Prolactin family 8, subfamily a, member 2 (PRL8A2; also called decidual prolactin-related protein; dPRP) is a member of the expanded prolactin family. PRL8A2 is expressed in the uterine decidua and contributes to pregnancy-dependent adaptations to hypoxia. The purpose of this study was to identify gene targets for PRL8A2 action within the uteroplacental compartment. Affymetrix DNA microarray analysis was performed for RNA samples from WT and Prl8a2 null tissues. Validation of the DNA microarray was performed using quantitative RT-PCR. Nine genes were confirmed with decreased expression in Prl8a2 null tissues (e.g. Klk7, Rimklb, Arhgef6, Calm4, Sprr2h, Prl4a1, Ccl27, Lipg, and Htra3). These include potential decidual, endothelial and trophoblast cell targets positively regulated by PRL8A2. A significant upregulation of Derl3, Herpud1, Creld2, Hsp90b1, Ddit3 and Hspa5 was identified in Prl8a2 null tissues, reflecting an increased endoplasmic reticulum (ER) stress response. ER stress genes were prominently expressed in the uterine decidua. We propose that PRL8A2 is a mediator of progesterone-dependent modulation of intrauterine responses to physiological stressors.
The mouse possesses an expanded prolactin (PRL) gene family that encodes hormones/cytokines (Wiemers et al. 2003, Soares et al. 2007). In some species the expansion was robust such as occurred in the mouse, rat, guinea pig and cow (Wiemers et al. 2003, Alam et al. 2006, 2010, Ushizawa & Hashizume 2006), whereas evidence for an expansion in other species such as the human and dog is lacking (Cooke & Liebhaber 1995, Lindblad-Toh et al. 2005). These hormones and cytokines are associated with pregnancy and are produced by the anterior pituitary, uterine decidua and/or trophoblast cells (Soares 2004). The biological activities of PRL are well described and include profound effects on the reproductive axis and lactation (Horseman et al. 1997, Bole-Feysot et al. 1998, Horseman & Gregerson 2014); however, the actions of the remaining PRL family paralogs are less well appreciated. Roles for these PRL related proteins in regulating blood vessel and hematopoietic cell development have been demonstrated (Jackson et al. 1994, Lin & Linzer 1999, Bittorf et al. 2000). Based on mouse mutagenesis experiments, the biological activities of at least some expanded PRL family paralogs include modulation of uteroplacental adaptations to physiological stressors (Ain et al. 2004, Alam et al. 2007, Soares et al. 2007). PRL also participates in homeostatic responses to stress (Dorshkind & Horseman 2001).
Hemochorial placentation is associated with differentiation of uterine stromal cells into epithelial-like cells called decidual cells possessing extensive secretory capabilities and essential roles in the establishment and maintenance of pregnancy (Aplin 2000, Gellersen et al. 2007, Herington & Bany 2009, Teklenburg et al. 2010a,b). Decidual cells effectively create an environment within the uterus compatible with development of the placenta and fetus. Among the factors secreted by decidual cells are members of the PRL family (Orwig et al. 1997a, Jabbour & Critchley 2001). Human decidual cells produce PRL, while the mouse and rat produce PRL and an additional three PRL family paralogs (Soares 2004, Soares et al. 2007).
Biological roles for decidual PRL family hormones/cytokines are not well understood (Jabbour & Critchley 2001). Among the decidual PRL family paralogs in the mouse and rat is a protein referred to as PRL family 8, subfamily a, member 2 (PRL8A2; also referred to as decidual PRL-related protein dPRP; Roby et al. 1993). PRL8A2 is abundantly expressed in the uterine decidua (Roby et al. 1993, Gu et al. 1994, Rasmussen et al. 1996, 1997, Lin et al. 1997, Orwig et al. 1997a,b,c, Orwig & Soares 1999, Bany & Cross 2006, Alam et al. 2008), binds to heparin and although it is structurally similar to PRL it does not appear to signal through the PRL receptor (Rasmussen et al. 1996, Wang et al. 2000, Alam et al. 2008). PRL8A2 deficiency interferes with pregnancy-dependent adaptations to hypoxia resulting in pregnancy failure (Alam et al. 2007).
The purpose of this study was to identify candidate targets for PRL8A2 action within the uteroplacental compartment.
