Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Signaling from the secretory granule to the nucleus: Uhmk1 and PAM

ABSTRACT: Neurons and endocrine cells package peptides in secretory granules (large dense core vesicles; LDCVs) for storage and stimulated release. Studies of PAM, an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by Uhmk1 and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble cytosolic domain (CD) fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously over-expressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wildtype mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels while expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent. Three separate clones of AtT-20 mouse corticotrope tumor cells stably transfected with doxycycline-inducible rat PAM-1 (clones 14, 6_1, 6_2) were treated with cotrol or doxycycline medium for 48 hours. Total mRNA was isolated and hybridized to an Illumina MouseWG-6 v 2.0 whole genome BeadChip.

ORGANISM(S): Mus musculus

SUBMITTER: Chitra Rajagopal

PROVIDER: E-GEOD-22509 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3 449 STAT3 binding sites, whereas 2 396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation: 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response. Experiment Overall Design: 1) Pituitary corticotrophs cell line (AtT-20) response to glucocorticoids and LIF: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control, LIF for 18h, Dex+LIF for 3h and Dex+LIF for 18h. Each treatment were made in duplicate. [GSM471246-GSM471253] Experiment Overall Design: 2) Pituitary corticotrophs cell line (AtT-20) response to LIF after 3h treatment: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control or LIF for 3h. Each treatment were made in duplicate. [GSM471254-GSM471257] Experiment Overall Design: 3) Pituitary corticotrophs cell line (AtT-20) response to Dex after 3h treatment: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control or Dex for 3h. Each treatment were made in duplicate. [GSM471258-GSM471261] Experiment Overall Design: 4) Pituitary corticotrophs cell line (AtT-20) response to Dex after 18h treatment: Experiment Overall Design: AtT-20 cells mRNA extracted after treatment with PBS as control or Dex for 18h. Each treatment were made in duplicate. [GSM471262-GSM471269]

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Signaling from the secretory granule to the nucleus: Uhmk1 and PAM

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