Investigating crosstalk between Hedgehog signalling and compartmentalized ciliary specific cAMP-PKA signalling in the regulation of 3T3-L1 adipogenesis
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Authors
Loureiro, Angela
Date
2025-09-30
Type
thesis
Language
eng
Keyword
signalling
Alternative Title
Abstract
The primary cilium (PC) is a tiny and complex antenna-like organelle present on most animal cells which functions as a signalling hub to organize information encoded by numerous systems. For instance, ciliary Hedgehog (Hh) and PKA signalling systems interact to regulate many events, including cell differentiation. While previous unpublished work in the Maurice laboratory showed that selective inhibition of ciliary PKA reduced adipogenesis in response to Free Fatty Acid Receptor 4 (FFAR4) agonism in of 3T3-L1 preadipocytes, a model cell line used in adipogenesis research, this work was silent on the identity of the PKA isoform involved. Very recently, a ciliary G-protein-coupled receptor (GPCR) (GPR161) was shown to encode a C-terminal A-Kinase anchoring protein (AKAP) domain with PKA regulatory subunit type I (i.e., RI) specificity. In this work, we hypothesize that the GPR161-anchored RI-regulated PKA is involved in the pro-adipogenic effects of ciliary FFAR4 agonism. We tested this hypothesis using pharmacological and molecular approaches.
The impact of ciliary Hh activation by the Smo agonist (SAG) on FFAR4-dependent adipogenesis was determined. Data from these studies showed that SAG reduced FFAR4 agonist (i.e., TUG-891)-induced 3T3-L1 adipogenesis. To investigate whether GPR161-anchored PKA was important in these effects, a ciliary targeted GPR161-based AKAP displacing peptide construct (AKAP-DP WT), and its leucine to proline negative control (AKAP-DP L-P), were created and used in RI-displacement type experiments. Consistent with the size and construction of these constructs, their expression yielded yellow-fluorescent protein (YFP)-tagged proteins which targeted the cilium, and which were of the expected molecular size. With respect to the impact of these constructs on adipogenesis of 3T3-L1 cells, when expressed transiently, both the AKAP-DP WT and AKAP-DP L-P constructs inhibited adipogenesis. Since these findings were inconsistent with our hypothesis but could be due to excessive expression of these fusion proteins in these cells, stable cell lines encoding either AKAP-DP WT or AKAP-DP L-P were created. Although preliminary, our findings do suggest that cultures of stable 3T3-L1 cells expressing AKAP-DP WT were less likely to differentiate in the presence of TUG-891 than those expressing the AKAP-DP L-P.
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Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution 4.0 International