Materials and methods
Animals and tissue preparation
C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed in an environmentally controlled facility, with lights on from 0600 to 2000 h, and allowed free access to food and water. Timed matings of animals were conducted by placing females with fertile males. The day when a seminal plug was found in the vagina of female mice was designated as day 0.5 of pregnancy. Placentation sites, including uterus, decidual and placental tissues, were dissected from pregnant animals. Harvested tissues were snap-frozen in liquid nitrogen for RNA and protein analyses. For in situ hybridization analyses, tissues were frozen in dry ice-cooled heptane. All tissue samples were stored at −80 °C until used. Protocols for the preceding procedures have been described (Ain et al. 2006, Deb et al. 2006, Alam et al. 2007, 2008). The University of Kansas Medical Center Animal Care and Use Committee approved all procedures for handling and experimentation with rodents.
WT and Prl8a2 null mice were mated and sacrificed on gestation day 7.5. Decidual–placental-embryonic tissues were dissected from implantation sites and homogenized. Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol (Invitrogen). RNA extractions were pooled to form three groups of three for each group in nuclease-free water at a concentration of 1.0 μg/μl. RNA samples were hybridized to the Affymetrix 430 2.0 DNA microarray chip using the GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA, USA). Washing and staining of the hybridized chips were conducted using the GeneChip Fluidics Station 450 (Affymetrix). Chips were scanned using the Affymetrix GeneChip Scanner 3000 (Affymetrix) with autoloader by the KUMC Biotechnology Support Facility. Hybridization signals were normalized with internal controls. Expression data sets were analyzed using the expression analysis software GeneSpring 7.0 and R Statistics Software (http://www.r-project.org/) with BioConductor Software (http://www.bioconductor.org/) packages. The RMA method from the BioConductor Software was used for background correction, normalization and summarization of the DNA microarray data. Statistical comparisons of expression values between two groups were determined with a moderated t-test. Pathway analysis was performed with AltAnalyze (http://altanalyze.org) and PathVisio (http://www.pathvisio.org).
cDNAs were synthesized with total RNA (1 μg) from each sample using M-MLV reverse transcriptase (Invitrogen), diluted five times with water and subjected to quantitative RT-PCR (qRT-PCR) to quantify mRNA levels of the genes identified from the DNA microarray. Primers were designed using Primer Express 2.0 (Applied Biosystems). Primer sequences can be found in Table 1. Real-time PCR amplification of cDNAs was carried out in a reaction mixture (10 μl) containing SYBR GREEN PCR Master Mix (Applied Biosystems) and primers (600 nM each). Amplification and fluorescence detection were carried out using the ABI Prism 7500 Real Time PCR System (Applied Biosystems). Cycling conditions included an initial hold step (95 °C for 10 min) and 40 cycles of a two-step PCR (92 °C for 15 s, then 60 °C for 1 min), followed by a dissociation step (95 °C for 15 s, 60 °C for 15 s, and then 95 °C for 15 s). qRT-PCR for each query mRNA was validated, including determining amplification efficiencies and co-linearity of the query mRNAs and 18S rRNA. The comparative CT method was used for relative quantification of the amount of mRNA for each sample normalized to 18S RNA.
Primer sequences for transcripts regulated by PRL8A2.
|Gene||GenBank accession no.||Forward primer||Reverse primer|
In situ hybridization
The localization of mRNAs within tissues was performed as described previously (Ain et al. 2003, Wiemers et al. 2003). Cryosections (10 μm) of tissues were prepared and stored at −80 °C until used. Plasmids containing cDNAs for mouse Rimklb, Derl3, Hspa5 and Hsp90b1 were used as templates to synthesize sense and antisense digoxigenin labeled riboprobes according to the manufacturer's instructions (Roche Molecular Biochemicals). Images were captured using a Leica MZFIII stereomicroscope (Leica Microsystems GmbH, Welzlar, Germany) or a Nikon Eclipse 55i microscope (Nikon Instruments, Inc., Melville, NY, USA), both equipped with Leica CCD cameras (Leica).
Statistical analyses were performed using the R Statistical Software (http://www.r-project.org). Statistical comparisons between two means were determined with Student's t-test or Welch's t-test, depending on the homogeneity of variances.
Mice possessing null mutations at the Prl8a2 locus reproduce within the normal range but unlike WT mice do not effectively adapt when exposed to hypoxic conditions during pregnancy (Alam et al. 2007). This mutant mouse model was used as a tool to identify downstream actions of PRL8A2 signaling. We used DNA microarray analysis to examine the consequences of PRL8A2 deficiency on gene expression at gestation day 7.5. Gestation day 7.5 is associated with robust Prl8a2 expression and represents a pivotal time point in decidual development and the establishment of the placenta. Probe sets for 57 transcripts exhibited a greater than or equal to twofold change in expression between Prl8a2 null and WT tissues. Thirty-four transcripts were significantly downregulated and 23 transcripts were significantly upregulated in the Prl8a2 null tissues (P<0.05, Tables 2 and 3). The complete dataset has been deposited at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE60220). Pathway analyses of the transcriptome data were not informative.
