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Class I PI3K regulatory subunits control differentiation of dendritic cell subsets and regulate Flt3L mediated signal transduction.

ABSTRACT: Dendritic cells (DCs) play pivotal roles in initiating and shaping both innate and adaptive immune responses. The spatiotemporal expression of transcription factor networks and activation of specific signal transduction pathways determine the specification, distribution and differentiation of DC subsets. Even though pioneering studies have established indispensable roles for specific catalytic subunits (p110δ and p110γ) in immune cells, functions of the regulatory subunits, particularly of Class I PI3K, within the hematopoietic system remain incompletely understood. In the study presented here, we deleted the key regulatory subunits-p85α and p85β of the Class I_A PI3K in hematopoietic cells and studied its impact on DC differentiation. Our studies identify that a deficiency of p85 causes increased differentiation of conventional DC (cDC) 2 and plasmacytoid DC (pDC) subsets in the spleen. On the other hand, DC numbers in the bone marrow (BM), thymus and lymph nodes were decreased in p85 mutant mice. Analysis of DC-specific progenitors and precursors indicated increased numbers in the BM and spleen of p85 deficient mice. In-vitro differentiation studies demonstrated augmented DC-differentiation capacities of p85 deficient BM cells in the presence of GM-CSF and Flt3L. BM chimera studies established that p85 deficiency affects DC development through cell intrinsic mechanisms. Molecular studies revealed increased proliferation of DCs and common DC progenitors (CDPs) in the absence of p85 and altered signal transduction pathways in p85 mutant DC subsets in response to Flt3L. In essence, data presented here, for the first time, unequivocally establish that the P85α subunit of class I_A PI3Ks has an indispensable role in the development and maintenance of DCs.

SUBMITTER: Thummar K

PROVIDER: S-EPMC9296662 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.

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Class I PI3K regulatory subunits control differentiation of dendritic cell subsets and regulate Flt3L mediated signal transduction.

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