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

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 mug/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

Identification and Characterization of the Lactating Mouse Mammary Gland Citrullinome

Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, alpha-tubulin, and beta-casein. Given the importance of prolactin to lactation, we next tested if it stimulates PAD-catalyzed citrullination using mouse mammary epithelial CID-9 cells. Stimulation of CID-9 cells with 5 microg/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in alpha-tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes beta-casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased beta-casein and butyrophilin mRNA expression; however, this increase was significantly inhibited by the pan-PAD inhibitor, BB-Cl-amidine (BB-ClA). We also examined the effect of tubulin citrullination on the overall polymerization rate of microtubules. Our results show that citrullinated tubulin had a higher maximum overall polymerization rate. Our work suggests that protein citrullination is an important PTM that regulates gene expression and microtubule dynamics in mammary epithelial cells

Citrullination regulates the expression of insulin-like growth factor-binding protein 1 (IGFBP1) in ovine uterine luminal epithelial cells.

There are five peptidylarginine deiminase (PAD) isozymes designated as PADs 1, 2, 3, 4 and 6, and many are expressed in female reproductive tissues. These enzymes post-translationally convert positively charged arginine amino acids into neutral citrulline residues. Targets for PAD-catalyzed citrullination include arginine residues on histone tails, which results in chromatin decondensation and changes in gene expression. Some of the first studies examining PADs found that they are localized to rodent uterine epithelial cells. Despite these findings, the function of PAD-catalyzed citrullination in uterine epithelial cells is still unknown. To address this, we first examined PAD expression in uterine cross-sections from pregnant ewes on gestation day 25 (d25). Immunohistochemistry revealed that the levels of PADs 2 and 4 are robust in luminal and glandular epithelia compared with those of PADs 1 and 3. As PADs 2 and 4 have well-characterized roles in histone citrullination, we next hypothesized that PADs citrullinate histones in these uterine cells. Examination of caruncle lysates from pregnant ewes on gestation d25 and an ovine luminal epithelial (OLE) cell line shows that histone H3 arginine residues 2, 8, 17 and 26 are citrullinated, but histone H4 arginine 3 is not. Using a pan-PAD inhibitor, we next attenuated histone citrullination in OLE cells, which resulted in a significant decrease in the expression of insulin-like growth factor-binding protein 1

The human <i>PAD3</i> gene promoter is prolactin responsive in CID-9 cells -276/+41 and -94/+41 base pairs of the human <i>PAD3</i> gene promoter were cloned from genomic DNA and fused to the cDNA of luciferase.

<p>CID-9 cells were transfected overnight with hPAD3–276/+41-Luc, hPAD3–94/+41-Luc, pGL3 empty, and CMV-β-galactosidase. The next morning cells were treated with vehicle or 5 μg/mL of prolactin for 24 hours. Cellular lysates were assayed for luciferase and β-galactosidase activity. Luciferase values were corrected for β-galactosidase activity, and data are expressed as adjusted light units (top panel A) or fold change in adjusted luciferase activity (bottom panel B). The inset figure in top panel A illustrates that the hPAD3–94/+41-Luc construct has significant transcriptional activity compared to the pGL3 empty. Values represent the mean ± SEM. Means were separated using Student’s T-Test with * designating significant differences with treatment (P<0.05).</p

Prolactin stimulation of CID-9 cells increases PAD3 mRNA and protein expression.

<p>Equal numbers of CID-9 cells were grown in phenol red free media with charcoal-stripped FBS overnight. The following morning cells were treated with vehicle or 5 μg/mL of prolactin for 24 or 48 hours. (A) Prolactin treatment significantly increases PAD3 mRNA. Total RNA was extracted from CID-9 cells, reverse transcribed, and resulting cDNA examined by qPCR with intron spanning primers specific for PAD3 or GAPDH as the reference gene control. All data values were normalized to vehicle treated control and data are presented as means ± SEM. Means were separated using SNK ANOVA and * indicates significant difference (P<0.05). (B) Prolactin treatment increases PAD3 protein levels. The top panel shows a representative western blot, while the graph in the bottom panel represents the quantification of western blots using BioRad Image Lab 4.0 (n = 4, P<0.05). Protein concentrations of cell lysates were determined by BCA assay and equal amounts loaded and examined by western blot. Membranes were probed with a rabbit anti-PAD3 antibody or with anti-β-actin as a loading control. The positive control was generated by overexpressing human PAD3 in CID-9 cells.</p

Prolactin stimulates the JAK2/STAT5 signaling pathway to upregulate PAD3 expression in CID-9 cells.