List of transcripts downregulated (greater than or equal to twofold) in implantation sites of the PRL8A2 deficient mouse.
|Gene name||Symbol||GenBank accession no.||Function||Ratio (null/WT)|
|Prolactin family 8, subfamily A, member 2||Prl8a2||Hormone/cytokine||0.00|
|Ribosomal modification protein rimK-like family member B||Rimklb||ATP binding, amino acid ligase activity, glutathione synthase activity||0.10|
|Kallikrein related peptidase 7||Klk7||Trypsin-like serine protease||0.22|
|Midline 1||Mid1||Microtubule associated||0.25|
|Chemokine (C-C motif) ligand 27a||Ccl27a||Chemokine, leukocyte recruitment||0.28|
|Predicted gene, EG633640||EG633640||Unknown||0.28|
|Proline rich 9||A030004J04Rik||Unknown||0.30|
|Orosomucoid 1||Orm1||Transporter activity/immune-related||0.30|
|Calmodulin 4||Calm4||Calcium signaling||0.35|
|Porcupine homolog||Porcn||Wnt signaling pathway||0.35|
|Predicted gene 9780||MGI:3710532||Unknown||0.36|
|Expressed sequence tag||–||Unknown||0.40|
|Orosomucoid 2||Orm2||Transporter activity/immune-related||0.40|
|Cellular retinoic acid binding protein||Crabp2||Retinoic acid transport||0.40|
|Expressed sequence tag||–||Unknown||0.40|
|Lipase, endothelial||Lipg||Lipid metabolism||0.41|
|A disintegrin-like and metalloproteinase with thrombospondin type 1 motif, 5||Adamts5||Integrin-mediated signaling, metalloproteinase||0.41|
|Prolactin family 4, subfamily A, member 1||Prl4a1||Hormone/cytokine||0.42|
|HtrA serine peptidase 3||Htra3||Serine protease||0.43|
|Expressed sequence tag||–||Unknown||0.43|
|Small proline-rich protein 2H||Sprr2h||Epithelial barrier||0.43|
|Neuromedin U||Nmu||Neuropeptide signaling||0.43|
|PR domain containing 16||Prdm16||Transcription coregulator||0.44|
|Endogenous retroviral sequence 3||Erv3||Unknown||0.44|
|Carcinoembryonic antigen-related cell adhesion molecule 9||Ceacam9||Immune-related||0.44|
|Expressed sequence tag||–||Unknown||0.45|
|Expressed sequence tag||–||Unknown||0.45|
|Guanylate cyclase activator 2a||Guca2a||Activator of guanylate cyclase||0.45|
|Calmodulin-like 3||Calml3||Calcium signaling||0.46|
|Shisa homolog 3||Shisa3||FGF and WNT signaling||0.46|
|Histidine ammonia lyase||Hal||Histidine catabolism||0.46|
|LRRN4 C-terminal like||Lrrn4cl||Unknown||0.46|
|Predicted gene 9746||D14Ertd449e||Unknown||0.48|
|Rac/Cdc42 guanine nucleotide exchange factor 6||Arhgef6||Rho GTPase guanine nucleotide exchange factor||0.50|
List of transcripts upregulated (greater than or equal to twofold) in implantation sites of the PRL8A2 deficient mouse.
