Pregnancy-associated Plasma protein-A Expression Research Articles

Effects of GH treatment in GH-deficient adults on adiponectin, leptin and pregnancy-associated plasma protein-A.

GH deficiency (GHD) in adults is associated with adverse effects on metabolism and increased cardiovascular risk. Pregnancy-associated plasma protein-A (PAPP-A) is a protease that promotes IGF-I availability in vascular tissues. PAPP-A levels appear to correlate with carotid intima-media thickness and have been proposed as an early predictor of cardiac events. The aim of our study was to evaluate PAPP-A levels in GHD adults at baseline and after GH replacement and correlate them with changes in body composition, lipid profile, glucose homeostasis, inflammatory markers and in leptin and adiponectin. Fourteen GHD adults were evaluated at baseline and after 1 year of GH therapy. All patients were compared at baseline with 28 age-, sex- and body mass index (BMI)-matched control subjects. At baseline, GHD adults showed higher PAPP-A levels (P=0.03) and higher leptin (P=0.04), fibrinogen (P=0.002) and highly sensitive C-reactive protein (P=0.01) values than controls. Therapy with GH reduced PAPP-A (P=0.03) and fibrinogen levels (P=0.002) while increased BMI (P=0.01) and reduced waist-hip ratio (WHR; P=0.05) were observed. Insulin and homeostasis model assessment of insulin resistance index increased after treatment (P<0.004/P=0.007), without changes in leptin or adiponectin levels. PAPP-A values correlated positively with BMI and WHR and negatively with adiponectin before and after treatment, with no correlation with glucose homeostasis parameters, lipid profile or leptin. Our study suggests that PAPP-A expression is increased in GHD adults, and that 1 year of GH replacement therapy is able to reduce PAPP-A levels in this population. However, further studies are required to determine whether this decrease correlates with an improvement in atherosclerosis.

Transgenic Overexpression of Pregnancy-Associated Plasma Protein-A Increases the Somatic Growth and Skeletal Muscle Mass in Mice

Although IGFs are indispensable to skeletal muscle development, little information is available regarding the mechanisms regulating the local action of IGFs in skeletal muscle tissues. Here we tested the hypothesis that pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, promotes skeletal muscle formation in vivo through degrading IGF binding proteins (IGFBPs), which increases the bioavailability of IGFs. Expression of PAPP-A is significantly increased in muscle five days after muscle injury in mice. Targeted overexpression of PAPP-A using a muscle-specific promoter significantly increased the prenatal/postnatal growth, skeletal muscle weight, and muscle fiber area in mice. These anabolic effects were reproduced using F2/F3 progeny. Free IGF-I concentration was severalfold higher in the conditioned medium (CM) of ex vivo cultured muscle from the transgenic mice, compared with the wild-type littermate muscle. Accordingly, the proliferation of C2C12 myoblasts was significantly increased in the presence of CM from cultured skeletal muscle of the transgenic mice, compared with the controls. This observed increase in myoblast proliferation was abolished on addition of noncleavable IGFBP-4 peptide, which reduced free IGF-I concentration back to the basal level of the wild-type CM. Furthermore, proliferation and differentiation of C2C12 myoblasts was increased by transient overexpression of proteolytically active PAPP-A but not by inactive mutant PAPP-A (E483/A). Collectively, we identified PAPP-A as a novel regulator of prenatal/postnatal growth and skeletal muscle formation in vivo. Moreover, our studies provide the first experimental evidence that IGFBP degradation is a key determinant in modulating the local action of IGFs in muscle.

