Dataset Information

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland.

ABSTRACT: Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 ?g/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.

SUBMITTER: Li G

PROVIDER: S-EPMC4723263 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland.

Li Guangyuan G Hayward Isaac N IN Jenkins Brittany R BR Rothfuss Heather M HM Young Coleman H CH Nevalainen Marja T MT Muth Aaron A Thompson Paul R PR Navratil Amy M AM Cherrington Brian D BD

PloS one 20160122 1

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived fro ...[more]

PMID: 26799659

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Project description:BackgroundPenetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process.MethodsFor our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development.ResultsOur results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland.ConclusionTogether, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration.

Project description:BackgroundMicroRNAs (miRNA) are small endogenous non-coding RNA involved in the post-transcriptional regulation of specific mRNA targets. The first whole goat genome sequence became available in 2013, with few annotations. Our goal was to establish a list of the miRNA expressed in the mammary gland of lactating goats, thus enabling implementation of the goat miRNA repertoire and considerably enriching annotation of the goat genome.ResultsHere, we performed high throughput RNA sequencing on 10 lactating goat mammary glands. The bioinformatic detection of miRNA was carried out using miRDeep2 software. Three different methods were used to predict, quantify and annotate the sequenced reads. The first was a de novo approach based on the prediction of miRNA from the goat genome only. The second approach used bovine miRNA as an external reference whereas the last one used recently available goat miRNA. The three methods enabled the prediction and annotation of hundreds of miRNA, more than 95% were commonly identified. Using bovine miRNA, 1,178 distinct miRNA were detected, together with the annotation of 88 miRNA for which corresponding precursors could not be retrieved in the goat genome, and which were not detected using the de novo approach or with the use of goat miRNA. Each chromosomal coordinate of the precursors determined here were generated and depicted on a reference localisation map. Forty six goat miRNA clusters were also reported. The study revealed 263 precursors located in goat protein-coding genes, amongst which the location of 43 precursors was conserved between human, mouse and bovine, revealing potential new gene regulations in the goat mammary gland. Using the publicly available cattle QTL database, and cow precursors conserved in the goat and expressed in lactating mammary gland, 114 precursors were located within known QTL regions for milk production and composition.ConclusionsThe results reported here represent the first major identification study on miRNA expressed in the goat mammary gland at peak lactation. The elements generated by this study will now be used as references to decipher the regulation of miRNA expression in the goat mammary gland and to clarify their involvement in the lactation process.

Dataset Information

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland.

Publications

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland.

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