|Gene name||Symbol||GenBank accession no.||Function||Ratio (null/WT)|
|Platelet-derived growth factor receptor-like||Pdgfrl||Similarity to ligand binding domain of Pdgfr||11.48|
|Der1-like domain family, member 3||Derl3||Endoplasmic reticulum stress response||6.29|
|Expressed sequence tag||–||Unknown||4.65|
|SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1||Smarcb1||Chromatin remodeling||3.84|
|Nicotinamide nucleotide transhydrogenase||Nnt||Mitochondrial enzyme, production of NADPH||3.20|
|CDC14 cell division cycle 14 homolog B||Cdc14b||Protein tyrosine phosphatase, cell cycle control||3.10|
|Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1||Herpud1||Endoplasmic reticulum stress response||3.00|
|EF-hand calcium binding domain 7||Efcab7||Calcium binding||2.91|
|Expressed sequence tag||–||Unknown||2.84|
|Cysteine-rich with EGF-like domains 2||Creld2||Endoplasmic reticulum stress response/calcium binding||2.75|
|Expressed sequence tag||–||Unknown||2.69|
|Rab9 effector protein with kelch motifs||Rabepk||Facilitates transport of mannose 6-phosphate receptor||2.60|
|Arrestin domain containing 3||Arrdc3||Associated with G protein-coupled receptor signaling||2.44|
|γ-aminobutyric acid A receptor, subunit α 2||Gabra2||GABA-A receptor, ligand-gated chloride channel||2.39|
|Immunoglobulin κ constant||Igkc||Light chain of antibodies||2.38|
|Predicted gene, EG665955||EG665955||Unknown||2.23|
|DNA segment, Chr 13, ERATO Doi 666, expressed||D13Ertd666e||Unknown||2.17|
|DNA-damage inducible transcript 3||Ddit3||Endoplasmic reticulum stress response||2.13|
|Heat shock protein 5||Hspa5||Endoplasmic reticulum stress response||2.07|
|Uroplakin 1B||Upk1b||Member of the tetraspanin family, signal transduction||2.06|
|Expressed sequence tag||–||Unknown||2.04|
|Heat shock protein 90, β, member 1||Hsp90b1||Endoplasmic reticulum stress response||2.00|
Nine genes were confirmed with decreased expression in gestation day 7.5 Prl8a2 null implantation sites (e.g. Klk7, Rimklb, Ccl27, Calm4, Prl4a1, Lipg, Sprr2h, Htra3, Arhgef6; Table 2, Fig. 1). These include potential decidual, endothelial and trophoblast cell targets positively regulated by PRL8A2. Rmklb transcripts were localized to a subset of cells within the anti-mesometrial decidual compartment of the gestation day 7.5 implantation site (Fig. 2).
Six genes were confirmed with increased expression in gestation day 7.5 Prl8a2 null implantation sites (Derl3, Herpud1, Creld2, Hsp90b1, Ddit3, and Hspa5; Table 3, Fig. 3). Each of these transcripts encodes proteins that participate in the endoplasmic reticulum (ER) stress response. Derl3, Hspa5, and Hsp90b1 transcripts were localized to the anti-mesometrial decidual compartment and were dramatically upregulated in the Prl8a2 null mouse (Fig. 4).
The uterine deciduum is a transitory tissue with the responsibilities of modulating hemochorial placentation. A PRL-related cytokine, PRL8A2, is expressed in a temporally- and spatially-precise pattern within the uterine deciduum during the establishment of pregnancy. PRL8A2 facilitates pregnancy-associated uterine adaptations to physiological stressors (Alam et al. 2007). In this report, we identified potential targets of PRL8A2 action and determined that PRL8A2 acts in a pathway that restrains activation of decidual cell ER stress.
The ER stress response is a cellular process facilitating adaptations to harmful conditions, including cellular damage, and if severe or prolonged leads to cell death (Xu et al. 2005, Yoshida 2007, Zhang & Kaufman 2008). Implantation of the embryo within the uterus elicits many of the hallmarks of an inflammatory response (Finn 1986, Mor et al. 2011). Inflammation leads to cellular injury and activation of ER stress (Zhang & Kaufman 2008). An assortment of pregnancy-related disorders, including early pregnancy loss, preeclampsia and intrauterine growth restriction, are associated with increased decidual cell ER stress responses (Lian et al. 2011, Liu et al. 2011, Loset et al. 2011, Gao et al. 2012). Pregnancy related disease occurs when the harmful inflammatory stimuli are excessive or the decidual cell adaptations are inadequate. Consequently, during the establishment of a successful pregnancy mechanisms must exist to thwart excessive or prolonged ER stress responses, which could compromise embryo survival.
