Analysis of Pregnancy-Associated Plasma Protein A Production in Human Adult Cardiac Progenitor Cells

IGF-binding proteins (IGFBPs) and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A) is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs) and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily, thus positively affecting IGF signalling. Therefore, our results strongly support the importance of PAPP-A for the IGFs/IGFBPs/PAPP-A axis in CPCs biology.

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Selection of the Dominant Follicle and Insulin-Like Growth Factor (IGF)-Binding Proteins: Evidence that Pregnancy-Associated Plasma Protein A Contributes to Proteolysis of IGF-Binding Protein 5 in Bovine Follicular Fluid

Endocrinology ◽

10.1210/en.2002-220657 ◽

2003 ◽

Vol 144 (2) ◽

pp. 437-446 ◽

Cited By ~ 49

Author(s):

G. M. Rivera ◽

J. E. Fortune

Keyword(s):

Proteolytic Activity ◽

Plasma Protein ◽

Follicular Fluid ◽

Binding Proteins ◽

Protein A ◽

Positive Control ◽

Dominant Follicle ◽

Igf Binding Proteins ◽

Igf Binding ◽

Igf Binding Protein

Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn2+ metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band (∼400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.

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

Scientific Reports ◽

10.1038/s41598-019-53957-x ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

J. A. Bøtkjær ◽

P. R. Noer ◽

Claus Oxvig ◽

Claus Yding Andersen

Keyword(s):

Proteolytic Activity ◽

Plasma Protein ◽

Binding Proteins ◽

Protein A ◽

Common Variant ◽

Igf Binding Proteins ◽

Igf Binding

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

Scientific Reports ◽

10.1038/s41598-019-49626-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Jane Alrø Bøtkjær ◽

Pernille Rimmer Noer ◽

Claus Oxvig ◽

Claus Yding Andersen

Keyword(s):

Plasma Protein ◽

Binding Proteins ◽

Specific Activity ◽

Protein A ◽

P Value ◽

Surface Binding ◽

Cleavage Rate ◽

Igf Binding Proteins ◽

Single Nucleotide ◽

Igf Binding

Abstract 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.

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Insulin-Like Growth Factor (IGF) Binding Protein-4 Is Both a Positive and Negative Regulator of IGF Activity in Vivo

Molecular Endocrinology ◽

10.1210/me.2007-0536 ◽

2008 ◽

Vol 22 (5) ◽

pp. 1213-1225 ◽

Cited By ~ 78

Author(s):

Yun Ning ◽

Alwin G. P. Schuller ◽

Cheryl A. Conover ◽

John E. Pintar

Keyword(s):

Plasma Protein ◽

Protein A ◽

Postnatal Growth ◽

Negative Regulator ◽

Igf Binding Proteins ◽

Prenatal Growth ◽

Igf Binding ◽

Growth Deficit

Abstract IGFs are required for normal prenatal and postnatal growth. Although actions of IGFs can be modulated by a family of IGF-binding proteins (IGFBPs) in vitro, these studies have identified a complicated pattern of stimulatory and inhibitory IGFBP effects, so that understanding relevant aspects of IGFBP action in vivo has been limited. Here we have produced a null mutation of one specific IGFBP, IGFBP-4, which is coexpressed with IGF-II early in development. Surprisingly, mutation of IGFBP-4, believed from in vitro studies to be exclusively inhibitory, leads to a prenatal growth deficit that is apparent from the time that the IGF-II growth deficit first arises, which strongly suggests that IGFBP-4 is required for optimal IGF-II-promoted growth during fetal development. Mice encoding a mutant IGFBP-4 protease (pregnancy-associated plasma protein-A), which facilitates IGF-II release from an inactive IGF-II/IGFBP-4 complex in vitro, are even smaller than IGFBP-4 mutant mice. However, the more modest IGFBP-4 growth deficit is completely restored in double IGFBP-4/pregnancy-associated plasma protein-A-deficient mice. Taken together these results indicate not only that IGFBP-4 functions as a local reservoir to optimize IGF-II actions needed for normal embryogenesis, but also establish that IGFBP-4 proteolysis is required to activate most, if not all, IGF-II mediated growth-promoting activity.

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Abstract 12613: Apurinic/apyrimidinic Endonuclease/redox Factor-1 Gene Enhances Anti-apoptotic Function of Cardiac Progenitor Cells via TAK1-Activation and Promotes Cardiac Regeneration in Myocardial Infarction

Circulation ◽

10.1161/circ.132.suppl_3.12613 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Tatsuya Aonuma ◽

Naofumi Takehara ◽

Keisuke Maruyama ◽

Maki Kabara ◽

Motoki Matsuki ◽

...

