PAPP-A as a Potential Target in Thyroid Eye Disease.

CONTEXT: Proptosis in Thyroid Eye Disease (TED) can result in facial disfigurement and visual dysfunction. Treatment with Insulin-like growth factor I receptor (IGF-IR) inhibitors has been shown to be effective in reducing proptosis but with side effects. OBJECTIVE: To test the hypothesis that inhibition of IGF-IR indirectly and more selectively with PAPP-A inhibitors attenuates IGF-IR signaling in TED. DESIGN: Informed consent was obtained from TED patients undergoing surgery, and retro-orbital tissue collected for fibroblast isolation and culture. SETTING: Surgeries were performed in Mayo Clinic operating suites. Cell culture was performed in a sterile tissue culture facility. PATIENT SAMPLES: Retro-orbital tissue was collected from 19 TED patients. INTERVENTIONS: Treatment of TED fibroblasts with pro-inflammatory cytokines. Flow separation of CD34- and CD34+ orbital fibroblasts, the latter representing infiltrating fibrocytes into the orbit in TED. MAIN OUTCOME MEASURES: PAPP-A expression and proteolytic activity, IGF-I stimulation of phosphatidylinositol 3 kinase/Akt pathway and inhibition by immuno-neutralizing antibodies against PAPP-A, CD34+ status and associated PAPP-A and IGF-IR expression. RESULTS: Pro-inflammatory cytokines markedly increased PAPP-A expression in TED fibroblasts. IGF-IR expression was not affected by cytokine treatment. Inhibition of PAPP-A's proteolytic activity suppressed IGF-IR activation in orbital fibroblasts from TED patients. TED fibroblasts that were CD34+ represented ∼80% of the cells in culture and accounted for ∼70% of PAPP-A and IGF-IR expressing cells. CONCLUSIONS: These results support a role for PAPP-A in TED pathogenesis and indicate the potential for novel therapeutic targeting of the IGF axis.

The Pregnancy-Associated Plasma Protein-A (PAPP-A) Story.

Pregnancy-associated plasma protein-A (PAPP-A) was first identified in the early 1970s as a placental protein of unknown function, present at high concentrations in the circulation of pregnant women. In the mid-to-late 1990s, PAPP-A was discovered to be a metzincin metalloproteinase, expressed by many nonplacental cells, that regulates local insulin-like growth factor (IGF) activity through cleavage of high-affinity IGF binding proteins (IGFBPs), in particular IGFBP-4. With PAPP-A as a cell surface-associated enzyme, the reduced affinity of the cleavage fragments results in increased IGF available to bind and activate IGF receptors in the pericellular environment. This proteolytic regulation of IGF activity is important, since the IGFs promote proliferation, differentiation, migration, and survival in various normal and cancer cells. Thus, there has been a steady growth in investigation of PAPP-A structure and function outside of pregnancy. This review provides historical perspective on the discovery of PAPP-A and its structure and cellular function, highlights key studies of the first 50 years in PAPP-A research, and introduces new findings from recent years.

The Stanniocalcin-PAPP-A-IGFBP-IGF Axis.

The pappalysin metalloproteinases, PAPP-A and PAPP-A2, have emerged as highly specific proteolytic enzymes involved in the regulation of insulin-like growth factor (IGF) signaling. The only known pappalysin substrates are a subset of the IGF binding proteins (IGFBPs), which bind IGF-I or IGF-II with high affinity to antagonize receptor binding. Thus, by cleaving IGFBPs, the pappalysins have the potential to increase IGF bioactivity and hence promote IGF signaling. This is relevant both in systemic and local IGF regulation, in normal and several pathophysiological conditions. Stanniocalcin-1 and -2 were recently found to be potent pappalysin inhibitors, thus comprising the missing components of a complete proteolytic system, the stanniocalcin-PAPP-A-IGFBP-IGF axis. Here, we provide the biological context necessary for understanding the properties of this molecular network, and we review biochemical data, animal experiments, clinical data, and genetic data supporting the physiological operation of this branch as an important part of the IGF system. However, although in vivo data clearly illustrate its power, it is a challenge to understand its subtle operation, for example, multiple equilibria and inhibitory kinetics may determine how, where, and when the IGF receptor is stimulated. In addition, literally all of the regulatory proteins have suspected or known activities that are not directly related to IGF signaling. How such activities may integrate with IGF signaling is also important to address in the future.

Senescence induces proteolytically-active PAPP-A secretion and association with extracellular vesicles in human pre-adipocytes.

Senescence is a cellular response to various stressors characterized by irreversible cell cycle arrest, resistance to apoptosis and expression of a senescence-associated secretory phenotype (SASP). Interestingly, studies where senescent cells were deleted in mice produced beneficial effects similar to those where the zinc metalloproteinase, PAPP-A, was deleted in mice. In this study, we investigated the effect of senescence on PAPP-A secretion and activity in primary cultures of adult human pre-adipocytes. Cultured pre-adipocytes were isolated from subcutaneous (Sub) and omental (Om) fat. Senescence was induced with low dose etoposide. PAPP-A protein was measured by an ultrasensitive PAPP-A ELISA. PAPP-A proteolytic activity was measured by a specific substrate cleavage assay. Senescence significantly increased PAPP-A levels in both Sub and Om conditioned medium (CM) 8- to 15-fold over non-senescent CM. Proteolytic activity reflected PAPP-A protein with 12- to 18-fold greater activity in senescent CM versus non-senescent CM. Furthermore, PAPP-A was found at high levels on the surface of extracellular vesicles secreted by senescent pre-adipocytes and was proteolytically active. In conclusion, we identified enzymatically active PAPP-A as a component of human pre-adipocyte SASP. This recognition warrants further investigation of PAPP-A as a new biomarker for senescence and a potential therapeutic target to control of the spread of senescence in adipose tissue.

Circulating insulin-like growth factor system adaptations in hibernating brown bears indicate increased tissue IGF availability.

Brown bears conserve muscle and bone mass during 6 mo of inactive hibernation. The molecular mechanisms underlying hibernation physiology may have translational relevance for human therapeutics. We hypothesize that protective mechanisms involve increased tissue availability of insulin-like growth factors (IGFs). In subadult Scandinavian brown bears, we observed that mean plasma IGF-1 and IGF-2 levels during hibernation were reduced to 36 ± 10% and 56 ± 15%, respectively, compared with the active state (n = 12). Western ligand blotting identified IGF-binding protein (IGFBP)-3 as the major IGFBP in the active state, whereas IGFBP-2 was codominant during hibernation. Acid labile subunit (ALS) levels in hibernation were reduced to 41±16% compared with the active state (n = 6). Analysis of available grizzly bear RNA sequencing data revealed unaltered liver mRNA IGF-1, IGFBP-2, and IGFBP-3 levels, whereas ALS levels were significantly reduced during hibernation (n = 6). Reduced ALS synthesis and circulating levels during hibernation should prompt a shift from ternary IGF/IGFBP/ALS to smaller binary IGF/IGFBP complexes, thereby increasing IGF tissue availability. Indeed, size-exclusion chromatography of bear plasma demonstrated a shift to lower molecular weight IGF-containing complexes in the hibernating versus the active state. Furthermore, we note that the major IGF-2 mRNA isoform expressed in livers in both Scandinavian brown bears and grizzly bears was an alternative splice variant in which Ser29 is replaced with a tetrapeptide possessing a positively charged Arg residue. Homology modeling of the bear IGF-2/IGFBP-2 complex showed the tetrapeptide in proximity to the heparin-binding domain involved in bone-specific targeting of this complex. In conclusion, this study provides data which suggest that increased IGF tissue availability combined with tissue-specific targeting contribute to tissue preservation in hibernating bears.NEW & NOTEWORTHY Brown bears shift from circulating ternary IGF/IGFBP/ALS complexes in the active state to binary IGF/IGFBP complexes during hibernation, indicating increased tissue IGF-bioactivity. Furthermore, brown bears use a splice variant of IGF-2, suggesting increased bone-specific targeting of IGF anabolic signaling.

Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions. METHODS AND RESULTS: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2. CONCLUSION: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment.

Pregnancy-associated plasma protein-A (PAPP-A) is a key component of an interactive cellular mechanism promoting pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with few effective treatment options. We found a highly significant correlation between pregnancy-associated plasma protein (PAPP)-A expression in IPF lung tissue and disease severity as measured by various pulmonary and physical function tests. PAPP-A is a metalloproteinase that enhances local insulin-like growth factor (IGF) activity. We used primary cultures of normal adult human lung fibroblasts (NHLF) to test the hypothesis that PAPP-A plays an important role in the development of pulmonary fibrosis. Treatment of NHLF with pro-fibrotic transforming growth factor (TGF)-ß stimulated marked increases in IGF-I mRNA expression (>20-fold) and measurable IGF-I levels in 72-h conditioned medium (CM). TGF-ß treatment also increased PAPP-A levels in CM fourfold (p = 0.004) and proteolytic activity ~2-fold. There was an indirect effect of TGF-ß to stimulate signaling through the PI3K/Akt pathway, which was significantly inhibited by both IGF-I-inactivating and PAPP-A inhibitory antibodies. Induction of senescence in NHLF increased PAPP-A levels in CM 10-fold (p = 0.006) with attendant increased proteolytic activity. Thus, PAPP-A is a novel component of the senescent lung fibroblast secretome. In addition, NHLF secreted extracellular vehicles (EVs) with surface-bound active PAPP-A that were increased fivefold with senescence. Regulation of PAPP-A and IGF signaling by TGF-ß and cell senescence suggests an interactive cellular mechanism underlying the resistance to apoptosis and the progression of fibrosis in IPF. Furthermore, PAPP-A-associated EVs may be a means of pro-fibrotic, pro-senescent communication with other cells in the lung and, thus, a potential therapeutic target for IPF.

Brain-specific PAPP-A knock-out mice?

PAPP-A knock-out (KO) mice are a valuable model for investigating the effects of down-regulating localized insulin-like growth factor (IGF) action, which has been shown to extend lifespan and healthspan when the PAPP-A gene is globally deleted. Based on previous mouse models of brain-specific reduction in IGF signaling associated with longevity, we sought to generate brain-specific PAPP-A KO mice and determine effects on metabolism and lifespan. Mice with the PAPP-A gene floxed (fPAPP-A) were crossed with Nestin promoter-driven Cre recombinase transgenic mice. This cross-breeding of mice for Nestin-Cre and mice with other floxed target alleles has been used extensively to investigate brain-specific effects. Our cross-breeding generated four genotypes for study: fPAPP-A/Nestin positive (brain-specific PAPP-A KO); fPAPP-A/Nestin negative (Control for floxed PAPP-A); WT/Nestin positive (Control for Nestin-Cre); WT/Nestin negative (Wild-type Control). The basic genotype screen of neonatal tail snip DNA clearly indicated PAPP-A gene status and the presence (pos) or absence (neg) of Nestin-Cre. We then determined tissue specificity of PAPP-A gene excision. We had expected fPAPP-A/pos mice to be relatively brain-specific for PAPP-A gene deletion and the controls (fPAPP-A/neg, WT/neg and WT/pos mice) to show no effect on PAPP-A expression in brain or other tissues. However, in fPAPP-A/neg mice we found evidence of PAPP-A excision in all tissues examined, i.e., in the presumed absence of Nestin-Cre, indicating germline recombination. We further found that fPAPP-A/pos mice showed near complete excision of the PAPP-A gene in brain, but some also showed germline recombination affecting all tissues tested. To determine if the level of excision indicated by tissue genotyping approximated PAPP-A mRNA expression, we performed RT-qPCR. fPAPP-A/pos mice that showed markedly decreased whole brain PAPP-A mRNA expression (~80%), with little or no effect on expression in the other tissues tested, were designated as "brain-specific" PAPP-A KO. fPAPP-A/pos mice that showed germline recombination had similar decreases in PAPP-A expression in brain but also showed 40-65% decreased PAPP-A mRNA expression in other tissues as well, which was especially striking in kidney, tibia, thymus and spleen. These were designated as "non-specific" PAPP-A KO mice. With unknown and unpredictable specificity until harvest, we chose to assess a surrogate marker of lifespan i.e., thymic involution, in 15- to 18-month-old fPAPP-A/pos and WT/pos mice, the latter an important control for a possible effect of Nestin-Cre per se. Diminished thymic involution as indicated by increased thymic weight (135%, P = 0.035) and decreased histological disruption was seen in "non-specific" PAPP-A KO mice, similar to what was previously reported in 18-month-old global PAPP-A KO mice. There was no significant difference between "brain-specific" PAPP-A KO and control mice. This study highlights the importance of thorough characterization of assumed tissue-specific mouse models and awareness of potential germline recombination for proper data interpretation.

Genetic and Pharmacological Inhibition of PAPP-A Protects Against Visceral Obesity in Mice.

Pathogenicity of visceral adipose tissue (VAT) has been linked to the metabolic stress of enlarging mature adipocytes and a limited ability to recruit new adipocytes. One of the major distinguishing features of VAT preadipocytes is the high expression of the zinc metalloprotease, pregnancy-associated plasma protein-A (PAPP-A), when compared to subcutaneous adipose tissue (SAT). In this study we used 2 different approaches to investigate the effect of PAPP-A inhibition on different fat depots in mice on a high-fat diet (HFD) for 15 weeks. Conditional knockdown of PAPP-A gene expression in female adult mice resulted in significant decreases of 30% to 40% in adipocyte size in VAT (mesenteric and pericardial depots) compared to control mice. There was no effect on SAT (inguinal) or intra-abdominal perigonadal fat. Liver lipid was also significantly decreased without any effect on heart and skeletal muscle lipid. We found similar effects when using a pharmacological approach. Weekly injections of a specific immunoneutralizing monoclonal antibody (mAb-PA 1/41) or isotype control were given to male and female wild-type mice on HFD for 15 weeks. Adipocyte size was significantly decreased (30%-50%) only in VAT with mAb-PA 1/41 treatment. In this model, cell number was significantly increased in mesenteric fat in mice treated with mAb-PA 1/41, suggesting hyperplasia along with reduced hypertrophy in this VAT depot. Gene expression data indicated a significant decrease in F4/80 (macrophage marker) and interleukin-6 (proinflammatory cytokine) and a significant increase in adiponectin (anti-inflammatory adipokine with beneficial metabolic effects) in mesenteric fat compared to inguinal fat in mice treated with mAb-PA 1/41. Furthermore, there was significantly decreased liver lipid content with mAb-PA 1/41 treatment. Thus, using 2 different models systems we provide proof of principle that PAPP-A inhibition is a potential therapeutic target to prevent visceral obesity and its metabolic sequelae, such as fatty liver.

Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in Autoimmunity.

Appropriate balance of T helper 17 (Th17) and regulatory T (Treg) cells maintains immune tolerance and host defense. Disruption of Th17-Treg cell balance is implicated in a number of immune-mediated diseases, many of which display dysregulation of the insulin-like growth factor (IGF) system. Here, we show that, among effector T cell subsets, Th17 and Treg cells selectively expressed multiple components of the IGF system. Signaling through IGF receptor (IGF1R) activated the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, increased aerobic glycolysis, favored Th17 cell differentiation over that of Treg cells, and promoted a heightened pro-inflammatory gene expression signature. Group 3 innate lymphoid cells (ILC3s), but not ILC1s or ILC2s, were similarly responsive to IGF signaling. Mice with deficiency of IGF1R targeted to T cells failed to fully develop disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Thus, the IGF system represents a previously unappreciated pathway by which type 3 immunity is modulated and immune-mediated pathogenesis controlled.

Metalloproteinase PAPP-A regulation of IGF-1 contributes to polycystic kidney disease pathogenesis.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of Pkd1RC/RC mice, a model of ADPKD. Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that cleaves inhibitory IGF binding proteins (IGFBPs), increasing the local bioactivity of IGF-1, was highly induced in the kidney of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed that PAPP-A transcription in ADPKD was mainly regulated through the cAMP/CREB/CBP/p300 pathway. Pappa deficiency effectively inhibited the development of cysts in the Pkd1RC/RC mice. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with a monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1RC/RC mice and ex vivo in embryonic kidneys. These data indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a therapeutic strategy for ADPKD that involves the inhibition of PAPP-A.

Effects of suppressing bioavailability of insulin-like growth factor on age-associated intervertebral disc degeneration.

Suppression of the insulin-like growth factor-1 (IGF-1) signaling pathway reduces age-related disorders and increases lifespan across species, making the IGF-1 pathway a key regulator of aging. Previous in vitro intervertebral disc cell studies have reported the pro-anabolic effect of exogenously adding IGF-1 on matrix production. However, the overall effects of suppressing IGF-1 signaling on age-related intervertebral disc degeneration (IDD) is not known. Here, the effects of suppressing IGF-1 signaling on age-related IDD in vivo were examined using PAPPA -/- mice. These are animals with targeted deletion of pregnancy-associated plasma protein A (PAPPA), the major protease that cleaves inhibitory IGF binding proteins that control bioavailability of IGF-1 for cell signaling. Compared to age-matched wild-type (Wt) littermates, reduced levels of matrix proteoglycan (PG) and aggrecan were seen in discs of 23-month old PAPPA -/- mice. Decreased aggrecanolysis and expression of two key catabolic markers, matrix metalloproteinase-3 and a disintegrin and metalloproteinase with thrombospondin motifs-4, were also observed in discs of old PAPPA -/- mice compared to Wt littermates. Suppressing IGF-1 signaling has been implicated to shift cellular metabolism toward maintenance rather than growth and decreasing cellular senescence. Along this line, discs of old PAPPA -/- mice also exhibited lower cellular senescence, assessed by p53 and lamin B1 markers. Collectively, the data reveal complex regulation of disc matrix homeostasis by PAPPA/IGF-1 signaling during chronologic aging, that is, reduced IGF-1 bioavailability confers the benefit of decreasing disc cellular senescence and matrix catabolism but also the disadvantage of decreasing disc PG matrix anabolism. This pathway requires further mechanistic elucidation before IGF-1 could be considered as a therapeutic growth factor for treating IDD.

Overcoming platinum resistance in ovarian cancer by targeting pregnancy-associated plasma protein-A.

OBJECTIVES: Inhibition of pregnancy-associated plasma protein-A (PAPP-A), an upstream activator of the insulin-like growth factor (IGF) pathway, is known to augment sensitivity to platinum-based chemotherapy. This study further tests the efficacy of PAPP-A inhibition with a monoclonal antibody inhibitor (mAb-PA) in ovarian cancer (OC) platinum-resistant patient-derived xenograft (PDX) models. METHODS: PAPP-A expression was quantitated in platinum-resistant PDX models by ELISA. A subset with High (n = 5) and Low (n = 2) expression were revived in female SCID/beige mice for studies with either saline, carboplatin/paclitaxel (CP) + mAb-PA, or CP + IgG2a. The primary endpoint was tumor area by ultrasound on day 28 relative to baseline. Conversion to platinum-sensitive was defined by average tumor regression below baseline. Statistical analyses included linear mixed effects modeling and Kaplan Meier curves. Response to therapy was correlated with changes in the ratio of phosphorylated/total AKT and ERK 1/2 using Wes analysis. RESULTS: The addition of mAb-PA to CP induced tumor regression below baseline in one High PAPP-A PDX model; another three models exhibited notable growth inhibition relative to CP + IgG2a. None of the Low PAPP-A PDX models regressed below baseline. The PDX model with the greatest magnitude of tumor regression from baseline after combination therapy was maintained on single agent mAb-PA or IgG2a, but no benefit was observed. Decreased phosphorylation of ERK1/2 correlated with conversion to platinum-sensitive. CONCLUSIONS: The addition of mAb-PA to CP overcame platinum-resistance in one of five High PAPP-A PDX models; three other models demonstrated improved platinum-response. This supports further clinical development of this novel therapeutic.

PAPP-A and the IGF system in atherosclerosis: what's up, what's down?

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase with a well-established role in releasing bioactive insulin-like growth factor-1 (IGF-1) from IGF-binding protein-2, -4, and -5 by proteolytic processing of these. The IGF system has repeatedly been suggested to be involved in the pathology of atherosclerosis, and both PAPP-A and IGF-1 are proposed biomarkers and therapeutic targets for this disease. Several experimental approaches based on atherosclerosis mouse models have been undertaken to obtain causative and mechanistic insight to the role of these molecules in atherogenesis. However, reports seem conflicting. The literature suggests that PAPP-A is detrimental, while IGF-1 is beneficial. This raises important questions that need to be addressed. Here we summarize the various studies and discuss potential underlying explanations for this seemingly inconsistency with the objective of better understanding complexities and limitations when manipulating the IGF system in mouse models of atherosclerosis. A debate clarifying what's up and what's down is highly warranted going forward with the ultimate goal of improving atherosclerosis therapy by targeting the IGF system.

Cellular characterization of human epicardial adipose tissue: highly expressed PAPP-A regulates insulin-like growth factor I signaling in human cardiomyocytes.

Little is known about the cellular biology of fat surrounding the human heart. In this study, we obtained paired samples of epicardial fat, the visceral fat depot attached to the heart, and subcutaneous skin fat from patients undergoing open heart surgery to test the hypothesis that human epicardial fat cells differentially express bioactive molecules that have the potential to affect cardiac function. First, we characterized the free fatty acids (FFAs), adipocytokines, and growth factors secreted by isolated adipocytes and preadipocytes in cell culture. There was little to distinguish the fat cell secretory products in terms of FFAs and adipocytokines. The most striking finding was that preadipocytes from epicardial adipose tissue expressed high levels of pregnancy-associated plasma protein-A (PAPP-A), a novel metalloproteinase that enhances local insulin-like growth factor (IGF) action through cleavage of inhibitory IGF binding protein-4 (IGFBP-4). PAPP-A levels were 15-fold higher in conditioned medium from epicardial preadipocytes than from subcutaneous preadipocytes (P < 0.0001). PAPP-A was not expressed in mature adipocytes. Next we determined whether PAPP-A could affect IGF-I signaling in a human cardiomyocyte cell line. IGF-I activated receptor-mediated auto-phosphorylation, and this was blocked by wild-type and protease-resistant IGFBP-4. Addition of PAPP-A induced cleavage of wild-type, but not protease-resistant, IGFBP-4 thereby restoring IGF-I action. A proteolytically defective PAPP-A had no effect. IGF-I receptor-mediated signaling through the phosphatidylinositol 3-kinase pathway was similarly inhibited by IGFBP-4 and restored by PAPP-A. Thus, human epicardial fat cells differentially express PAPP-A, which has the potential to affect IGF signaling in the heart.

Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion.

BACKGROUND: Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling. METHODS: By comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided. RESULTS: EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways. CONCLUSION: This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.

Characterization of mouse pericardial fat: regulation by PAPP-A.