<p>(A) JAK2 mediates prolactin induced PAD3 upregulation. CID-9 cells were pre-treated for 4 hours with DMSO or 3 μM JAK2 inhibitor (SD-1029). Following pre-treatment, cells were stimulated with vehicle or 5 μg/mL of prolactin for 48 hours. After the first 24 hours, cells received fresh inhibitor. Protein concentrations of cell lysates were determined by BCA assay and equal amounts examined by western blot. Membranes were probed with anti-PAD3, p-JAK2, total JAK2 antibodies or with anti-β-actin as a loading control. (B) STAT5 mediates prolactin induced PAD3 upregulation. CID-9 cells were infected overnight with 10 MOI of an adenovirus expressing GFP (Ad-GFP) or an adenovirus expressing a dominant-negative form of STAT5 (Ad-DN-STAT5) as indicated. The following morning cells were treated with vehicle or 5 μg/mL of prolactin for 24 or 48 hours. Protein concentrations of cell lysates were determined by BCA assay and equal amounts examined by western blot. Membranes were probed with anti-PAD3, GFP, p-STAT5, total STAT5 or β-actin antibodies.</p

Inhibition of PAD3 decreases the level of citrullinated proteins in CID-9 cells.

<p>CID-9 cells were treated with vehicle or 50 μM Cl4-amidine overnight. The following morning cells were lysed and equal concentrations of protein lysates examined by western blot following the AMC protocol. The positive control is <i>in vitro</i> citrullinated bulk histones.</p

PAD3 is the most highly expressed PAD isoform in mouse mammary epithelial CID-9 cells.

<p>PAD3 mRNA levels are significantly higher in CID-9 cells compared to all other PAD isoforms. Total RNA was extracted from CID-9 cells, reverse transcribed, and resulting cDNA examined by qPCR with intron spanning primers specific for PAD1, PAD2, PAD3, PAD4 or GAPDH as the reference gene control. All data values were normalized to PAD1 to yield fold change, and data are expressed as means ± SEM. Means were separated using Tukey’s test ANOVA and letters indicate significant differences (P< 0.05).</p

Citrullinated proteins are present in the lactating mammary gland and are citrullinated, in part, by PAD3.

<p>Mammary glands were collected on L2 and 9, homogenized and sonicated in modified RIPA buffer. (A) Citrullinated proteins are present in L2 and L9 mammary glands. Equal concentrations of protein lysates were examined by western blot following the AMC protocol. The positive control is <i>in vitro</i> citrullinated bulk histones. (B) Citrullinated histone tail arginine residues 2, 8, and 17 are present in L2 and L9 mammary glands. Equal concentrations of protein lysates were examined by western blot using an anti-citrullinated histone H3 arginine 2, 8, and 17 antibody. (C) Citrullinated proteins are detected in L2 and L9 mammary tissue. Mouse mammary tissue from lactation day 2 (a, b, c), and day 9 (d, e, f) was harvested, fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned. 5 μm mammary tissue sections were subject to a standard IHC protocol using a rabbit anti-citrulline antibody or an equal amount of non-specific rabbit IgG as a control. Images were taken with 20 and 40X objectives, and DAB staining represents citrullinated proteins. (D) Cl4-amidine decreases PAD3 activity in L9 mammary gland lysates. Equal concentrations of L9 mammary gland lysates were incubated with 100 μM Cl4-amidine for 15 minutes then subject to COLDER PAD activity assay. Values represent the mean ± SEM. Means were separated using Student’s T-Test with * designating significant differences with treatment (P<0.05).</p

PAD3 expression in mouse mammary secretory cells initiates during late pregnancy and is highest during lactation.

<p>(A) PAD3 expression is first detected at P18 and increases from L2 through L9. Mouse mammary tissue from pregnancy day 12 (a, b), day 18 (c, d), lactation day 2 (e, f), and day 9 (g, h) was harvested, fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned. 5 μm mammary tissue sections were subject to a standard IHC protocol using a rabbit anti-PAD3 antibody or an equal amount of non-specific rabbit IgG as a control. Images were taken with 20 and 40X objectives, and DAB staining represents PAD3 expression. (B) PAD3 mRNA and protein expression increases two-fold between L2 and L9. Total RNA was extracted from L2 and L9 mouse mammary glands, reverse transcribed, and resulting cDNA examined by qPCR with intron spanning primers specific for PAD3 or EE2F as the reference gene control. Data values were normalized to L2 to yield fold change, and data are expressed as means ± SEM. L2 and 9 mammary glands were homogenized in RIPA buffer and equal concentrations subject to western blot. The positive control was generated by overexpressing PAD3 in CID-9 cells. Western blots were quantified using BioRad Image Lab 4.0. Data values were normalized to L2 to yield fold change, and data are expressed as means ± SEM. Means were separated using Student’s T-test and * indicates a significant difference (P< 0.05).</p