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Kinase pathways in cilia disassembly signalling

ABSTRACT: Aim: To determine the major protein kinase pathways altered upon serum- or LPA-induced cilia disassembly Primary cilia are dynamic structures which must disassemble for a cell to go through mitosis and re-assemble in G0 to transduce essential signals. Also, in non-cycling cells (such as renal epithelial cells), there is evidence to suggest that deregulated cilia disassembly is of critical importance, for example in instances with an excess of a cilia disassembly stimuli (such as Aurora kinase A expression and activation, a key disassembly factor) which is associated with ciliopathy phenotypes and a net loss of ciliated cells. Serum, growth factor or lysophosphatidic acid (LPA) stimulation of ciliated cells are well established experimental paradigms for the induction of cilia disassembly. The signalling network activated by these stimuli that culminates in cilia disassembly is complex and poorly understood. Studies have generally focused on a single or small group of proteins of interest to define their roles in cilia disassembly and the key pathways and kinases activated upon serum stimulation to induce cilia disassembly have not been defined. Moreover, to the best of our knowledge an unbiased analysis of the major pathways activated during cilia disassembly has not been performed. Ciliated hTERT-RPE1 cells were treated with 10% serum or 2 M LPA for 15’ or 2 h to initiate cilia disassembly + pan-class I PI3K inhibitor GDC-0941, followed by label-free phosphoproteomics.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Silvia Surinova

LAB HEAD: Silvia Surinova

PROVIDER: PXD046462 | Pride | 2024-07-25

REPOSITORIES: Pride

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Project description:Brown adipose tissue (BAT) is a thermogenic organ that dissipates stored energy as heat to maintain body temperature in infants and small mammals. This process may also provide protection from development of diet-induced obesity. We found that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, while potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibited reduced expression of BAT-related genes in peripheral white adipose tissue and accumulated significantly more fat than wild-type controls when fed a high fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and suggest that a decrease in peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice. Primary BAT cell cultures were established as previously reported with some minor modifications (Cannon B., 2001; NÃ©chad M., 1983). Four- to 6-week-old mice were euthanized under aseptic conditions and interscapular BAT (IBAT) dissected, finely minced in digestion buffer containing 123 mM NaCl, 5mM HCl, 1.3 mM CaCl2, 5mM glucose, 1.5% (w/v) crude bovine serum albumin fraction V, 100 mM HEPES, 0.2% collagenase type II (Sigma-Aldrich Corp., St. Louis, MO), pH 7.4, and digested in 10 ml of the same buffer for 30 min at 37Â°C. The supernatant obtained was then filtered using a 250 Âµm nylon mesh and kept in ice for 15 minutes to allow fat droplets and mature cells to float. The resulting infranatant was filtered through a 30 Âµm nylon mesh to enrich the fraction with precursor cells and centrifuged at 700g for 10 min. The pellet obtained was washed once in warm DMEM and resuspended in DMEM medium containing 10% newborn calf serum, 10 mM HEPES, 4mM glutamine, 25 Âµg/ml sodium ascorbate (Sigma-Aldrich Corp., St. Louis, MO)., 100U/ml penicillin and 100mg/ml streptomycin. Cells were seeded onto 35mm dish plates. Insulin treatment was started on day 1 at 50 nM and the concentration increased to 100 nM at day 5 and 200 nM at day 7. Differentiated/confluent cells were collected for biochemical analysis (~day 9). To identify genes regulated by ATX and LPA signaling, 9 DIV primary BAT cultures were treated with the vehicle (DMSO), specific ATX inhibitor HA155 (2.5ÂµM), or LPA (1-Oleoly-LPA, 5ÂµM) (Avanti Polar Lipids, AL, USA) during differentiation. Treatment of the BAT cultures did not affect confluence or markers of proliferation. RNA was extracted from three cultures per condition using TRIzol reagent (Invitrogen, Carlsbad, CA) as described previously (Blalock EM., 2003).

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Kinase pathways in cilia disassembly signalling

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