novel single nucleotide polymorphisms (SNPs) associated with OA and mapping to the COG5 gene cluster. The cluster contains six genes, including the orphan G-protein coupled receptor type 22 (GPR22). Importantly, GPR22 was induced in the joints of osteoarthritic mouse models, which led to the hypothesis that GPR22 is involved in the disease process. However, the biological function and regulation of GPR22 remains poorly investigated and its role in the disease process is still debated. Since it has been shown that a reactivation of embryonic pathways is involved in OA, investigating the biological function of GPR22 in a developmental model can be of relevance for the understanding of the molecular basis of the disease. Consequently, we used the well-established zebrafish model to study the cellular processes and signaling pathways regulated by GPR22 in a simple and well-described in vivo context by gain- and loss of function studies.

We have shown that morpholino knock down or overexpression of gpr22 led to defective left-right (LR) axis formation in the zebrafish embryo. Specifically, defective LR patterning included randomization of the left-specific lateral plate mesodermal genes (LPM) (lefty1, lefty2, southpaw and pitx2a), resulting in randomized cardiac looping. Furthermore, gpr22 inactivation in the Kupffer’s vesicle (KV) alone was still able to generate the phenotype, indicating that Gpr22 mainly regulates LR asymmetry through the KV. Analysis of the KV cilia by immunofluorescence and transmission electron microscopy (TEM), revealed that gpr22 knock down or overexpression resulted in changes of cilia length and structure. Deregulation of gpr22 led to defects in other ciliated organs as well, such as the inner ear, the neuromasts and the olfactory system, which might indicate a more general role for Gpr22 in ciliogenesis. Nevertheless, the exact function of Gpr22 in regulating cilia length or structure of these ciliated organs remains unclear.

Our finding that Gpr22 is involved in ciliogenesis, is supported by the evidence that many GPCRs and their effectors are targeted to the cilia to regulate ciliogenesis. The lack of a zebrafish Gpr22 antibody, however, prevented us from investigating the subcellular localization of the Gpr22 protein in zebrafish, but we were able to detect porcine Gpr22 in cilia protein extracts isolated from a porcine kidney epithelial cell line (CL4) (in collaboration with Wim Annaert and Applonia Rose, Laboratory for Membrane Transport, University of Leuven). In addition, the Gpr22 protein sequence contains conserved cilia targeting motifs, such as the VxPx and AxxxQ motifs. It is important to point out, however, that many regulators of ciliogenesis are not targeted to the cilium per se, such as transcription factors, miRNAs, proteins associated with the centrosome, the basal bodies, actin etc.

It has been shown that the FGF signaling pathway is an important regulator of ciliogenesis in a wide range of epithelia. For example, Fgf signaling regulates cilia length in the zebrafish Kupffer’s vesicle by inducing the expression of the master cilia regulators, foxj1a and rfx2. Our data indicate that Gpr22 does not alter the expression or transcriptional activity of neither of these transcription factors. However, these results do not exclude a potential interaction of Gpr22 with the Fgf signaling pathway. Indeed, overexpression of gpr22 resulted in ectopic Fgf signaling in the embryonic midline and in the cells surrounding the KV, suggesting an interaction between the Gpr22 and Fgf signaling in these tissues. Furthermore, gpr22 overexpression rescued the shortened cilia phenotype observed in embryos treated with the Fgfr1 inhibitor SU5402. Based on our observations, it is plausible that the Gpr22 and Fgf signaling pathways interact downstream of their receptors through a yet unknown common regulator to control KV ciliogenesis.

Overexpression of GPR22 in HEK-293 cells led to the inhibition of adenylate cyclase and consequently, a decrease in PKA activity. Previous studies report that PKA promotes ciliogenesis and an activation of PKA by activating adenylate cyclase results in lengthening of the KV cilia in zebrafish. Based on these data, embryos overexpressing gpr22 are expected to have a decrease in PKA activity and consequently, a reduction in cilia length. However, they displayed a lengthening of the KV cilia. Therefore, it is likely that Gpr22 signals in a PKA-independent manner to regulate KV cilia length. Indeed, we found that the treatment of zebrafish embryos with PKA modulators could not mimic the KV cilia phenotype resulting from gpr22 deregulation. In addition, the shorter cilia phenotype of gpr22 morphants could not be rescued by an inhibition of PKA. Thus, based on the literature and our own preliminary data, it is likely that there is no direct interaction between Gpr22 and PKA in KV ciliogenesis.

Since we could not observe a reduction in the number of KV cilia and our preliminary results indicate that the basal bodies were not affected, it is likely that Gpr22 controls the growth or maintenance of the cilium rather than the induction of ciliogenesis. Despite the growing list of genes and pathways affecting ciliogenesis, the actual mechanism of maintaining cilia length and structure is still poorly investigated. An appealing theoretical model is the ‘balance-point’ mechanism. In this model, the cilia length is depending on the efficiency of the intraflagellar transport (IFT). Gpr22, together with genes already identified in screens for cilia length mutants, might control the IFT and therefore, cilia length and structure. This hypothesis is supported by our notion that the KV cilia appeared to be swollen and filled with electro dense material and that the ‘9+0/9+2’ ultrastructure was affected in embryos with deregulated gpr22 expression. These cilia defects are often associated with genetic mutations in genes encoding components or regulators of the IFT system, like the IFT proteins, the BBS proteins and the IFT motor-proteins. Evidence that GPCRs are able to regulate ciliogenesis by controlling the IFT transport system, came from a study in which they demonstrate that the GPCR EP4 (enzyme cyclooxygenase-1 receptor) promotes ciliogenesis by enhancing the anterograde IFT transport. Nevertheless, further experiments are required to determine the exact effect of gpr22 deregulation on the IFT system.

Although the potential role of GPR22 in OA is still debated, it is plausible that GPR22 influences the disease process by affecting ciliogenesis. Indeed, many studies indicate that cilia are essential for proper skeletal development and maintenance, mainly because of their essential role in the Hedgehog and Wnt signaling pathways, which are critical for the development and homeostasis of bone and cartilage. In addition, cilia act as mechano- and chemosensors in the skeleton, allowing the tissue to respond properly to changes in the extracellular environment.

In summary, our research identified a novel player in the field of ciliogenesis and raises some interesting questions concerning the function of GPR22 and cilia in human skeletal and cilia-related disorders, such as osteoarthritis and the ciliopathies.

Date:1 Oct 2010 →  30 Mar 2015
Keywords:Bcap29, Gpr22, Osteoarthritis, Joint development, Pathway analysis, Chick embryo, Zebrafish embryo
Disciplines:Orthopaedics, Genetics, Gynaecology and obstetrics, Molecular and cell biology, Morphological sciences
Project type:PhD project