Pregnancy-associated Plasma protein-A Protein Research Articles

A common variant of the pregnancy-associated plasma protein-A (PAPPA) gene encodes a protein with reduced proteolytic activity towards IGF-binding proteins

Pregnancy-associated plasma protein-A (PAPP-A) is a key regulator of insulin-like growth factor (IGF) bioactivity, by releasing the IGFs from their corresponding IGF-binding proteins (IGFBPs). The minor allele of the single nucleotide polymorphism (SNP), rs7020782 (serine < tyrosine), in PAPPA has previously been associated with recurrent pregnancy loss as well as with significant reduced levels of PAPP-A protein in human ovarian follicles. The aim of the present study was to reveal a possible functional effect of the rs7020782 SNP in PAPPA by comparing recombinant PAPP-A proteins from transfected human embryonic kidney 293 T cells. The proteolytic cleavage of IGFBP-4 was shown to be affected by the rs7020782 SNP in PAPPA, showing a significantly reduced cleavage rate for the serine variant compared to the tyrosine variant (p-value < 0.001). The serine variant also showed a trend towards reduced cleavage rates, that was not significant, towards IGFBP-2 and IGFBP-5 compared to the tyrosine variant. No differences were found when analysing cell surface binding, complex formation between PAPP-A and STC2 or proMBP, nor when analysing STC1 inhibition of PAPP-A-mediated IGFBP-4 cleavage. Regulation of IGF bioactivity in reproductive tissues is important and the rs7020782 SNP in PAPPA may disturb this regulation by altering the specific activity of PAPP-A.

Abstract 4817: Regression of malignant ascites via PAPP-A inhibition in ovarian cancer patient-derived xenograft model

Abstract Malignant ascites is one of the most common causes of morbidity in end stage ovarian cancer patients with negative impact on quality of life. Novel non-invasive palliative therapeutic options are lacking for such patients. The zinc metalloprotease, pregnancy-associated plasma protein-A (PAPP-A), plays a key role in the insulin-like growth factor (IGF) pathway, promoting ovarian cancer cellular transformation, growth and invasiveness. Furthermore, patient primary malignant ascites is known to contain high levels of PAPP-A by ELISA. Preliminary data shows that inhibition of PAPP-A through a neutralizing monoclonal PAPP-A antibody (mAb-PA) inhibits the accumulation and promote the regression of ascites in an ovarian cancer patient-derived xenograft (PDX) models. In the current study, we investigated whether mAb-PA can promote ascites regression in an additional ovarian PDX model with measurable ascites. Patient derived ascites xenograft (PDAX) models were defined as SCID mice that developed ascites after intraperitoneal heterotransplantation of patient solid tumor collected at the time of primary cytoreduction. Ascites from these models (n=51) was screened for human PAPP-A protein by ELISA and models were divided into two groups by the relative concentration of PAPPA-A: high (n=18) and low (n=33). PAPP-A High PDAX model PH438 was re-established intraperitoneal in 20 SCID mice by ascites injection (0.1 ml per mouse). When ascites area reached a threshold of &amp;gt;0.60 cm2 by ultrasound, mice were treated with 60mg/Kg of mAb-PA (n=10) or IgG2a control (n=10) on day one and three. On day four, a second ultrasound measurement was obtained and the mice were euthanized. Ascites burden was measured at necropsy. Personnel involved with the acquisition of ultrasounds measurements, subsequent ascites harvests, and post hoc analyses were blinded to the treatments. The ratio of mean ascites (mAb-PA/IgG2a in grams) collected at necropsy was 2.04/2.51, indicating that the mAb-PA arm had less ascites compared to control. These data were consistent with pre- and post-treatment ultrasound measurements of ascites burden; a statistically significant (p=0.0283 by paired t test) reduction of ascites burden of 52.3% was observed, compared to the starting baseline, indicating that mAb-PA treatment causes ascites regression. Ascites weight at necropsy was compared to the area of greatest fluid echogenicity by ultrasound and the Pearson correlation R value achieved was of 0.859 (p&amp;lt;0.0001). These PDAX data implicate PAPP-A attenuation as a potential strategy to treat malignant ascites in OC. Additional PDAX models are under examination to confirm these findings and support the translational development of PAPP-A as a new therapeutic target for women with refractory ascites. Citation Format: Valentina Zanfagnin, Laurie K. Bale, Marc A. Becker, Xiaonan Hou, Diogo Torres, Cheryl A. Conover, Saravut J. Weroha. Regression of malignant ascites via PAPP-A inhibition in ovarian cancer patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2017-4817

Interplay between PAPP-A, inflammation and adiposity in patients with angiographically proven acute coronary syndrome (ACS).

Introduction Studies conducted in the recent past have demonstrated the role of inflammation, obesity and dysfunctional insulin signaling as contributing factors in the pathogenesis of acute coronary syndrome (ACS). However, pharmacological interventions targeting a single pathway have not proven useful in the long run. This indicates that a synergism occurs between the various risk factors and hence calls for a combinatorial approach. This study was planned to study the interplay, if any, between pregnancy associated plasma protein-A (PAPP-A), inflammation and adiposity in patients with ACS. Materials and methods The study was conducted in a tertiary care hospital in Delhi. The study population consisted of 128 subjects, divided into two groups. The control group consisted of 64 healthy subjects without ACS. Cases consisted of 64 subjects with angiographically proven ACS cases. PAPP-A and high sensitivity C-reactive protein (hs-CRP) were estimated by enzyme-linked immunosorbent assay (ELIZA) kits. Results The mean level of PAPP-A and hs-CRP were significantly higher in cases as compared to the controls. A positive correlation of PAPP-A was observed with hs-CRP, insulin, ApoB and Lp(a). The relative risk for ACS was 14.2 with a p value of <0.001 when all the three parameters - hs-CRP, PAPP-A and body mass index (BMI) were considered together. This was significantly higher when each risk factor was assessed standalone. Conclusions Our study results suggest a possible interplay between chronic inflammation, obesity and plaque instability among patients with ACS. This interaction can accelerate the process of plaque rupture in patients with increased BMI as compare to those patients with low/normal BMI.

Effect of pregnancy-associated plasma protein-A (PAPP-A) single-nucleotide polymorphisms on the level and activity of PAPP-A and the hormone profile in fluid from normal human small antral follicles.

To reveal a possible relationship between two single nucleotide polymorphisms (SNPs) in PAPP-A-1224 (rs7020782) and 327 (rs12375498)-and the level and activity of PAPP-A in follicular fluid (FF) of human small antral follicles, and to analyze the intrafollicular hormone levels. Laboratory investigation. University hospital. Fifty volunteer women who contributed a total of 210 samples of FF from normal small antral follicles. Genotyping and measurement of antigen levels of steroids, PAPP-A, stanniocalcin-2 (STC2), and antimüllerian hormone (AMH) plus activity of PAPP-A toward insulin-like growth factor binding protein 4 (IGFBP-4). Measurement of PAPP-A levels and hormones with enzyme-linked immunosorbent assay (ELISA) and PAPP-A activity toward radiolabeled IGFBP-4. Women homozygous for the minor C allele of the 1224 SNP showed a statistically significantly lower level of PAPP-A protein and activity in FF compared with women carrying the major A allele. These women also displayed nonsignificant reduced levels of estradiol and increased levels of AMH and androgen. A statistically significant correlation between FF levels of PAPP-A activity and the molar ratio of PAPP-A/STC2 was obtained. The 327 SNP did not show statistically significant associations. This study presents a statistically significant effect of the 1224 SNP on the level and activity of PAPP-A in human follicles, suggesting that the FF level of bioactive insulin-like growth factor depends on the genotype. We observed STC2 to be an important regulator of PAPP-A in human FF. The 1224 SNP has previously been associated with recurrent pregnancy loss, so further evaluation of an underlying mechanism including aberrant control of insulin-like growth factor activity is warranted.

Abstract P4-02-11: Circulating methylated DNA of pregnancy-associated plasma protein A as a clinically useful biomarker in breast cancer

Abstract Background: A recent report suggests that Pregnancy-Associated Plasma Protein A (PAPPA) plays a pivotal role in normal cell division and is targeted early in breast cancer development. Epigenetic silencing of PAPPA is highly prevalent in precursor lesions and invasive breast cancer, correlating with loss of PAPPA protein expression. In normal breast tissue PAPPA promoter methylation was not observed (Journal of Pathology 344-356: 2014). We sought to test the hypothesis that PAPPA promoter hypermethylation can be detected in cell-free tumour specific DNA (ctDNA) in the bloodstream of patients with metastatic breast cancer. Methods: Plasma derived from 12 patients with metastatic breast cancer was examined for ctDNA. A semi-quantitative real-time PCR based MethyLight assay was used for detection of PAPPA methylation. Spiking experiments with breast cancer cell lines were performed to develop the assay and confirm linearity of sodium bisulfite treatment. Results: Percentage methylated reference (PMR) values from spiking experiments with PAPPA methylated and non-methylated DNA in plasma (matrix) demonstrate good linearity for the PAPPA gene and the endogenous control Col2A. PAPPA methylation was detected in 10 out of 12 (83%) of the metastatic breast cancer samples. In stark contrast, epigenetic silencing of PAPPA was not detected in any of the plasma samples from non-cancer bearing patients (n=10). Conclusions: Given the potential clinical impact of our data, this study is being extended to include early stage breast cancer patients with poor prognostic clinic-pathological indicators. Our proof-of-concept findings indicate that testing for PAPPA promoter methylation in ctDNA may be used as a biomarker of disseminated disease, helping clinicians with treatment decisions and adjusting those choices as conditions change. By developing and refining more sensitive techniques, PAPPA methylation testing in ctDNA could also find practical utility in breast cancer screening. Citation Format: Richard Sainsbury, Karsten Koehler, Catherine Page, Saroj Velamakanni. Circulating methylated DNA of pregnancy-associated plasma protein A as a clinically useful biomarker in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-11.

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.

Pregnancy-associated plasma protein A regulates mitosis and is epigenetically silenced in breast cancer.

Aberrant mitosis is a common feature of cancer, yet little is known about the altered genes causing mitotic defects. We screened human tumours for cells with morphological signatures of highly specific mitotic defects previously assigned to candidate genes in a genome-wide RNA interference screen carried out in HeLa cells (www.mitocheck.org). We discovered a striking enrichment of early mitotic configurations indicative of prophase/prometaphase delay in breast cancer. Promoter methylation analysis of MitoCheck candidate genes assigned to the corresponding 'mitotic delay' class linked this defect to epigenetic silencing of the gene encoding pregnancy-associated plasma protein-A (PAPPA), a secreted protease. PAPPA silencing was highly prevalent in precursor lesions and invasive breast cancer. Experimental manipulation of PAPPA protein levels in human mammary epithelial cells and in breast cancer cell lines demonstrates that progression through early mitosis is dependent on PAPPA function, and that breast cancer cells become more invasive after down-regulation of this protease. PAPPA regulates mitotic progression through modulating the IGF-1 signalling pathway resulting in activation of the forkhead transcription factor FoxM1, which drives a transcriptional cluster of essential mitotic genes. Our results show that PAPPA has a critical function in normal cell division and is targeted early in breast cancer development.

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.

Abstract P3-02-01: Pregnancy-associated plasma protein A regulates mitosis and is epigenetically silenced in breast cancer

Abstract Using Phosphohistone-H3, a marker of cells in mitosis, we identified mitotic delay in 95% of invasive breast cancers with a significant proportion of cells in prophase/prometaphase (58%). This delay was seen in breast cancer tissues only with other malignancies and normal tissues showing a normal mitotic phase distribution (23% of mitotic cells in prophase/prometaphase). We could detect a clear mitotic delay phenotype already in 80% (55 out of 69 evaluable patients) of precursor ductal carcinoma in situ (DCIS) lesions. This strong phenotype has been concealed for over a century because tinctorial stains routinely used in diagnostic practice do not allow cells in prophase or prometaphase to be distinguished from interphase cells. Promoter methylation analysis of candidate genes linked this defect to epigenetic silencing of the gene encoding Pregnancy-Associated Plasma Protein-A (PAPPA), a secreted protease. PAPPA silencing was highly prevalent in precursor lesions and invasive breast cancer. Experimental manipulation of PAPPA protein levels in human mammary epithelial cells and in breast cancer cell lines demonstrates that progression through early mitosis is dependent on PAPPA function, and that breast cancer cells become more invasive after PAPPA down regulation. We discovered that PAPPA regulates mitotic progression through its known role in modulating the IGF-1 signalling pathway, resulting in activation of the forkhead transcription factor FoxM1 which drives a transcriptional cluster of essential mitotic genes. Our results identify PAPPA as a candidate tumour suppressor in breast cancer. PAPPA loss is an early event in breast tumourigenesis increasing tumour cell invasiveness and may contribute to the transition from precursor lesions to invasive disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-02-01.

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.

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 predicts outcomes of percutaneous coronary intervention in patients with non–ST-elevation acute coronary syndrome

Pregnancy-associated plasma protein A (PAPP-A) may play an important role in the development of acute coronary syndrome. This study aimed to investigate the relationship between the levels of circulating PAPP-A and the mid-term outcomes of percutaneous coronary intervention (PCI) in patients with non-ST-elevation acute coronary syndrome. The circulating PAPP-A levels and high-sensitivity C-reactive protein before PCI were measured in 129 patients with single coronary artery stenosis. The end point of clinical follow-up was cardiac death, nonfatal myocardial infarction, target vessel revascularization, and rehospitalization for angina. During the follow-up of an average of 20.3 ± 5.2 months, a cardiac event was recorded in 25 patients (19.4%). The levels of PAPP-A (29.85 ± 19.51 mIu/L vs 20.47 ± 14.33 mIu/L, P = .007) and high-sensitivity C-reactive protein (5.63 ± 2.13 mg/L vs 4.11 ± 1.28 mg/L, P = .014) in patients with cardiac events were higher than in those without cardiac events. PAPP-A ≥ 11.33 mIu/L has a strong predictive value for a combined end point (risk ratio = 4.1; 95% confidence interval, 1.0-16.2; P = .037). Patients with lower PAPP-A levels (<11.33 mIu/L) had higher event-free survivals than patients with higher PAPP-A levels (log rank = 9.334, P = .025). Circulating PAPP-A levels predict the mid-term outcomes of PCI in patients with non-ST-elevation acute coronary syndrome and single-vessel stenosis.

Transgenic overexpression of pregnancy-associated plasma protein-A in murine arterial smooth muscle accelerates atherosclerotic lesion development

Pregnancy-associated plasma protein-A (PAPP-A) increases local IGF-I bioavailability through cleavage of inhibitory IGF binding protein (IGFBP)-4 in a variety of systems, including the cardiovascular system. To test the hypothesis that expression of PAPP-A promotes the development of atherosclerotic lesions, we generated transgenic mice that express human PAPP-A in arterial smooth muscle. Four founder lines were characterized for transgenic human PAPP-A mRNA and protein expression, IGFBP-4 protease activity, and tissue specificity. In study I, apolipoprotein E knockout (ApoE KO) mice, a well-characterized mouse model of atherosclerosis, and ApoE KO mice expressing the human PAPP-A transgene at relatively high levels (ApoE KO/Tg) were fed a high-fat diet. At harvest, aortas were dissected and opened longitudinally for en face staining of lipid-rich lesions. Lesion area was increased 3.5-fold in aortas from ApoE KO/Tg compared with ApoE KO mice (P < 0.001), but no significant difference was seen in lesion number. In study II, replacement of PAPP-A expression in arterial smooth muscle of double ApoE KO/PAPP-A KO mice resulted in a 2.5-fold increase in lesion area (P = 0.002), without an effect on lesion number. PAPP-A transgene expression was associated with a significant increase in an IGF-responsive gene (P < 0.001), suggesting increased local IGF-I action. We therefore conclude that expression of human PAPP-A localized to arterial smooth muscle accelerates lesion progression in a mouse model of atherosclerosis. These data provide further evidence for the importance of PAPP-A in the cardiovascular system and suggest PAPP-A as a potential therapeutic target in the control of atherosclerosis.

Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle

To determine intrafollicular hormone levels and characterize the mRNA expression of the insulin-like growth factor (IGF) receptors, IGF binding proteins (IGFBP), and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells before and after an ovulatory hCG stimulus. Experimental animal study. Academic medical center. Adult rhesus macaques. Animals received exogenous FSH to promote the development of multiple preovulatory follicles. Follicles were aspirated before (0 hours) or 3, 6, 12, or 24 hours after an ovulatory hCG bolus. IGF1, IGF2, and insulin levels in follicular fluid were determined by radioimmunoassay. Messenger RNA (mRNA) levels in granulosa cells were determined by real-time reverse transcriptase-polymerase chain reaction. IGFBPs and PAPP-A in follicular fluid were determined by Western blot analysis and enzyme-linked immunosorbent assay. IGF1, IGF2, and insulin in follicular fluid did not change during luteinization. IGF1R, IGFBP1, and IGFBP2 mRNAs were unchanged by hCG. IGF2R, IGFBP3, -5, and -6 and PAPP-A mRNA levels increased after hCG administration, while insulin receptor and IGFBP4 mRNA levels decreased after hCG administration. IGFBP3 and -6 and PAPP-A protein increased after hCG administration. Dynamic changes in the expression of the IGFBPs and PAPP-A suggest tight regulation of IGF action during ovulation and corpus luteum formation.

Stress-Activated Signaling Pathways Mediate the Stimulation of Pregnancy-Associated Plasma Protein-A Expression in Cultured Human Fibroblasts

Pregnancy-associated plasma protein-A (PAPP-A) is an IGF binding protein protease that appears to function as a posttranslational modulator of IGF bioavailability in response to injury. A previous study indicated that the proinflammatory cytokines, TNFalpha and IL-1beta, were potent stimulators of PAPP-A expression in cultured human fibroblasts. In this study, we investigated the intracellular signaling pathways mediating cytokine-stimulated PAPP-A expression. Treatment of human fibroblasts with TNFalpha and IL-1beta (1 nm) had little or no effect on phosphatidylinositol 3-kinase and Erk1/2 activation, pathways commonly associated with proliferation. On the other hand, TNFalpha and IL-1beta induced p38, c-Jun N-terminal kinase (JNK), and nuclear factor (NF)kappaB activation, pathways more closely related to stress response. An inhibitor of p38 activation (SB203580) had no effect on TNFalpha- or IL-1beta-stimulated PAPP-A expression. The JNK inhibitor, SP600125, had no effect on IL-1beta- or TNFalpha-stimulated PAPP-A mRNA expression. However, SP600125 effectively inhibited IL-1beta-induced PAPP-A protein expression. MG-132, a proteasome inhibitor that blocked degradation of the intrinsic NFkappaB inhibitor, IkappaB, and thereby prevented NFkappaB activation, was a potent inhibitor of both TNFalpha- and IL-1beta-stimulated PAPP-A mRNA and protein expression and IGF binding protein-4 protease activity. MG-132 had no effect on JNK phosphorylation or p38 activation, and SB203580 and SP600125 had no effect on IkappaB degradation, documenting inhibitor specificity. BAY11-7082, another inhibitor of NFkappaB activation, also inhibited TNFalpha- and IL-1beta-stimulated PAPP-A expression and IGF binding protein-4 protease activity. These data indicate that NFkappaB activation is the primary mediator of cytokine-stimulated PAPP-A expression in human fibroblasts.

Pregnancy-Associated Plasma Protein-A Is Involved in Matrix Mineralization of Human Adult Mesenchymal Stem Cells and Angiogenesis in the Chick Chorioallontoic Membrane

Pregnancy-associated plasma protein A (PAPP-A) is an IGF binding protein 4 protease that can function to increase local IGF-I bioavailability. Aside from its assumed role during pregnancy, in vitro and in vivo studies have indicated roles for PAPP-A in IGF-I-mediated wound healing, vascular repair, and bone formation. Because bone morphogenetic protein 2 (BMP-2) is known to up-regulate Igf-I gene expression, we hypothesized that PAPP-A may be involved in BMP-2 mechanisms in bone formation. To test this hypothesis, we quantified gene expression of Papp-A in response to BMP-2 treatment and runt-related transcription factor 2, Osterix, and Igf-I in response to PAPP-A protein treatment in human adult mesenchymal stem cells. Our results demonstrate that BMP-2 directly up-regulated Papp-A gene and protein expression. Purified PAPP-A protein directly up-regulated runt-related transcription factor 2 and Igf-I gene expression but not Osterix. When added in combination with recombinant human BMP-2, PAPP-A increased matrix mineralization in the absence of dexamethasone. PAPP-A further demonstrated an angiogenic effect in the chick chorioallontoic membrane, which implicates a critical developmental role and possible therapeutic potential. Our findings suggest that PAPP-A functions in the formation of mineralized tissues through direct up-regulation of key genes. Furthermore, PAPP-A is involved in the formation of new blood vessels, which is essential for proper bone regeneration.

Pregnancy-associated plasma protein-a production in rat granulosa cells: stimulation by follicle-stimulating hormone and inhibition by the oocyte-derived bone morphogenetic protein-15.

Pregnancy-associated plasma protein-A (PAPP-A) is the major IGF binding protein-4 (IGFBP-4) protease in follicular fluid, consistent with its proposed role in folliculogenesis. Despite growing interest, almost nothing is known about how PAPP-A expression is regulated in any tissue. Here we show that FSH and oocytes regulate PAPP-A expression in granulosa cells (GCs). By in situ hybridization, ovary PAPP-A mRNA was markedly increased by pregnant mare serum gonadotropin treatment, and the message was localized to the membrana GCs but not cumulus GCs (CGCs) of dominant follicles. To explore the mechanism, we used primary cultures of rat GCs. Control (untreated) cells produced little or no PAPP-A spontaneously. Conversely, FSH markedly stimulated PAPP-A mRNA and protein in a dose- and time-dependent fashion. Interestingly, PAPP-A expression in isolated CGCs was also strongly induced by FSH, and the induction was inhibited by added oocytes. To investigate the nature of the inhibition, we tested the effect of oocyte-derived bone morphogenetic protein-15 (BMP-15). BMP-15 alone had no effect on basal levels of PAPP-A expression by cultures of membrana GCs or CGCs. However, BMP-15 markedly inhibited the FSH stimulation of PAPP-A production in a dose-dependent manner. The cleavage of IGFBP-4 by conditioned media from FSH-treated GCs was completely inhibited by anti-PAPP-A antibody, indicating the IGFBP-4 protease secreted by GCs is PAPP-A. These results demonstrate stimulatory and inhibitory roles for FSH and BMP-15, respectively, in regulating PAPP-A production by GCs. We propose that FSH and oocyte-derived BMP-15 form a controlling network that ensures the spatiotemporal pattern of GC PAPP-A expression in the dominant follicle.

Pregnancy-associated plasma protein-A (PAPP-A) expression and insulin-like growth factor binding protein-4 protease activity in normal and malignant ovarian surface epithelial cells.

Pregnancy-Associated Plasma Protein-A (PAPP-A) proteolyses insulin-like growth factor binding protein-4 (IGFBP-4), thereby regulating local IGF availability. Reduced PAPP-A mRNA expression has been reported in ovarian cancer specimens compared to normal ovarian surface epithelial cells (OSE). To characterize PAPP-A expression and proteolytic activity in OSE, we developed a lifespan-extended human cell model using a temperature-sensitive mutant of the SV40 large T antigen (SV40LT). These OSE(tsT) cells proliferate at 34 degrees C (i.e., when SV40LT-positive), but not at 39 degrees C, a temperature at which the SV40LT is unstable (SV40LT-negative). Proteolysis of radiolabeled IGFBP-4 in conditioned media from OSE(tsT) lines was IGF-dependent and blocked by anti-PAPP-A antisera. Temperature shifts that eliminated stable SV40LT induced a 7-fold increase in PAPP-A mRNA and a 4-fold increase in protein. The converse experiment (shifting to SV40LT-positive conditions) resulted in decreased levels of PAPP-A mRNA but little change in PAPP-A protein. Nevertheless, there was a marked reduction in IGF-BP-4 proteolytic activity in medium of SV40LT-positive OSE-(tsT) cells. This decreased PAPP-A activity coincided with a nearly 20-fold increase in mRNA encoding a physiological inhibitor of PAPP-A, the precursor form of eosinophil Major Basic Protein (proMBP), and 4- to 5-fold increases in proMBP protein. Primary cultures of unmodified OSE expressed high levels of PAPP-A and undetectable proMBP, and therefore produced abundant IGFBP-4 protease activity. Short-term ovarian tumor cell cultures expressed variable levels of PAPP-A and high levels of proMBP, and consequently secreted little or no IGFBP-4 protease activity. The concurrent regulation of PAPP-A and its inhibitor, proMBP, suggests that IGFBP-4 proteolysis and local regulation of IGF availability may be altered in malignant ovarian epithelial cells.

Regulation of pregnancy-associated plasma protein-A expression in cultured human osteoblasts

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase secreted by cultured human osteoblasts that has been implicated in the regulation of local insulin-like growth factor (IGF) bioavailability during bone growth and remodeling. However, very little is known about the regulation of PAPP-A expression in bone. In this study, we determined the effect of systemic and local osteoregulatory factors on PAPP-A mRNA and protein expression in normal human osteoblasts (hOB cells). Treatment of hOB cells with particular peptide growth factors (basic fibroblast growth factor, epidermal growth factor), steroid hormones (dexamethasone, 1,25-dihydroxyvitamin D 3), and cytokines [interleukin-6 (IL-6), IL-13, oncostatin M] with known involvement in bone cell physiology had no significant effect on PAPP-A expression. Agents that increase intracellular cyclic AMP (forskolin, prostaglandin E 2) increased PAPP-A mRNA and protein expression ∼3-fold. Tumor necrosis factor α (TNFα), IL-1β, and IL-4 also increased PAPP-A expression 3- to 4-fold. Transforming growth factor β (TGFβ) was previously shown to stimulate PAPP-A expression in hOB cells. The effects of TGFβ, TNFα, and IL-1β were additive, whereas the effects of TGFβ and IL-4 were synergistic. In summary, TNFα, IL-1β, and IL-4 were identified as potent stimulators of PAPP-A expression in primary cultures of human osteoblasts. These findings suggest a mechanism whereby cytokines present in bone and bone marrow could augment IGF bioavailability during skeletal growth and remodeling.