Although implicated in cardiovascular disease, little is known about the fat surrounding the heart. In humans, epicardial fat is the visceral fat depot of the heart, which directly contacts the myocardium. This strategically placed fat depot is thought to produce bioactive molecules that could affect cardiac function. A major limitation in understanding the biology of epicardial fat is its restricted access in humans and its seeming absence in commonly-used experimental animal models. Although laboratory mice do not have epicardial fat per se, they do have a fat depot around the heart. In this study, we found that mouse pericardial fat has the molecular signature, small adipocyte size, and resistance to differentiation consistent with visceral fat. In addition, we show that mouse pericardial fat is regulated by pregnancy-associated plasma protein-A (PAPP-A), a key modulator of local insulin-like growth factor bioavailability. PAPP-A is highly expressed in mouse pericardial fat at levels equivalent to those in mesenteric visceral fat and 10-fold higher than in subcutaneous inguinal fat (P = .0003). Cultured pre-adipocytes isolated from pericardial fat show 2-fold increased PAPP-A secretion compared to pre-adipocytes isolated from inguinal fat. Furthermore, PAPP-A knock-out mice fed a high fat diet for 20 weeks have significantly reduced pericardial fat (by 60%; P < .0001) compared to wild-type littermates. There was no significant difference in inguinal fat between wild-type and PAPP-A knock-out mice. These data characterize a new mouse model of visceral-like pericardial fat and lay a foundation for understanding its role in human heart disease.

PAPP-A and cancer.

The zinc metalloproteinase, PAPP-A, enhances local insulin-like growth factor (IGF) action through cleavage of inhibitory IGF-binding proteins, thereby increasing IGF available for IGF receptor-mediated cell proliferation, migration and survival. In many tumors, enhanced IGF receptor signaling is associated with tumor growth, invasion and metastasis. We will first discuss PAPP-A structure and function, and post-translational inhibitors of PAPP-A expression or proteolytic activity. We will then review the evidence supporting an important role for PAPP-A in many cancers, including breast, ovarian and lung cancer, and Ewing sarcoma.

The IGF-p53 connection in cancer.

Healthy tissue growth depends on a well-controlled and context-appropriate balance of cellular proliferation, cell cycle arrest, and programmed cell death (apoptosis). Disturbance of this balance by activation of oncogenes, inactivation/mutation of tumor suppressor genes, or inhibition of apoptosis can promote tumorigenesis. This mini-review will focus on evidence for the contribution of insulin-like growth factor (IGF) signaling and its regulation by the transcription factor, p53, to tumor development and progression.

Inducible knockdown of pregnancy-associated plasma protein-A gene expression in adult female mice extends life span.

Pregnancy-associated plasma protein-A (PAPP-A) knockout (KO) mice, generated through homologous recombination in embryonic stem cells, have a significantly increased lifespan compared to wild-type littermates. However, it is unknown whether this longevity advantage would pertain to PAPP-A gene deletion in adult animals. In the present study, we used tamoxifen (Tam)-inducible Cre recombinase-mediated excision of the floxed PAPP-A (fPAPP-A) gene in mice at 5 months of age. fPAPP-A mice, which were either positive (pos) or negative (neg) for Tam-Cre, received Tam treatment with quarterly boosters. Only female mice could be used with this experimental design. fPAPP-A/neg and fPAPP-A/pos mice had similar weights at the start of the experiment and showed equivalent weight gain. We found that fPAPP-A/pos mice had a significant extension of life span (P = 0.005). The median life span was increased by 21% for fPAPP-A/pos compared to fPAPP-A/neg mice. Analysis of mortality in life span quartiles indicated that the proportion of deaths of fPAPP-A/pos mice were lower than fPAPP-A/neg mice at young adult ages (P = 0.002 for 601-800 days) and higher than fPAPP-A/neg mice at older ages (P = 0.004 for >1000 days). Thus, survival curves and age-specific mortality indicate that female mice with knockdown of PAPP-A gene expression as adults have an extended healthy life span.