PAPP-A: A possible pathogenic link to the instability of atherosclerotic plaque

The rupture of coronary atherosclerotic plaque and subsequent thrombus formation are major events underlying acute coronary syndromes (ACS). Pregnancy associated plasma protein A (PAPP-A) is a member of the metzincin superfamily of metalloproteinases originally identified in the serum of pregnant women. Recent studies indicate that ACS is associated with elevated serum concentrations of PAPP-A. PAPP-A level is not only a marker of plaque instability favoring the progression to myocardial infarction, but is indicative of a poor prognosis even after the occurrence of an acute ischemic event caused by plaque instability. Why PAPP-A expression in unstable plaques is high is a puzzling problem. We hypothesized that PAPP-A is a possible cause of the instability of atherosclerotic plaque which plays a role in ACS. Studies found that PAPP-A was abundantly expressed in both eroded and ruptured plaques, but was only minimally expressed in stable plaques. Other studies have also demonstrated that patients with hyperechoic or isoechoic carotid plaques exhibit significantly higher PAPP-A levels than those with hypoechoic early carotid lesions. If the hypothesis is confirmed, administration of PAPP-A monoclonal antibodies may be used to eliminate the pathogen. It will be a new target point to treat ACS.

Surface association of pregnancy-associated plasma protein-A accounts for its colocalization with activated macrophages

Intense immunostaining for pregnancy-associated plasma protein-A (PAPP-A), a newly characterized metalloproteinase in the insulin-like growth factor system, colocalizes with activated macrophages in human atherosclerotic plaque. To determine macrophage regulation of PAPP-A expression, we developed two models of human macrophages with basal and activated phenotypes. THP-1 cells and peripheral blood monocytes could be differentiated into macrophages and activated upon specific treatment regimens with phorbol myristate acetate, macrophage colony-stimulating factor, and interleukin-1beta. Activation was assessed by cell secretion of tumor necrosis factor-alpha, which increased 30- to 100-fold with activation. Activated macrophages also secreted matrix metalloproteinase-9. However, no PAPP-A mRNA or PAPP-A antigen could be detected in these cells under any condition. Upon incubation with recombinant PAPP-A, we found that activated macrophages bound and internalized more PAPP-A than unactivated macrophages or monocytes. Internalization accounted for at least 50% of macrophage-associated PAPP-A, as assessed in studies with cytochalasin B. Membrane-bound PAPP-A retained protease activity, whereas internalized PAPP-A had little or no activity. Similar experiments carried out with a mutated variant of PAPP-A, which retains functionality as a protease but is unable to bind surface-associated glycosaminoglycan, showed no macrophage association or internalization. Absence of PAPP-A expression was confirmed in activated macrophages isolated from a hypercholesterolemic rabbit model of atherosclerosis. We therefore conclude that PAPP-A is not synthesized in, but rather is bound and internalized by, macrophages. Our findings likely account for the observed intense immunostaining for PAPP-A colocalizing with activated macrophages and may have physiological significance in the development of vulnerable plaque.

Modulation of PAPP-A Expression By PPARγ in Human First Trimester Trophoblast

Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin metalloproteinase that cleaves the insulin-like growth factor (IGF)-dependent binding protein-4 and increases in maternal serum during pregnancy. In human placenta PAPP-A is expressed both in villous cytotrophoblasts (VCT) that cover the chorionic villi and in extravillous cytotrophoblasts (EVCT) of the anchoring villi. Due to the key role of PPARgamma in human trophoblast differentiation such as syncytiotrophoblast formation and EVCT invasion, we studied the effect of PPARgamma activation on PAPP-A expression using our in vitro model of EVCT and VCT primary cultures isolated from the same first trimester chorionic villi. First, we demonstrated that invasive EVCT expressed and secreted 10 times more PAPP-A than VCT did. Then, we showed that activation of PPARgamma inhibited PAPP-A gene expression and secretion in EVCT, whereas it had no effect in VCT. Since we have previously shown that PPARgamma agonist inhibits EVCT invasion in vitro, we suggest that PPARgamma-mediated inhibition of PAPP-A might decrease the amount of bioactive IGFII, a factor known to promote trophoblast invasion.

