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NfκB signaling dynamics and their target genes differ between mouse blood cell types and induce distinct cell behavior

ABSTRACT: Cells can use signaling pathway activity over time (i.e., dynamics) to control cell fates. However, little is known about the potential existence and function of signaling dynamics in primary hematopoietic stem and progenitor cells (HSPCs). Here, we use time-lapse imaging and tracking of single murine HSPCs from GFP-p65/H2BmCherry reporter mice to quantify their nuclear factor κB (NfκB) activity dynamics in response to TNFα and IL1β. We find response dynamics to be heterogeneous between individual cells, with cell type specific dynamics distributions. Transcriptome sequencing of single cells physically isolated after live dynamics quantification shows activation of different target gene programs in cells with different dynamics. Finally, artificial induction of oscillatory NfκB activity causes changes in GMP behavior. Thus, HSPC behavior can be influenced by signaling dynamics, which are tightly regulated during hematopoietic differentiation and enable cell type specific responses to the same signaling inputs.

ORGANISM(S): Mus musculus

PROVIDER: GSE199404 | GEO | 2022/07/20

REPOSITORIES: GEO

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Project description:Gonadotrophin-releasing hormone (GnRH) significantly inhibits proliferation of a proportion of cancer cell lines by activating GnRH receptor-G protein signaling. Therefore, manipulation of GnRH receptor signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GnRH receptor activation in sensitive cells (HEK293-GnRHR, SCL60) in in vitro and in vivo settings, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic SCL60 response to the GnRH super-agonist Triptorelin. Early and mid-phase changes (0.5-1.0 h) comprised mainly transcription factors. Later changes (8-24 h) included a GnRH target gene, CGA, and up or down-regulation of transcripts encoding signaling and cell division machinery. Pathway analysis exposed identified altered mitogen-activated protein kinase and cell cycle pathways, consistent with occurrence of G2/M arrest and apoptosis. NFκB pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenografts in vivo during Triptorelin anti-proliferation. Increased phosphorylated NFκB (p65) occurred in SCL60 in vitro, and p-NFκB and IκBε were higher in treated xenografts than controls after 4 days Triptorelin. NFκB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GnRH receptor signaling, identifies potential anti-proliferative target genes and implicates the NFκB survival pathway as a node for enhancing GnRH agonist-induced anti-proliferation. 55 samples: 35 SCL60 (15 Control, 20 Treated), 20 HEK293 (12 Control, 8 Treated). Samples collected after 0, 0.5, 1, 2, 8 or 24h after treatment with Triptorelin (100nM) or vehicle control (20% Propylene Glycol solution). SCL60 cells are HEK293 cells stably transfected with a high level of functional rat GnRHR.

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NfκB signaling dynamics and their target genes differ between mouse blood cell types and induce distinct cell behavior

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