Pregnancy-associated Plasma protein-A Knock-out Mice Research Articles

Recapitulation of anti-aging phenotypes by global, but not by muscle-specific, deletion of PAPP-A in mice.

Deletion of pregnancy-associated plasma protein-A (PAPP-A), a protease that cleaves some but not all IGF1 binding proteins, postpones late-life diseases and extends lifespan in mice, but the mechanism of this effect is unknown. Here we show that PAPP-A knockout (PKO) mice display a set of changes, in multiple tissues, that are characteristic of other varieties of slow-aging mice with alterations in GH production or GH responsiveness, including Ames dwarf, Snell dwarf, and GHRKO mice. PKO mice have elevated UCP1 in brown and white adipose tissues (WAT), and a change in fat-associated macrophage subsets that leads to diminished production of inflammatory cytokines. PKO mice also show increased levels of muscle FNDC5 and its cleavage product, the myokine irisin, thought to cause changes in fat cell differentiation. PKO mice have elevated production of hepatic GPLD1 and plasma GPLD1, consistent with their elevation of hippocampal BDNF and DCX, used as indices of neurogenesis. In contrast, disruption of PAPP-A limited to muscle ("muPKO" mice) produces an unexpectedly complex set of changes, in most cases opposite in direction from those seen in PKO mice. These include declines in WAT UCP1, increases in inflammatory macrophages and cytokines in WAT, and a decline in muscle FNDC5 and plasma irisin. muPKO mice do, however, resemble global PKO mice in their elevation of hippocampal BDNF and DCX. The data for the PKO mice support the idea that these changes in fat, macrophages, liver, muscle, plasma, and brain are consistent and biologically significant features of the slow-aging phenotype in mice. The results on the muPKO mice provide a foundation for further investigation of the complex, local, and global circuits by which PAPP-A modulates signals ordinarily controlled by GH and/or IGF1.

Comparative gene expression and phenotype analyses of skeletal muscle from aged wild-type and PAPP-A-deficient mice

Mice deficient in pregnancy-associated plasma protein-A (PAPP-A) have extended lifespan associated with decreased incidence and severity of degenerative diseases of age, such as cardiomyopathy and nephropathy. In this study, the effect of PAPP-A deficiency on aging skeletal muscle was investigated. Whole-genome expression profiling was performed on soleus muscles from 18-month-old wild-type (WT) and PAPP-A knock-out (KO) mice of the same sex and from the same litter (‘womb-mates’) to identify potential mechanisms of skeletal muscle aging and its retardation in PAPP-A deficiency. Top genes regulated in PAPP-A KO compared to WT muscle were associated with increased muscle function, increased metabolism, in particular lipid metabolism, and decreased stress. Fiber cross-sectional area was significantly increased in solei from PAPP-A KO mice. In vitro contractility experiments indicated increased specific force and decreased fatigue in solei from PAPP-A KO mice. Intrinsic mitochondrial oxidative capacity was significantly increased in skeletal muscle of aged PAPP-A KO compared to WT mice. Moreover, 18-month-old PAPP-A KO mice exhibited significantly enhanced endurance running on a treadmill. Thus, PAPP-A deficiency in mice is associated with indices of healthy skeletal muscle function with age.

Pregnancy-associated plasma protein-A deficiency improves survival of mice on a high fat diet

Obesity is on the rise in westernized countries, and visceral obesity in particular is associated with enhanced risk of developing metabolic disease and accelerated aging. Various dietary restriction regimens have been shown to extend healthy lifespan in a variety of species. However, identification of alternative approaches that could be more acceptable to humans is actively being pursued. We have shown previously that mice deficient in pregnancy-associated plasma protein-A (PAPP-A) have an extended healthy lifespan on a regular chow diet. In this study, we determined the lifespan of PAPP-A knock-out (KO) and wild-type (WT) littermates fed a high fat diet (HFD) starting at 12months of age. PAPP-A KO and WT mice had equivalent weight gain as measured over 25weeks on HFD. However, PAPP-A KO mice on HFD had a significant increase in lifespan (P=0.018). Body composition and tissue pathology were assessed in a separate cohort of mice after 30weeks on HFD. Percent body fat was equivalent in the two groups. However, there was a decrease in visceral fat depot weights and an increase in serum adiponectin levels in PAPP-A KO compared to WT mice. Major pathological differences were seen in kidney, heart and testes, with PAPP-A KO mice having little, if any, evidence of inflammation, mineralization, or degeneration in these tissues compared to WT mice. Thus, PAPP-A is a novel drug target with the potential to promote healthy longevity without a need for dietary restriction.

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.

IGF binding protein-4 is required for the growth effects of glucagon-like peptide-2 in murine intestine.

Glucagon-like peptide-2 (GLP-2) is an enteroendocrine hormone that stimulates the growth of the intestinal epithelium. We have previously demonstrated that GLP-2 exerts its intestinotropic effect through an indirect mechanism that requires both IGF-1 and the intestinal epithelial IGF-1 receptor. However, the biological activity of IGF-1 is modulated by IGF binding proteins (IGFBPs), including IGFBP-4, which is highly expressed in the intestine. To determine the role of IGFBP-4 in the tropic effects of GLP-2, IGFBP-4 knockout (KO) and control mice were treated with degradation-resistant GLP-2 or vehicle for 10 days. Comparable levels of IGFBP-1-3/5-7 mRNAs were observed in the intestinal mucosa of all animals. IGFBP-4 KO mice had greater small intestinal weight and length, and deeper crypts (P < .05) as compared with controls, suggesting that IGFBP-4 has an inhibitory role in basal intestinal growth. However, small intestinal weight, crypt-villus height and crypt cell proliferation increased in response to GLP-2 in control mice (P < .05), and these changes were abrogated with IGFBP-4 KO. In contrast, pregnancy-associated plasma protein-A KO mice, which have increased levels of circulating IGFBP-4, demonstrated a normal intestinotropic response to GLP-2. Finally, GLP-2 treatment of control mice significantly increased IGFBP-4 mRNA expression in the jejunal mucosa (P < .05), a finding that was recapitulated by GLP-2 treatment of fetal rat intestinal cells in culture (10(-8)M for 2 h; P < .05). Collectively, these results indicate that the IGF-I-modulating protein, IGFBP-4, exerts a negative effect on basal intestinal growth but plays a positive regulatory role in the intestinotropic actions of GLP-2.

Motor and memory testing of long-lived pregnancy-associated plasma protein—A knock-out mice

Mice deficient in pregnancy-associated plasma protein-A (PAPP-A), an IGF binding protein protease, have been shown to be resistant to experimentally induced atherosclerosis and diabetic nephropathy, and, in the laboratory environment, live 30–40% longer than wild-type littermates in association with delayed incidence and occurrence of age-related neoplasms and degenerative diseases. ObjectivePAPP-A is highly expressed in the cerebellum and hippocampus of the mouse brain. Therefore, the studies presented here were aimed at determining motor behavior, learning and retention in PAPP-A knock-out (KO) mice compared to wild-type (WT) littermates with age. DesignBalance and coordination were assessed using an accelerating rotarod; learning and memory were assessed in a Stone T-maze. ResultsTime on the rotarod decreased with age but there was no significant difference between PAPP-A KO and WT mice at any of the testing ages. Latency to reach the goal box and number of errors committed in the Stone T-maze did not change with age and there were no significant differences between PAPP-A KO and WT mice. ConclusionLack of PAPP-A in mice did not impact central regulation of coordination, learning or memory.

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.

Insulin-like growth factor (IGF)-I and IGF-II contribute differentially to the phenotype of pregnancy associated plasma protein-A knock-out mice

Context Insulin-like growth factor (IGF) signaling is essential for achieving optimal body size during fetal development, peak bone mass during puberty, and maximal fecundity in the reproductive period. IGF-II is considered the main fetal IGF, whereas IGF-I is more important postnatally. Pregnancy-associated plasma protein-A (PAPP-A) enhances local IGF signaling through cleavage of inhibitory IGF binding proteins. Conversely, inhibition of PAPP-A results in reduced local IGF action. Thus, PAPP-A knock-out (KO) mice are born as proportional dwarfs due to the dysregulation of IGF-II signaling during early embryogenesis that impacts body size. Relaxation of IgfII imprinting through mutation of a reciprocally imprinted downstream gene, H19, which allowed transcription of IGF-II from the normally silent maternal allele, rescued the dwarf phenotype of PAPP-A KO mice. Objective To determine the effect of increased IGF-II expression on postnatal phenotypes of PAPP-A KO mice. Design Young adult wild-type (WT), PAPP-A KO, H19 mutant (ΔH19/WT) and ΔH19/PAPP-A KO mice were characterized for skeletal phenotype (peripheral quantitative computed tomography at the midshaft and distal metaphysis of the femur) and reproductive phenotype (time to first litter, time between litters, pups per litter). Results Serum IGF-II levels were significantly increased in ΔH19/WT and ΔH19/PAPP-A KO mice compared to WT and PAPP-A KO mice; serum IGF-I levels were not affected by H19 mutation. PAPP-A KO mice had reductions in cortical thickness and in cortical and trabecular area, bone mineral content and bone mineral density compared to WT mice. There were no significant differences between PAPP-A KO and ΔH19/PAPP-A KO mice in any of the bone parameters. PAPP-A KO crossed with (×) PAPP-A KO had a longer time until first litter, normal time between subsequent litters, and significantly reduced number of pups per litter compared to WT × WT. ΔH19/PAPP-A KO × ΔH19/PAPP-A KO had an even longer time to first litter, but also longer time between litters. This phenotype was associated with female ΔH19/PAPP-A KO mice. Furthermore, these ΔH19/PAPP-A KO mouse mothers failed to care for their pups. Conclusions An increase in IGF-II expression did not rescue the skeletal and reproductive deficiencies associated with reduced local IGF-I signaling in PAPP-A KO mice. In addition, the data suggest a potential new role for genomic imprinting at the IgfII/ H19 locus affecting maternal behavior.

Metabolic consequences of pregnancy-associated plasma protein-A deficiency in mice: exploring possible relationship to the longevity phenotype

Mice born with the deletion of the gene for pregnancy-associated plasma protein-A (PAPP-A), a model of reduced local IGF activity, live approximately 30% longer than their wild-type (WT) littermates. In this study, we investigated metabolic consequences of PAPP-A gene deletion and possible relationship to lifespan extension. Specifically, we determined whether 18-month-old PAPP-A knockout (KO) mice when compared with their WT littermates have reduced energy expenditure and/or altered glucose-insulin sensitivity. Food intake, and total energy expenditure and resting energy expenditure as measured by calorimetry were not different between PAPP-A KO and WT mice when subjected to the analysis of covariance with body weight as the covariate. However, there was an increase in spontaneous physical activity in PAPP-A KO mice. Both WT and PAPP-A KO mice exhibited mild insulin resistance with age, as assessed by fasting glucose/insulin ratios. Oral glucose tolerance and insulin sensitivity were not significantly different between the two groups of mice, although there appeared to be a decrease in the average size of the pancreatic islets in PAPP-A KO mice. Thus, neither reduced 'rate of living' nor altered glucose-insulin homeostasis can be considered key determinants of the enhanced longevity of PAPP-A KO mice. These findings are discussed in the context of those from other long-lived mouse models.

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

Impact of Pregnancy-Associated Plasma Protein-A Deletion on the Adult Murine Skeleton

The metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A) functions to enhance local insulin-like growth factor (IGF)-I bioavailability through cleavage of inhibitory IGF binding proteins. Because IGF-I is an important regulator of skeletal growth and remodeling and PAPP-A is highly expressed by osteoblastic cells, we hypothesized that, in the absence of PAPP-A, bone physiology would be compromised because of a blunting of local IGF-I action even in the presence of normal circulating IGF-I levels. pQCT, muCT, histomorphometry, and mechanical strength testing were performed on bones from PAPP-A knockout (KO) mice and wildtype (WT) littermates at 2-12 mo of age. IGF-I levels and bone formation and resorption markers were determined in sera from these animals. Volumetric BMD in PAPP-A KO mice measured by pQCT at the femoral midshaft, which is primarily cortical bone, was 10% less than WT at 2 mo. This difference was maintained at 4, 6, and 12 mo. Cortical thickness at this site was similarly decreased. On the other hand, trabecular bone at the distal femur (pQCT) and in the tibia (muCT) showed age-progressive decreases in bone volume fraction in PAPP-A KO compared with WT mice. Tibial muCT indicated a 46% relative decrease in trabecular bone volume/total volume (BV/TV) and a 28% relative decrease in trabecular thickness in PAPP-A KO compared with WT mice at 6 mo. These trabecular deficiencies in PAPP-A KO mice corresponded to a weakening of the bone. Serum markers and bone histomorphometry indicated that the primary impact of PAPP-A is on skeletal remodeling resulting in a state of low-turnover osteopenia in adult PAPP-A KO mice. Circulating IGF-I levels were not altered in PAPP-A KO mice. PAPP-A is a bone growth regulatory factor in vivo and, in its absence, mice show skeletal insufficiency in mass, density, architecture, and strength. The data suggest a primary role for PAPP-A in modulating local IGF bioavailability for trabecular bone remodeling.