The PRL family is part of a conserved decidual cell adaptation regulatory pathway. PRL and a subgroup of PRL related genes are expressed in decidua cells of the rat, mouse, and human (Orwig et al. 1997a, Telgmann & Gellersen 1998). PRL has a decidua-protective role in the rat and mouse. It inhibits the expression of decidual genes that interfere with the maintenance of pregnancy (Tessier et al. 2001, Bao et al. 2007). Complementary observations are apparent in the human. PRL is produced by decidua and its production is impaired in decidua from patients with recurrent pregnancy loss (Salker et al. 2010, Teklenburg et al. 2010a,b) and correlates with failures in optimal embryo recognition (Brosens & Gellersen 2010, Weimar et al. 2012). PRL8A2, a PRL-related protein, is abundantly expressed in decidua of the mouse and rat, especially within anti-mesometrial decidua (Orwig et al. 1997a,b,c, Rasmussen et al. 1997). In the absence of PRL8A2, transcripts associated with ER stress are significantly upregulated in the anti-mesometrial decidua. Insights into the mechanism of PRL8A2 action are modest. PRL8A2 is a secreted heparin-binding cytokine (Rasmussen et al. 1996, Wang et al. 2000, Alam et al. 2008). Although PRL8A2 is structurally related to PRL, it does not bind the PRL receptor (Rasmussen et al. 1996). Collectively, the results suggest that the decidua-protective functions associated with PRL may extend to other members of the PRL family, including PRL8A2.
DDIT3 is a component of the ER stress response targeted by PRL8A2 and may represent a critical modulator of the integrity of decidual cells. DDIT3 is also known as CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and is a negative modulator of C/EBP transcriptional regulation (Ron & Habener 1992, Tang & Lane 2000). C/EBPβ is a key transcriptional mediator of the actions of progesterone on decidual cell differentiation (Bagchi et al. 2006, Mantena et al. 2006, Wang et al. 2010, Ramathal et al. 2011). Uterine stromal cells of C/EBPβ null female mice fail to undergo decidualization and are unresponsive to the actions of progesterone (Bagchi et al. 2006, Mantena et al. 2006). Progesterone signaling and C/EBPβ also synergize in the differentiation of primate endometrial stromal cells to decidual cells (Pohnke et al. 1999, Christian et al. 2002a,b, Kannan et al. 2010). This leads us to speculate that by restraining DDIT3 expression, PRL8A2 effectively facilitates the actions of progesterone and C/EBPβ on decidual cell development and integrity.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This work was supported in part by research grants from the National Institutes of Health (HD020676, HD055523, HD066406).
ChristianMPohnkeYKempfRGellersenBBrosensJJ2002aFunctional association of PR and CCAAT/enhancer-binding protein β isoforms: promoter-dependent cooperation between PR-B and liver-enriched inhibitory protein, or liver-enriched activatory protein and PR-A in human endometrial stromal cells. Molecular Endocrinology16141–154. (doi:10.1210/mend.16.1.0763)
ChristianMZhangXSchneider-MerckTUntermanTGGellersenBWhiteJOBrosensJJ2002bCyclic AMP-induced forkhead transcription factor, FKHR, cooperates with CCAAT/enhancer-binding protein β in differentiating human endometrial stromal cells. Journal of Biological Chemistry27720825–20832. (doi:10.1074/jbc.M201018200)
LianIALosetMMundalSBFenstadMHJohnsonMPEideIPBjorgeLFreedKAMosesEKAustgulenR2011Increased endoplasmic reticulum stress in decidual tissue from pregnancies complicated by fetal growth restriction with and without pre-eclampsia. Placenta32823–829. (doi:10.1016/j.placenta.2011.08.005)
LiuA-XHeW-HYinL-JLvP-PZhangYShengJ-ZLeungPCKHuangH-F2011Sustained endoplasmic stress as a cofactor of oxidative stress in decidual cells from patients with early pregnancy loss. Journal of Clinical Endocrinology and Metabolism96E493–E497. (doi:10.1210/jc.2010-2192)
RamathalCWangWHuntEBagchiICBagchiMK2011Transcription factor CCAAT enhancer-binding protein β (C/EBPβ) regulates the formation of a unique extracellular matrix that controls uterine stromal differentiation and embryo implantation. Journal of Biological Chemistry28619860–19871. (doi:10.1074/jbc.M110.191759)
SalkerMTeklenburgGMolokhiaMLaverySTrewGAojanepongTMardonHJLokugamageAURaiRLandlesC2010Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss. PLoS ONE5e10287. (doi:10.1371/journal.pone.0010287)
TeklenburgGSalkerMMolokhiaMLaverySTrewGAojanepongTMardonHJLokugamageAURaiRLandlesC2010bNatural selection of human embryos: decidualizing endometrial stromals cells serve as sensors of embryo quality upon implantation. PLoS ONE5e10258. (doi:10.1371/journal.pone.0010258)
S M K Alam is now at Department of Biochemistry, Bagabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh
T Konno is now at Department of Agro-Environmental Sciences, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan