Pregnancy-associated Plasma protein-A mRNA Research Articles

The expression of pregnancy-associated plasma protein-A (PAPP-A) in human blastocoel fluid\u2013conditioned media: a proof of concept study

PurposeThe aim of this study was to determine if pregnancy-associated plasma protein-A (PAPP-A), typically measured in maternal serum and a potential predictor of adverse maternal and fetal outcomes such as spontaneous miscarriage, pre-eclampsia, and stillbirth, is expressed in blastocoel fluid–conditioned media (BFCM) at the embryonic blastocyst stage.DesignThis is an in vitro study.MethodsBFCM samples from trophectoderm-tested euploid blastocysts (n = 80) from in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) patients were analyzed for PAPP-A mRNA. BFCM was obtained from blastocyst stage embryos in 20 uL drops. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy prior to blastocyst vitrification and BFCM collection for snap freezing. cfDNA was synthesized using BFCM collected from 80 individual euploid blastocysts. Next, real-time qPCR was performed to detect expression of PAPP-A with GAPDH for normalization of expression in each sample.ResultsPAPP-A mRNA was detected in 45 of 80 BFCM samples (56.3%), with varying levels of expression across samples.ConclusionOur study demonstrates the expression of PAPP-A in BFCM. To our knowledge, this is the first study to report detection of PAPP-A mRNA in BFCM. Further studies are required and underway to investigate a greater number of BFCM samples as well as the possible correlation of PAPP-A expression with pregnancy outcomes of transferred euploid blastocysts. If found to predict IVF and obstetric outcomes, PAPP-A may provide additional information along with embryonic euploidy for the selection of the optimal blastocyst for embryo transfer.

Identification of Urothelial Cancer-Associated 1 (UCA1) as a diagnostic biomarker of pre-eclampsia via regulating microRNA-16 and its downstream signaling pathway

IntroductionIn this study, we studied the clinical value of using urothelial cancer-associated 1 (UCA1) and microRNA-16 (miR-16) as biomarkers for the diagnosis of pre-eclampsia (PE). Also, we compared the diagnostic values of miR-16, UCA1 and pregnancy-associated plasma protein-A (PAPP-A) in PE. Furthermore, we investigated the interaction between miR-16 and UCA1/PAPP-A.Material and methods128 PE patients and 172 healthy pregnant women were enrolled in this study. Receiver operating characteristic (ROC) analysis was carried out to predict the diagnostic values of UCA1, miR-16 and PAPP-A in PE. Enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (PCR), Western blot analysis, immunohistochemistry (IHC) assay, computational analysis, and luciferase assay were conducted to measure the differential expression of UCA1, miR-16, and PAPP-A while establishing a signaling pathway of UCA1/miR-16/PAPP-A.ResultsCompared with miR-16 and PAPP-A, UCA1 exhibited a better value in the diagnosis of PE. The expression of PAPP-A and UCA1 was down-regulated while the expression of miR-16 was up-regulated in patients with PE, especially in patients with HELLP pregnancies. Moreover, UCA1 was identified as a sponge of miR-16, while PAPP-A mRNA was identified as a virtual target gene of miR-16. Finally, a negative regulatory relationship was observed between the expression of miR-16 and UCA1 or PAPP-A, while the expression of UCA1 and PAPP-A were positively related.ConclusionsTaken together, UCA1 could be used as a more valuable biomarker in the diagnosis of PE. Meanwhile, the reduced expression of UCA1 could exert a positive effect by reducing the expression of PAPP-A in the pathogenesis of PE.

The potential role of pregnancy-associated plasma protein-A2 in angiogenesis and development of preeclampsia.

Compromised placentation strongly predisposes to preeclampsia (PE) which is a severe complication of pregnancy. Pregnancy-associated plasma protein-A2 (PAPP-A2) has higher expression in the placenta than in any other tissues. However, the possible role of PAPP-A2 in placental development and in the pathogenesis of PE remains unclear. In this study, we aimed at exploring placental expression of PAPP-A2 in early- and late-onset of severe PE and its role in the mechanism inducing the development of PE. We found that expression of PAPP-A2 mRNA and protein was elevated in placentas from women with severe PE compared to control placentas and was localized to differentiated trophoblasts; higher in early-onset PE than that in late-onset PE. PAPP-A2 was expressed in the cytoplasm of both primary trophoblasts and HTR-8/SVneo cells. Elevated PAPP-A2 attenuated migration, invasion, explant outgrowth and network formation of trophoblast cells in vitro without affecting cell proliferation and apoptosis. PAPP-A2 attenuated trophoblast invasion and migration by restraining epithelial-mesenchymal translations via downregulation of the Hedgehog signaling pathway. Overall, the increased expression of placental PAPP-A2 is specific to different period of PE onset and PAPP-A2 may contribute to poor placentation and inadequate angiogenesis thereby leading to the development of preeclampsia.

Differential expression of insulin-like growth factor family members in immature cumulus-oocyte complexes from dairy cows with different genotypes.

It has been evident the improvement of in vitro embryo production (IVEP) in dairy cows. Nevertheless, it is known that differences in the number and quality of oocytes between taurine and zebu females impact the efficiency and economic viability of IVEP. As the insulin-like growth factor (IGF) system is related to follicular and oocyte development, we aimed to quantify mRNA abundance of IGF system members and pregnancy-associated plasma protein-A (PAPPA) in the cumulus-oocyte complexes (COCs) of Gir, 1/2 Holstein×1/2 Gir and Holstein cows. Four pools of 30 immature COCs from Gir, 1/2 Holstein×1/2 Gir and Holstein cows were obtained by ovum pickup (OPU), and the oocytes and cumulus cells (CC) were mechanically separated and stored at -80°C. Total RNA was extracted from pools of 30 oocytes and their respective CC. Expression of target genes was assessed by real-time RT-PCR. In oocytes, the abundance of IGFR1 mRNA was higher (p<.05) in Gir cows compared with the other breeds. In contrast, in CC, mRNA encoding IGF2 (p<.05), IGFR2 (p<.05) and IGFBP4 (p<.01) was higher in Holstein donors compared with Gir and 1/2 Holstein×1/2 Gir cows. Additionally, the abundance of PAPPA mRNA was higher in oocytes (p<.001) and CC (p<.01) in Gir and 1/2 Holstein×1/2 Gir cows compared with the Holstein donors. In conclusion, the higher abundance of PAPPA mRNA in the oocytes and CC from Gir and cross-breed donors combined with the low expression of IGFBP4 in the CC suggests an enhancement of the bioavailability of IGF-free when compared with Holstein COCs.

Pregnancy-associated plasma protein-A modulates the anabolic effects of parathyroid hormone in mouse bone

Intermittent parathyroid hormone (PTH) is a potent anabolic therapy for bone, and several studies have implicated local insulin-like growth factor (IGF) signaling in mediating this effect. The IGF system is complex and includes ligands and receptors, as well as IGF binding proteins (IGFBPs) and IGFBP proteases. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease expressed by osteoblasts in vitro that has been shown to enhance local IGF action through cleavage of inhibitory IGFBP-4. This study was set up to test two specific hypotheses: 1) Intermittent PTH treatment increases the expression of IGF-I, IGFBP-4 and PAPP-A in bone in vivo, thereby increasing local IGF activity. 2) In the absence of PAPP-A, local IGF activity and the anabolic effects of PTH on bone are reduced. Wild-type (WT) and PAPP-A knock-out (KO) mice were treated with 80μg/kg human PTH 1-34 or vehicle by subcutaneous injection five days per week for six weeks. IGF-I, IGFBP-4 and PAPP-A mRNA expression in bone were significantly increased in response to PTH treatment. PTH treatment of WT mice, but not PAPP-A KO mice, significantly increased expression of an IGF-responsive gene. Bone mineral density (BMD), as measured by DEXA, was significantly decreased in femurs of PAPP-A KO compared to WT mice with PTH treatment. Volumetric BMD, as measured by pQCT, was significantly decreased in femoral midshaft (primarily cortical bone), but not metaphysis (primarily trabecular bone), of PAPP-A KO compared to WT mice with PTH treatment. These data suggest that stimulation of PAPP-A expression by intermittent PTH treatment contributes to PTH bone anabolism in mice.

Involvement of PAPP-A and IGFR1 in Cystic Ovarian Disease in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.

A novel neutralizing antibody targeting pregnancy-associated plasma protein-a inhibits ovarian cancer growth and ascites accumulation in patient mouse tumorgrafts.

The majority of ovarian cancer patients acquire resistance to standard platinum chemotherapy and novel therapies to reduce tumor burden and ascites accumulation are needed. Pregnancy-associated plasma protein-A (PAPP-A) plays a key role in promoting insulin-like growth factor (IGF) pathway activity, which directly correlates to ovarian cancer cell transformation, growth, and invasiveness. Herein, we evaluate PAPP-A expression in tumors and ascites of women with ovarian cancer, and determine the antitumor efficacy of a neutralizing monoclonal PAPP-A antibody (mAb-PA) in ovarian cancer using primary patient ovarian tumorgrafts ("Ovatars"). PAPP-A mRNA expression in patient ovarian tumors correlated with poor outcome and was validated as a prognostic surrogate in Ovatar tumors. Following confirmation of mAb-PA bioavailability and target efficacy in vivo, the antitumor efficacy of mAb-PA in multiple Ovatar tumor models was examined and the response was found to depend on PAPP-A expression. Strikingly, the addition of mAb-PA to standard platinum chemotherapy effectively sensitized platinum-resistant Ovatar tumors. PAPP-A protein in ascites was also assessed in a large cohort of patients and very high levels were evident across the entire sample set. Therefore, we evaluated targeted PAPP-A inhibition as a novel approach to managing ovarian ascites, and found that mAb-PA inhibited the development, attenuated the progression, and induced the regression of Ovatar ascites. Together, these data indicate PAPP-A as a potential palliative and adjunct therapeutic target for women with ovarian cancer.

LBA1 - A Novel Neutralizing Antibody Targeting PAPP-A Inhibits Tumor Growth and Ascites in Primary Patient Ovarian Tumorgrafts (Ovatars)

ABSTRACT The majority of ovarian cancer patients acquire resistance to standard platinum chemotherapy and novel therapies to reduce tumor burden and ascites accumulation are needed. Pregnancy-associated plasma protein-A (PAPP-A) plays a key role in promoting insulin-like growth factor (IGF) pathway activity, which directly correlates to ovarian cancer cell transformation, growth and invasiveness. Herein, we evaluate PAPP-A expression in tumors and ascites of women with ovarian cancer, and determine the anti-tumor efficacy of a neutralizing monoclonal PAPP-A antibody (mAb-PA) in ovarian cancer using primary patient ovarian tumorgrafts (“Ovatars”). PAPP-A mRNA expression in patient ovarian tumors correlated with poor outcome and was validated as a prognostic surrogate in Ovatar tumors. Following confirmation of mAb-PA bioavailability and target efficacy in vivo, the anti-tumor efficacy of mAb-PA in multiple Ovatar tumor models was examined and the response was found to depend on PAPP-A expression. Strikingly, the addition of mAb-PA to standard platinum chemotherapy effectively sensitized platinum-resistant Ovatar tumors. PAPP-A protein in ascites was also assessed in a large cohort of patients and very high levels were evident across the entire sample set. Therefore, we evaluated targeted PAPP-A inhibition as a novel approach to managing ovarian ascites, and found that mAb-PA inhibited the development, attenuated the progression and induced the regression of Ovatar ascites. Together, these data indicate PAPP-A as a potential palliative and adjunct therapeutic target for women with ovarian cancer.

Tissue-specific changes in pregnancy associated plasma protein-A expression with age in mice

Pregnancy-associated plasma protein-A (PAPP-A) is a novel zinc metalloproteinase that functions in many systems outside of pregnancy. Data in both humans and mice suggest a role for PAPP-A in aging and age-related diseases. However, our knowledge of tissue-specific PAPP-A expression and possible changes in this expression with age is limited. Thus, the aim of this study was to determine PAPP-A mRNA expression in multiple tissues with age in both male and female mice using real-time PCR. These included the heart, liver, kidney, bone, fat, skeletal muscle, gonads, brain, thymus and spleen. In young mice, PAPP-A mRNA was expressed at relatively high levels in all tissues examined except for liver. The only difference in expression between males and females was seen in the kidney, subcutaneous fat and gonads. The highest PAPP-A mRNA expression levels were found in visceral fat and these were 10-fold higher than in subcutaneous fat. PAPP-A expression significantly increased with age in kidney, brain and gonads. PAPP-A expression significantly deceased with age in bone and skeletal muscle. In the thymus, PAPP-A mRNA showed a biphasic response with age. There were no age-related changes in PAPP-A expression seen in any of the other tissues examined. Expression of IGFBP-5 mRNA, a marker of insulin-like growth factor-I (IGF-I) bioactivity known to be regulated by PAPP-A, paralleled the changes in PAPP-A expression with age in kidney, bone, skeletal muscle and thymus. Thus, tissue-specific PAPP-A expression in mice is differentially affected during aging, and may regulate local IGF-I bioactivity in certain tissues.

Preferential expression of PAPPA in human preadipocytes from omental fat

Fat distribution differs between individuals, and those with visceral fat predominance develop metabolic profiles that increase the risk of adverse cardiovascular events. This is due, in part, to the proinflammatory state associated with visceral obesity as well as depot-specific adipogenesis. The IGF system is important in adipose tissue development and metabolic function. Pregnancy-associated plasma protein A (PAPPA) is a novel zinc metalloproteinase that regulates local IGF availability. The first aim of this study was to characterize PAPPA mRNA and protein expression in primary cultures of human preadipocytes isolated from omental, mesenteric, and subcutaneous depots. PAPPA expression was significantly increased in omental preadipocytes compared with mesenteric and subcutaneous preadipocytes. The second aim of this study was to investigate the factors regulating PAPPA expression, focusing on proinflammatory cytokines and resveratrol that have been shown to have negative and positive effects, respectively, on metabolism and diet-induced obesity. Treatment of cultured primary human preadipocytes with tumor necrosis factor α and interleukin 1β led to significant increases in PAPPA expression. Activated pathways mediating cytokine-induced PAPPA expression include the nuclear factor κB pathway and the MAPK family, particularly c-Jun NH2-terminal kinase and p38 MAPK. Resveratrol, a polyphenolic compound with beneficial cardiometabolic effects, significantly downregulated PAPPA expression under basal and stimulated conditions. Effects of resveratrol on PAPPA appeared to be mediated through pathways independent of silent mating type information regulation 2 homolog 1 (SIRT1) and AMP kinase activation. Depot-specific PAPPA expression in human preadipocytes may contribute to a depot-specific function.

Preferential impact of pregnancy-associated plasma protein-A deficiency on visceral fat in mice on high-fat diet

Accumulation of visceral fat, more so than subcutaneous fat, is strongly associated with severe metabolic complications. However, the factors regulating depot-specific adipogenesis are poorly understood. In this study, we show differential expression of pregnancy-associated plasma protein-A (PAPP-A), a secreted regulator of local insulin-like growth factor (IGF) action, in adipose tissue of mice. PAPP-A mRNA expression was fivefold higher in visceral (mesenteric) fat compared with subcutaneous (inguinal, subscapular), perirenal, and brown fat of mice. To investigate the possible role of depot-specific PAPP-A expression in fat accumulation, wild-type (WT) and PAPP-A knockout (KO) mice were fed a high-fat diet (HFD) for up to 20 wk. Adipocyte size increased in subcutaneous and perirenal depots similarly in WT and PAPP-A KO mice. However, fat cell size and in vivo lipid uptake were significantly reduced in mesenteric fat of PAPP-A KO compared with WT mice. After 20 wk on HFD, phosphorylation of AKT, a downstream signaling intermediate of IGF-I and insulin receptor activation, was significantly decreased by 50% in mesenteric compared with subcutaneous fat in WT mice, but was significantly increased threefold in mesenteric compared with subcutaneous fat in PAPP-A KO mice. This appeared to be because of enhanced insulin-stimulated signaling in mesenteric fat of PAPP-A KO mice. These data establish fat depot-specific expression of PAPP-A and indicate preferential impact of PAPP-A deficiency on visceral fat in the mouse that is associated with enhanced insulin receptor signaling. Thus, PAPP-A may be a potential target for treatment and/or prevention strategies for visceral obesity and related morbidities.

A correlation analysis between the expression of pregnancy-associated plasma protein A in basal decidual cells and recurrent spontaneous abortion

The aim of this study was to investigate the correlation between the expression of pregnancy-associated plasma protein A (PAPP-A) in basal decidual cells and recurrent spontaneous abortion (RSA). A total of 39 patients with a history of RSA were enrolled into the RSA group. A further 30 females who had experienced normal pregnancy were enrolled into the control group. The mRNA expression of PAPP-A in basal decidual cells was analyzed using real-time PCR. The distribution and expression of PAPP-A protein levels in basal decidual cells were analyzed by immunohistochemistry. The correlation between PAPP-A protein levels and RSA was analyzed. The levels of PAPP-A mRNA in the RSA group were significantly decreased, compared with the control group (P<0.05). Consistent with the mRNA levels, the protein levels of PAPP-A were also significantly lower in the RSA group compared with the control group (P<0.05). Multivariate logistic analysis indicated that the suppression of PAPP-A was one of the risk factors for RSA. Furthermore, Hosmer-Lemeshow analysis suggested that the expression levels of PAPP-A is an important factor for predicting RSA. In conclusion, the expression levels of the PAPP-A protein were significantly reduced in basal decidual cells of the RSA group compared with the control group. Therefore, PAPP-A is likely to play an important role in RSA.

PAPP-A in Indian Water Buffalo (Bubalus bubalis) Ovary: Phylogeny, Expression, Hormonal Regulation, and Sequence Characterization

The insulin-like growth factor binding proteins and pregnancy-associated plasma protein-A (PAPP-A) within the future dominant follicle is a critical part of selection of one follicle. This study was aimed to find out the expression and hormonal regulation of PAPP-A mRNAs in the Indian water buffalo ovary. The results showed stage-specific expression of PAPP-A in the whole follicles and postovulatory structures, corpus luteum and corpus albicans. Expression of PAPP-A mRNA was found to be up regulated in corpus luteum. The 341 nucleotide partial cDNA sequence of buffalo PAPP-A was determined. Sequence analyses have revealed that bubaline PAPP-A belongs to Zinc-metzincin superfamily (zinc-dependent metalloprotease) and pappalysin-like subfamily. It also confirmed the stimulatory effect of follicle-stimulating hormone on PAPP-A mRNA expression. The PAPP-A sequences showed more than 90 % homology with reported sequences of cattle, sheep, pig, and human. The study was an attempt in understanding local regulatory mechanism of follicular selection and development in buffalo ovary.

PAPPA2 is increased in severe early onset pre-eclampsia and upregulated with hypoxia

Severe early onset pre-eclampsia is a serious pregnancy complication, believed to arise as a result of persistent placental hypoxia due to impaired placentation. Pregnancy-associated plasma protein A2 (PAPPA2) is very highly expressed in the placenta relative to all other tissues. There is some evidence that PAPPA2 mRNA and protein are increased in association with pre-eclampsia. The aim of the present study was to characterise the mRNA and protein expression, as well as localisation, of PAPPA2 in an independent cohort of severe early onset pre-eclamptic placentas. We also examined whether exposing placental explants to hypoxia (1% oxygen) changed the expression of PAPPA2. Expression of PAPPA2 mRNA and protein was upregulated in severe early onset pre-eclamptic placentas compared with preterm controls and localised to the syncytiotrophoblast. Interestingly, protein localisation was markedly reduced in term placenta. Syncytialisation of BeWo cells did not change PAPPA2 expression. However, hypoxia upregulated PAPPA2 mRNA and protein expression in primary placental explants. Together, our data suggest that PAPPA2 may be upregulated in severe pre-eclampsia and, functionally, this may be mediated via increased placental hypoxia known to occur with this pregnancy disorder.

Constitutive expression of pregnancy-associated plasma protein-A in arterial smooth muscle reduces the vascular response to injury in vivo

Pregnancy-associated plasma protein-A (PAPP-A) functions to increase local IGF-I bioactivity. In this study, we used transgenic mice that constitutively express human PAPP-A in arterial smooth muscle to test the hypothesis that overexpression of PAPP-A enhances vascular smooth muscle cell (SMC) response to IGF-I in vivo. PAPP-A transgenic (Tg) and wild-type (WT) mice underwent unilateral carotid ligation, a model of injury-induced SMC hyperplasia and neointimal formation. In both WT and PAPP-A Tg mice, endogenous PAPP-A mRNA expression showed peak elevation 5 days after carotid ligation. However, PAPP-A Tg mice had 70-75% less neointima than WT at 5 and 10 days postligation, with a significant reduction in occlusion of the ligated artery. WT and PAPP-A Tg mice had equivalent increases in medial area and vessel remodeling postligation. There was little change in medial area and no evidence of neointima in the contralateral carotid of WT or PAPP-A Tg mice. Both WT and PAPP-A Tg carotids exhibited signs of dedifferentiation of SMC, which precedes the increase in proliferation and migration that results in neointimal formation. However, the number of proliferating cells in the media and neointima of the ligated PAPP-A Tg artery was reduced by 90% on day 5 postsurgery compared with WT. This decrease was associated with a significant decrease in an in vivo marker of IGF-I bioactivity and reduced IGF-I-stimulated receptor phosphorylation ex vivo. These data suggest differential effects of chronic (transgenic) and transient (endogenous) PAPP-A expression on neointimal formation following vascular injury that may be due in part to the differential impact on IGF-I signaling.

CRP and TNF-α Induce PAPP-A Expression in Human Peripheral Blood Mononuclear Cells

Objective. The effects of C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α) on pregnancy-associated plasma protein-A (PAPP-A) expression in human peripheral blood mononuclear cells (PBMCs) require further investigation. Methods. The PAPP-A levels in culture supernatants, PAPP-A mRNA expression, and cellular PAPP-A expression were measured in human PBMCs isolated from fresh blood donations provided by 6 healthy volunteers (4 donations per volunteer). Analyses were conducted by ultrasensitive ELISA, western blotting, and RT-PCR following stimulation with CRP or TNF-α cytokines. Results. PAPP-A mRNA and protein levels after CRP stimulation peaked at 24 hours, whereas peak PAPP-A mRNA and protein levels were achieved after TNF-α stimulation at only 2 and 8 hours, respectively. These findings indicate the dose-dependent effect of CRP and TNF-α stimulation. Actinomycin D treatment completely prevented CRP and TNF-α induction of PAPP-A mRNA and protein expression. Additionally, nuclear factor- (NF-) κB inhibitor (BAY11-7082) potently inhibited both CRP and TNF-α stimulated PAPP-A mRNA and protein expression. Conclusions. Human PBMCs are capable of expressing PAPP-A in vitro, expression that may be regulated by CRP and TNF-α through the NF-κB pathway. This mechanism may play a significant role in the observed increase of serum PAPP-A levels in acute coronary syndrome (ACS).

Pregnancy-Associated Plasma Protein-A2 (PAPP-A2): Tissue Expression and Biological Consequences of Gene Knockout in Mice

Pregnancy-associated plasma protein-A2 (PAPP-A2) is a novel homolog of PAPP-A in the metzincin superfamily. However, compared with the accumulating data on PAPP-A, very little is known about PAPP-A2. In this study, we determined the tissue expression pattern of PAPP-A2 mRNA in wild-type (WT) mice and characterized the phenotype of mice with global PAPP-A2 deficiency. Tissues expressing PAPP-A2 in WT mice were more limited than those expressing PAPP-A. The highest PAPP-A2 mRNA expression was found in the placenta, with abundant expression in fetal, skeletal, and reproductive tissues. Heterozygous breeding produced the expected Mendelian distribution for the pappa2 gene and viable homozygous PAPP-A2 knockout (KO) mice that were normal size at birth. The most striking phenotype of the PAPP-A2 KO mouse was postnatal growth retardation. Male and female PAPP-A2 KO mice had 10 and 25-30% lower body weight, respectively, than WT littermates. Adult femur and body length were also reduced in PAPP-A2 KO mice, but without significant effects on bone mineral density. PAPP-A2 KO mice were fertile, but with compromised fecundity. PAPP-A expression was not altered to compensate for the loss of PAPP-A2 expression, and proteolysis of PAPP-A2's primary substrate, IGF-binding protein-5, was not altered in fibroblasts from PAPP-A2 KO embryos. In conclusion, tissue expression patterns and biological consequences of gene KO indicate distinct physiological roles for PAPP-A2 and PAPP-A in mice.

Regulation of pregnancy-associated plasma protein A2 (PAPPA2) in a human placental trophoblast cell line (BeWo)

BackgroundPregnancy-associated plasma protein A2 (PAPPA2) is an insulin-like growth factor-binding protein (IGFBP) protease expressed at high levels in the placenta and upregulated in pregnancies complicated by preeclampsia and HELLP (Hemolytic anemia, Elevated Liver enzymes, and Low Platelet count) syndrome. However, it is unclear whether elevated PAPPA2 expression causes abnormal placental development, or whether upregulation compensates for placental pathology. In the present study, we investigate whether PAPPA2 expression is affected by hypoxia, oxidative stress, syncytialization factors or substances known to affect the expression of PAPPA2's paralogue, PAPPA.MethodsBeWo cells, a model of placental trophoblasts, were treated with one of the following: hypoxia (2% O2), oxidative stress (20 microM hydrogen peroxide), forskolin (10 microM and 100 microM), TGF-beta (10 and 50 ng/mL), TNF-alpha (100 ng/mL), IL-1beta (100 ng/mL) or PGE2 (1 microM). We used quantitative RT-PCR (qRT-PCR) to quantify the mRNA levels of PAPPA2, as well as those of PAPPA and ADAM12 since these proteases have similar substrates and are also highly expressed in the placenta. Where we observed significant effects on PAPPA2 mRNA levels, we tested for effects at the protein level using an in-cell Western assay.ResultsHypoxia, but not oxidative stress, caused a 47-fold increase in PAPPA2 mRNA expression, while TNF-alpha resulted in a 6-fold increase, and both of these effects were confirmed at the protein level. PGE2 resulted in a 14-fold upregulation of PAPPA2 mRNA but this was not reflected at the protein level. Forskolin, TGF-beta and IL-1beta had no significant effect on PAPPA2 mRNA expression. We observed no effects of any treatment on PAPPA or ADAM12 expression.ConclusionOur study demonstrates that factors previously known to be highly expressed in preeclamptic placentae (PGE2 and TNF-alpha), contribute to the upregulation of PAPPA2. Hypoxia, known to occur in preeclamptic placentae, also increased PAPPA2 expression. These results are consistent with the hypothesis that PAPPA2 is upregulated as a consequence of placental pathology, rather than elevated PAPPA2 levels being a cause of preeclampsia.

Plasma mRNA concentrations of placenta‐specific 1 (PLAC1) and pregnancy associated plasma protein A (PAPP‐A) are higher in early‐onset than late‐onset pre‐eclampsia

Some mRNA concentrations are observed to increase in the maternal circulation in association with pre-eclampsia, including placenta-specific 1 (PLAC1) and pregnancy associated plasma protein A (PAPP-A), which were previously proposed as predictive markers for pre-eclampsia. Here, we investigated their concentrations in early-onset and late-onset pre-eclampsia maternal plasma to determine whether different mechanisms are involved in these two forms of the disorder. Peripheral blood and placental samples were collected from patients with pre-eclampsia. RNA was extracted and levels of PLAC1 and PAPP-A mRNAs were determined using real-time quantitative PCR. PLAC1 and PAPP-A mRNA levels were significantly increased in plasma from pregnant women with pre-eclampsia compared with those from healthy pregnant women. The median concentration of PLAC1 was 5.5 times higher (P<0.01) and that of PAPP-A was 5.1 times higher (P<0.01) in early-onset than in late-onset pre-eclampsia. The expression of these mRNAs in the placenta showed no significant difference in early-onset pre-eclampsia, late-onset pre-eclampsia and healthy pregnant women. These findings suggest that the concentration of mRNAs in maternal plasma reflect leakage from damaged villus cells rather than expression levels in the placenta. Injury to chorionic villus cells might be more severe in early-onset pre-eclampsia than in late-onset pre-eclampsia.

The Role of Luteinizing Hormone in Regulating Gene Expression During Selection of a Dominant Follicle in Cattle

At approximately 8.5 mm in diameter, the future dominant follicle is "selected" for continued growth in cattle. In the present study, cows were treated with a gonadotropin-releasing hormone receptor antagonist, acyline, just before follicle selection (near 7.8 mm) to investigate the role of LH in changing mRNA concentrations during selection of a dominant follicle. The ovaries containing the expected dominant follicle (EDF; first largest follicle) and expected largest subordinate follicle (ESF) were removed after 12 or 24 h of treatment. Real-time PCR was used to determine mRNA concentrations. ELISA was used to measure testosterone and 17beta-estradiol (E(2)) and radioimmunoassay to measure androstenedione (A(4)) in follicular fluid. Concentrations of E(2) were greater in EDF than in ESF of untreated cows near the time of follicle selection (12 h) or at 12 h after selection (24 h). Testosterone, E(2), and A(4) were all dramatically decreased by acyline treatment at both times. In theca cells, acyline treatment reduced CYP17A1 (P450 17alpha) in EDF and STAR (steroidogenic acute regulatory protein) in both EDF and ESF but did not alter CYP11A1 (P450scc). In granulosa cells (GCs), LHCGR (luteinizing hormone [LH] receptor) was much greater in EDF than in ESF at both time of selection (739% greater) and 12 h after selection (2837% greater) and was decreased by acyline in EDF (87% decrease). The mRNA for CYP19A1 (cytochrome P450 aromatase) and PAPPA (pregnancy-associated plasma protein-A) tended to be greater in EDF than in ESF at follicle selection, and both mRNAs were much greater at 12 h after selection, with acyline significantly decreasing PAPPA mRNA after 24 h of treatment. The mRNA for FSHR (follicle-stimulating hormone receptor) was not different in EDF versus ESF and was not altered by acyline. Thus, induction of LHCGR mRNA in GCs is an early event during the follicle selection process, and surprisingly, expression of LHCGR mRNA is dependent on circulating LH. Production of follicular A(4), testosterone, and E(2) are also acutely related to LH but due to changes in expression of STAR and CYP17A1 in TC.