Keyword(s):

Oxidative Stress ◽

Myocardial Infarction ◽

Cell Therapy ◽

Progenitor Cells ◽

Culture System ◽

Cardiac Progenitor Cells ◽

Cardiac Cell ◽

Cardiac Cell Therapy ◽

Dsred Gene

Introduction: Overcoming the poor survival of cell grafts is an indispensable mission in cell therapy. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1) is known as a multifunctional enzyme to encourage cell survival, whereas the role of APE1 in cardiac cell therapy is still unknown. Hypothesis: APE1 overexpression in cardiac progenitor cells (CPC) ameliorates the effect of cardiac cell therapy. Methods: CPCs from 8-10 week-old C57BL/6 mice hearts were transfected with APE1-DsRed gene (APE1-CPC) or DsRed gene (Control [Ct]-CPC). The apoptosis induced by oxidative stress was assessed in APE1-/Ct-CPCs, and in neonatal rat cardiomyocytes (NRCM) within the co-culture system of APE1-/Ct-CPCs. Western blot analysis indicated the cellular signal to protect CPC via APE1 enzyme. To evaluate the effect of APE1 overexpression in cell therapy, we transplanted APE1-CPCs and Ct-CPCs into the mice myocardial infarction (MI) model and assessed the pathophysiological role of APE1 with functional and histological analysis. Results: Under the oxidative stress condition, APE1 overexpression inhibited the apoptosis of CPCs and accelerated TAK1 activation (Ct-CPC : APE1-CPC = 1.5±0.4 : 3.3±1.6 fold, p<0.05), and consequently NfKB phosphorylation in CPCs. In the co-culture system, the apoptosis of NRCMs was inhibited with APE1-CPCs compared to that with Ct-CPCs. In vivo, in the mice MI model, the number of total CPC grafts and cardiac α-actinin-positive graft CPCs were significantly larger in APE1-CPC injected mice (APE1 mice) compared to Ct-CPC injected mice (Ct mice) at 7 days after implantation. Eventually, the left ventricular ejection fraction of APE1 mice was significantly improved compared to Ct mice (Ct mice : APE1 mice = +3.1±6.7 : +11.3±4.0%, p<0.05) and was accompanied with the attenuation of fibrosis at 28 days after implantation. Conclusions: APE1 gene inhibited the apoptosis of CPCs and host cells against oxidative stress via the activation of TAK1-NFkB pathway, which is a novel insight into the stress response of APE1 enzyme. Furthermore, APE1-CPC grafts that effectively survived in the ischemic heart restored cardiac dysfunction and attenuated myocardial infarct size, and may be an innovative strategy to reinforce cardiac cell therapy.

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Insulin-Like Growth Factor Bioactivity, Stanniocalcin-2, Pregnancy-Associated Plasma Protein-A, and IGF-Binding Protein-4 in Pleural Fluid and Serum From Patients With Pulmonary Disease

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2017-00033 ◽

2017 ◽

Vol 102 (9) ◽

pp. 3526-3534 ◽

Cited By ~ 16

Author(s):

Ulrick Skipper Espelund ◽

Mette Bjerre ◽

Rikke Hjortebjerg ◽

Torben Riis Rasmussen ◽

Anders Lundby ◽

...

Keyword(s):

Growth Factor ◽

Pleural Fluid ◽

Plasma Protein ◽

Binding Protein ◽

Protein A ◽

Insulin Like Growth Factor ◽

Igf System ◽

Igf Binding ◽

Stanniocalcin 2 ◽

Igf Binding Protein

Abstract Context Members of the insulin-like growth factor (IGF) system are primarily produced in the liver and secreted into the circulation, but they are also produced, recruited, and activated locally in tissues. Objective To compare activity and concentrations of IGF system components in pleural fluid and blood. Design Pathological pleural fluid, secondary to lung cancer or nonmalignant disease, and matching blood samples were collected from 24 patients ages 66.7 to 81.9 years. Methods IGF-related proteins and cytokine levels were measured by immunoassays or immunoblotting. Bioactive IGF was measured by an IGF-1 receptor phosphorylation assay. Results Total IGF-1 concentration did not differ between the compartments, but concentrations of free IGF-1 and bioactive IGF were more than threefold higher in pleural fluid than in corresponding serum samples (P = 0.0004), regardless of etiology. Median pregnancy-associated plasma protein-A (PAPP-A) and interleukin (IL)-6 levels were increased 47-fold and 143-fold, respectively, in pleural fluid compared with plasma (P < 0.0001). PAPP-A and IL-6 concentrations correlated positively (r = 0.46; P = 0.02). In pleural fluid, levels of PAPP-A–generated IGF binding protein-4 fragments correlated inversely with that of stanniocalcin-2 (r ≤ −0.42; P ≤ 0.05), a PAPP-A inhibitor; such correlations were absent in plasma. Conclusion Pathological pleural fluid is characterized by increased in vitro IGF bioactivity and elevated concentrations of PAPP-A, an IGF-activating proteinase. Thus, the tissue activity of the IGF system may differ substantially from that of the circulating IGF system. The correlation between IL-6 and PAPP-A indicates that inflammation plays a role in promoting local tissue IGF activity.

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