Genetic Deletion of Pregnancy-Associated Plasma Protein-A Is Associated With Resistance to Atherosclerotic Lesion Development in Apolipoprotein E–Deficient Mice Challenged With a High-Fat Diet

Pregnancy-associated plasma protein-A (PAPP-A), a metalloproteinase in the insulin-like growth factor (IGF) system, is markedly upregulated in human atherosclerotic plaque. To determine whether PAPP-A plays an active role in the development of atherosclerosis, we crossed mice lacking apolipoprotein E (ApoE) with PAPP-A-deficient mice, generating ApoE knock-out (KO), PAPP-A KO, wild-type (WT/WT), and ApoE/PAPP-A double KO (KO/KO) mice. These mice were fed a high-fat diet starting at 7 weeks of age. Total serum cholesterol levels were elevated similarly in the ApoE KO and KO/KO mice and were 10-fold higher than in the WT/WT and PAPP-A KO mice. WT/WT and PAPP-A KO mice showed little or no lesion development even after 20 weeks of diet. ApoE KO mice had a progressive increase in aortic lesion area over 20 weeks of diet. In comparison, lesion area was reduced 60% to 80% in KO/KO mice. Lesions of ApoE KO aortas had 8- to 20-fold increases in PAPP-A, IGFBP-4, and IGF-I mRNA levels compared with nonlesional areas, whereas IGF-I receptor levels were equivalent--conditions for enhanced lesional IGF activity. Consistent with this, an in vivo marker of IGF-I receptor-mediated action was increased 10-fold in lesions from ApoE KO compared with KO/KO aortas. These data indicate that PAPP-A plays a critical role in lesion development in a mouse model of atherosclerosis, at least in part, through amplification of local IGF-I bioavailability.

Pregnancy associated plasma protein-A is necessary for expeditious fracture healing in mice

Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that regulates IGF bioavailability in vitro through cleavage of inhibitory IGF-binding protein-4 (IGFBP-4), has been implicated in skeletal development and injury repair responses. However, direct in vivo data are lacking. In this study, we used PAPP-A knock-out (KO) mice to determine the role of PAPP-A in fracture repair. Stabilized mid-shaft fractures were produced in femurs of 3-month-old mice. At 14 days post-fracture, complete bony bridging of the fracture callus was seen radiographically in wild-type but not in PAPP-A KO mice. Histological examination 5 to 28 days post-fracture showed reductions in the amount of intramembranous bone formation, cartilage production, endochondral ossification and remodeling in PAPP-A KO compared with wild-type mice. However, fracture healing appeared similar in both groups at 42 days post-fracture when analyzed by histology. A similar degree of healing strength in wild-type and PAPP-A KO femurs was demonstrated by mechanical testing at 28 and 42 days post-fracture. Untreated cultures of day 5 fracture calluses from wild-type mice showed robust IGFBP-4 protease activity and IGF receptor phosphorylation, whereas fracture calluses from PAPP-A KO mice had no IGFBP-4 protease activity and reduced IGF receptor phosphorylation. These data demonstrate a marked delay in fracture healing in PAPP-A KO compared with wild-type mice, and suggest that PAPP-A is necessary in the early phases of the process for expeditious fracture repair. The ability of PAPP-A to enhance local IGF action may be an important mechanism for optimizing the fracture repair response.