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.

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.

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.

PAPP-A affects tendon structure and mechanical properties.

Pregnancy-associated plasma protein-A (PAPP-A) serves to increase local insulin-like growth factor (IGF) stimulation of proliferation and differentiation in many tissues through proteolysis of inhibitory IGF-binding proteins. The purpose of this study was to investigate the effects of PAPP-A on tendon structure and mechanical properties. A total of 30 tails from 6-month-old mice were tested with 10 tails in each of following groups: PAPP-A knockout (KO), skeletal-specific PAPP-A overexpressing transgenic (Tg) and wild type (WT). Morphologically, the total tail cross-sectional area (CSA), individual tissue CSAs of bone, muscle and tendon, and fascicle diameter were measured. A fascicle pullout test was performed to assess stiffness and strength of interfascicular structures. Fascicles were mechanically characterized through low and high displacement rate uniaxial tension tests providing modulus at each rate, hysteresis area and stress relaxation ratio. The KO mice had a smaller total tail CSA (p<0.05), fascicle diameter (p<0.05), absolute tendon CSA (p<0.05), fast and slow stiffness (p<0.05 for both) and larger hysteresis area (p<0.05) compared to WT and Tg mice. On the other hand, the Tg mice had a larger fascicle diameter (p<0.05), absolute tendon CSA (p<0.05), higher interfascicular strength and stiffness (p<0.05) and lower fascicular modulus at low displacement rates (p<0.05) compared to WT and KO mice. Tg mice also had larger total tail CSA area (p<0.05) and smaller hysteresis area (p<0.05) than KO mice, and larger normalized tendon CSA (p<0.05) than WT mice. Based on these data, we conclude that PAPP-A affects fascicle structure, thereby affecting tendon phenotype.

Pregnancy-associated plasma protein A (PAPP-A) modulates the early developmental rate in zebrafish independently of its proteolytic activity.

Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.

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.

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.

Constitutive expression of pregnancy-associated plasma protein-A in arterial smooth muscle reduces the vascular response to injury in vivo.

Pregnancy-associated plasma protein-A (PAPP-A) functions to increase local IGF-I bioactivity. In this study, we used transgenic mice that constitutively express human PAPP-A in arterial smooth muscle to test the hypothesis that overexpression of PAPP-A enhances vascular smooth muscle cell (SMC) response to IGF-I in vivo. PAPP-A transgenic (Tg) and wild-type (WT) mice underwent unilateral carotid ligation, a model of injury-induced SMC hyperplasia and neointimal formation. In both WT and PAPP-A Tg mice, endogenous PAPP-A mRNA expression showed peak elevation 5 days after carotid ligation. However, PAPP-A Tg mice had 70-75% less neointima than WT at 5 and 10 days postligation, with a significant reduction in occlusion of the ligated artery. WT and PAPP-A Tg mice had equivalent increases in medial area and vessel remodeling postligation. There was little change in medial area and no evidence of neointima in the contralateral carotid of WT or PAPP-A Tg mice. Both WT and PAPP-A Tg carotids exhibited signs of dedifferentiation of SMC, which precedes the increase in proliferation and migration that results in neointimal formation. However, the number of proliferating cells in the media and neointima of the ligated PAPP-A Tg artery was reduced by 90% on day 5 postsurgery compared with WT. This decrease was associated with a significant decrease in an in vivo marker of IGF-I bioactivity and reduced IGF-I-stimulated receptor phosphorylation ex vivo. These data suggest differential effects of chronic (transgenic) and transient (endogenous) PAPP-A expression on neointimal formation following vascular injury that may be due in part to the differential impact on IGF-I signaling.

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.

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.

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.

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.

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.

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.

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.

Lack of functional pregnancy-associated plasma protein-A (PAPPA) compromises mouse ovarian steroidogenesis and female fertility.

The insulin-like growth factor (IGF) system plays an important role in regulating ovarian follicular development and steroidogenesis. IGF binding proteins (IGFBP) mostly inhibit IGF actions, and IGFBP proteolysis is a major mechanism for regulating IGF bioavailability. Pregnancy-associated plasma protein-A (PAPPA) is a secreted metalloprotease responsible for cleavage of IGFBP4 in the ovary. The aim of this study was to investigate whether PAPPA plays a role in regulating ovarian functions and female fertility by comparing the reproductive phenotype of wild-type (WT) mice with mice heterozygous or homozygous for a targeted Pappa gene deletion (heterozygous and PAPP-A knockout [KO] mice, respectively). When mated with WT males, PAPP-A KO females demonstrated an overall reduction in average litter size. PAPP-A KO mice had a reduced number of ovulated oocytes, lower serum estradiol levels following equine chorionic gonadotropin administration, lower serum progesterone levels after human chorionic gonadotropin injection, and reduced expression of ovarian steroidogenic enzyme genes, compared to WT controls. In PAPP-A KO mice, inhibitory IGFBP2, IGFBP3, and IGFBP4 ovarian gene expression was reduced postgonadotropin stimulation, suggesting some compensation within the ovarian IGF system. Expression levels of follicle-stimulating hormone receptor, luteinizing hormone receptor, and genes required for cumulus expansion were not affected. Analysis of preovulatory follicular fluid showed complete loss of IGFBP4 proteolytic activity in PAPP-A KO mice, demonstrating no compensation for loss of PAPPA proteolytic activity by other IGFBP proteases in vivo in the mouse ovary. Taken together, these data demonstrate an important role of PAPPA in modulating ovarian function and female fertility by control of the bioavailability of ovarian IGF.

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.

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.

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.

Regional variation in adipogenesis and IGF regulatory proteins in the fetal baboon.

Intrauterine growth rate is associated with body distribution in adulthood suggesting differential response of fetal fat depots to nutritional modifications. We hypothesize that there is regional differences in fetal adipogenesis, in part, due to depot-specific regulation of the availability of insulin growth factors. In near-term baboon fetuses (n=3-5), the subcutaneous abdominal vs. omental preadipocytes had (1) more extensive lipid accumulation as assessed by BODIPY (lipid staining) to DAPI (nuclei) absorbance ratios (mean+/-SEM; 0.51+/-0.21, 0.35+/-0.09, p<0.05), (2) lower (p<0.05) secretion of IGF-binding protein 4 (9.6+/-1.2 vs. 17.4+/-2.8 ng/ml) and its protease pregnancy associated plasma protein A (24.6+/-1.9 vs. 39.1+/-6.3 microIU/ml), (3) lower protein expression of IGF2 "clearance" receptor in cell lysate (0.28+/-0.03 vs. 0.53+/-0.02 OD U/mm(2), p<0.05); all variables were intermediate in femoral preadipocytes. The regional variation of the adipogenesis and the IGF regulatory pathway set the stage for differential responsiveness of fat depots to external signals.