Laurie K. Bale

Metalloproteinase pregnancy-associated plasma protein A is a critical growth regulatory factor during fetal development

Pregnancy-associated plasma protein A (PAPPA) is a metzincin superfamily metalloproteinase in the insulin-like growth factor (IGF) system. PAPPA increases IGF bioavailability and mitogenic effectiveness in vitro through regulated cleavage of IGF-binding protein 4 (IGFBP4). To determine its function in vivo, we generated PAPPA-null mice by gene targeting. Mice homozygous for targeted disruption of the PAPPA gene were viable but 60% the size of wild-type littermates at birth. The impact of the mutation was exerted during the early embryonic period prior to organogenesis, resulting in proportional dwarfism. PAPPA, IGF2 and IGFBP4 transcripts co-localized in wild-type embryos, and expression of IGF2 and IGFBP4 mRNA was not altered in PAPPA-deficient embryos. However, IGFBP4 proteolytic activity was completely lacking in fibroblasts derived from PAPPA-deficient embryos, and IGFBP4 effectively inhibited IGF-stimulated mitogenesis in these cells. These results provide the first direct evidence that PAPPA is an essential growth regulatory factor in vivo, and suggest a novel mechanism for regulated IGF bioavailability during early fetal development.

Loss of pregnancy‐associated plasma protein A extends lifespan in mice

Genetic deletion in mice of pregnancy‐associated plasma protein A (PAPP‐A), a recently identified metalloproteinase in the insulin‐like growth factor system, extends by 30–40% both mean and maximum lifespan with no reduction in food intake or secondary endocrine abnormalities. Furthermore, these mice have markedly reduced incidence of spontaneous tumors. The findings implicate PAPP‐A as a critical regulator of lifespan and age‐related diseases, and suggest PAPP‐A as a possible target to promote longevity.

Transforming Growth Factor‐β Regulation of the Insulin‐Like Growth Factor Binding Protein‐4 Protease System in Cultured Human Osteoblasts

IGFBP‐4 is an inhibitor of IGF‐I in bone. We show that TGF‐β regulates IGFBP‐4 and enhances IGF‐I‐stimulated growth of cultured human bone cells through increased expression of an IGFBP‐4 protease, PAPP‐A. This effect of TGF‐β on IGF‐I bioavailability may promote local bone formation.

Longevity and Age-Related Pathology of Mice Deficient in Pregnancy-Associated Plasma Protein-A

The pregnancy-associated plasma protein-A knockout (PAPP-A KO) mouse is a model of reduced local insulin-like growth factor (IGF)-I activity with normal circulating IGF-I levels. In this study, PAPP-A KO mice had significantly increased mean (27%), median (27%), and maximum (35%) life span compared with wild-type (WT) littermates. End-of-life pathology indicated that the incidence of neoplastic disease was not significantly different in the two groups of mice; however, it occurred in older aged PAPP-A KO compared with WT mice. Furthermore, PAPP-A KO mice were less likely to show degenerative changes of age. Scheduled pathologies at 78, 104, and 130 weeks of age indicated that WT mice, in general, had more degenerative changes and tumors earlier than PAPP-A KO mice. This was particularly true for abnormalities in heart, testes, brain, kidney, spleen, and thymus. In summary, the major contributors to the extended life span of PAPP-A KO mice are delayed occurrence of fatal neoplasias and decreased incidence of age-related degenerative changes.

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.

Resistance to age-dependent thymic atrophy in long-lived mice that are deficient in pregnancy-associated plasma protein A

Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase that controls the tissue availability of insulin-like growth factor (IGF). Homozygous deletion of PAPPA in mice leads to lifespan extension. Since immune function is an important determinant of individual fitness, we examined the natural immune ecology of PAPPA−/− mice and their wild-type littermates reared under specific pathogen-free condition with aging. Whereas wild-type mice exhibit classic age-dependent thymic atrophy, 18-month-old PAPPA−/− mice maintain discrete thymic cortex and medulla densely populated by CD4+CD8+ thymocytes that are capable of differentiating into single-positive CD4 and CD8 T cells. Old PAPPA−/− mice have high levels of T cell receptor excision circles, and have bone marrows enriched for subsets of thymus-seeding progenitors. PAPPA−/− mice have an overall larger pool of naive T cells, and also exhibit an age-dependent accumulation of CD44+CD43+ memory T cells similar to wild-type mice. However, CD43+ T cell subsets of old PAPPA−/− mice have significantly lower prevalence of 1B11 and S7, glycosylation isoforms known to inhibit T cell activation with normal aging. In bioassays of cell activation, splenic T cells of old PAPPA−/− mice have high levels of activation antigens and cytokine production, and also elicit Ig production by autologous B cells at levels equivalent to young wild-type mice. These data suggest an IGF-immune axis of healthy longevity. Controlling the availability of IGF in the thymus by targeted manipulation of PAPPA could be a way to maintain immune homeostasis during postnatal development and aging.

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-A2s 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.

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°C (i.e., when SV40LT‐positive), but not at 39°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.

Effect of enhancement of cholesterol degradation during neonatal life of guinea pig on its subsequent response to dietary cholesterol

The effect of feeding cholestyramine to neonatal guinea pigs on their subsequent plasma cholesterol levels and response to dietary cholesterol were studied. Male neonatal guinea pigs were suckled for 6 days. One group was maintained on a 1.1% cholestyramine diet for 6 weeks and the control group weaned normally. Both groups of guinea pigs were then fed a standard diet of Guinea Pig Chow for 6 weeks. During the standard diet period bile acid and neutral sterol excretion rates were significantly higher in the group previously treated with cholestyramine than the control group despite the similarity in plasma cholesterol levels. When both groups of guinea pigs were subjected to a 0.5% cholesterol diet for 4 weeks, plasma cholesterol levels were significantly lower in the group previously treated with cholestyramine than the control group. The plasma cholesterol levels continued to be significantly lower in the group previously treated with cholestyramine after an additional four weeks on standard diet. These results suggest that stimulation of cholesterol catabolism in the neonatal period can influence the subsequent response to dietary cholesterol.

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. Mol Cancer Ther; 14(4); 973–81. ©2015 AACR.