Cytokine stimulation of pregnancy-associated plasma protein A expression in human coronary artery smooth muscle cells: inhibition by resveratrol

Through specific cleavage of proteins that bind and inhibit insulin-like growth factor-I (IGF-I), pregnancy-associated plasma protein-A (PAPP-A) enhances local IGF-I availability, and, consequently, receptor activation. PAPP-A expression is increased in experimental models of vascular injury and in human atherosclerotic plaque; however, little is known about the regulation of PAPP-A gene expression in vascular cells. In this study, we tested the hypothesis that proinflammatory cytokines involved in the vascular injury response stimulate PAPP-A gene expression in human coronary artery smooth muscle cells (hCASMC) in culture. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta stimulated PAPP-A gene expression in a time- and dose-dependent manner. The effect of these cytokines appears to be at the level of transcription because actinomycin D completely prevented the induction of PAPP-A gene expression. Accumulation of PAPP-A in cell-conditioned medium paralleled mRNA synthesis, as did proteolytic activity against IGF binding protein-4 (IGFBP-4). Interestingly, pretreatment of hCASMC with resveratrol, a polyphenol found in the skin of grapes and in red wine purported to underlie the "French paradox," inhibited TNF-alpha- and IL-1beta-induced PAPP-A expression and, hence, its IGFBP-4 proteolytic activity. Resveratrol had no effect on basal PAPP-A expression and protease activity. Our finding that PAPP-A gene expression in hCASMC is stimulated by TNF-alpha and IL-1beta suggests a mechanism for the regulation of PAPP-A in response to vascular injury that may contribute to the enhanced IGF-I bioactivity in intimal hyperplasia and atherosclerotic plaque development. Our results also suggest that PAPP-A may be a target of the cardiovascular system-protective effects of resveratrol.

Stress-Activated Signaling Pathways Mediate the Stimulation of Pregnancy-Associated Plasma Protein-A Expression in Cultured Human Fibroblasts

Pregnancy-associated plasma protein-A (PAPP-A) is an IGF binding protein protease that appears to function as a posttranslational modulator of IGF bioavailability in response to injury. A previous study indicated that the proinflammatory cytokines, TNFalpha and IL-1beta, were potent stimulators of PAPP-A expression in cultured human fibroblasts. In this study, we investigated the intracellular signaling pathways mediating cytokine-stimulated PAPP-A expression. Treatment of human fibroblasts with TNFalpha and IL-1beta (1 nm) had little or no effect on phosphatidylinositol 3-kinase and Erk1/2 activation, pathways commonly associated with proliferation. On the other hand, TNFalpha and IL-1beta induced p38, c-Jun N-terminal kinase (JNK), and nuclear factor (NF)kappaB activation, pathways more closely related to stress response. An inhibitor of p38 activation (SB203580) had no effect on TNFalpha- or IL-1beta-stimulated PAPP-A expression. The JNK inhibitor, SP600125, had no effect on IL-1beta- or TNFalpha-stimulated PAPP-A mRNA expression. However, SP600125 effectively inhibited IL-1beta-induced PAPP-A protein expression. MG-132, a proteasome inhibitor that blocked degradation of the intrinsic NFkappaB inhibitor, IkappaB, and thereby prevented NFkappaB activation, was a potent inhibitor of both TNFalpha- and IL-1beta-stimulated PAPP-A mRNA and protein expression and IGF binding protein-4 protease activity. MG-132 had no effect on JNK phosphorylation or p38 activation, and SB203580 and SP600125 had no effect on IkappaB degradation, documenting inhibitor specificity. BAY11-7082, another inhibitor of NFkappaB activation, also inhibited TNFalpha- and IL-1beta-stimulated PAPP-A expression and IGF binding protein-4 protease activity. These data indicate that NFkappaB activation is the primary mediator of cytokine-stimulated PAPP-A expression in human fibroblasts.

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles.

In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work, we showed that follicular fluid from late dominant equine follicles (35 mm diameter) contains a proteolytic activity against IGFBP-2. First follicular fluid from dominant follicles contained lower levels of native IGFBP-2 than the corresponding serum, as assessed by Western ligand blotting. In contrast, immunoblotting experiments showed much higher levels of a 12 kDa proteolytic fragment in dominant follicular fluid than in the serum. Moreover, equine dominant follicular fluid was able to induce proteolysis of exogenous recombinant bovine (rb)IGFBP-2, this degradation being dose-dependently enhanced by IGFs. The proteolytic activity against IGFBP-2 in equine follicles was partially immunoneutralized by a polyclonal antibody raised against human PAPP-A. Moreover, cleavage of rbIGFBP-2 by equine follicular fluid was dose-dependently inhibited by a peptide derived from the heparin-binding domain of IGFBP-5, as well as by peptides derived from other heparin-binding domain-containing proteins such as connective tissue growth factor, vitronectin and heparin-interacting protein, previously shown to inhibit PAPP-A. Finally, the proteolytic activity was very low in subordinate follicles, was high in both early (25 mm diameter) and late (35 mm diameter) dominant follicles, and was slightly lower in preovulatory follicles recovered 35 h after human chorionic gonadotropin (hCG) treatment.Overall, these data show that in the equine ovary, the selection of dominant follicles is associated with an increase of the proteolytic degradation of IGFBP-2 by PAPP-A, as for IGFBP-4, and potentially other protease(s), probably contributing to the increase in IGF bioavailability. In atretic subordinate follicles, the decrease in the proteolytic degradation of IGFBP-2, probably due in part to a direct inhibition by peptides containing heparin-binding domains, contributes to the increase in IGFBP-2 levels and the decrease in IGF bioavailability. The expression of PAPP-A and IGFBP-2 mRNA during folliculogenesis remain to be investigated in the mare.

Pregnancy-associated plasma protein-a production in rat granulosa cells: stimulation by follicle-stimulating hormone and inhibition by the oocyte-derived bone morphogenetic protein-15.

Pregnancy-associated plasma protein-A (PAPP-A) is the major IGF binding protein-4 (IGFBP-4) protease in follicular fluid, consistent with its proposed role in folliculogenesis. Despite growing interest, almost nothing is known about how PAPP-A expression is regulated in any tissue. Here we show that FSH and oocytes regulate PAPP-A expression in granulosa cells (GCs). By in situ hybridization, ovary PAPP-A mRNA was markedly increased by pregnant mare serum gonadotropin treatment, and the message was localized to the membrana GCs but not cumulus GCs (CGCs) of dominant follicles. To explore the mechanism, we used primary cultures of rat GCs. Control (untreated) cells produced little or no PAPP-A spontaneously. Conversely, FSH markedly stimulated PAPP-A mRNA and protein in a dose- and time-dependent fashion. Interestingly, PAPP-A expression in isolated CGCs was also strongly induced by FSH, and the induction was inhibited by added oocytes. To investigate the nature of the inhibition, we tested the effect of oocyte-derived bone morphogenetic protein-15 (BMP-15). BMP-15 alone had no effect on basal levels of PAPP-A expression by cultures of membrana GCs or CGCs. However, BMP-15 markedly inhibited the FSH stimulation of PAPP-A production in a dose-dependent manner. The cleavage of IGFBP-4 by conditioned media from FSH-treated GCs was completely inhibited by anti-PAPP-A antibody, indicating the IGFBP-4 protease secreted by GCs is PAPP-A. These results demonstrate stimulatory and inhibitory roles for FSH and BMP-15, respectively, in regulating PAPP-A production by GCs. We propose that FSH and oocyte-derived BMP-15 form a controlling network that ensures the spatiotemporal pattern of GC PAPP-A expression in the dominant follicle.

Pregnancy-associated plasma protein-A (PAPP-A) expression and insulin-like growth factor binding protein-4 protease activity in normal and malignant ovarian surface epithelial cells.

Pregnancy-Associated Plasma Protein-A (PAPP-A) proteolyses insulin-like growth factor binding protein-4 (IGFBP-4), thereby regulating local IGF availability. Reduced PAPP-A mRNA expression has been reported in ovarian cancer specimens compared to normal ovarian surface epithelial cells (OSE). To characterize PAPP-A expression and proteolytic activity in OSE, we developed a lifespan-extended human cell model using a temperature-sensitive mutant of the SV40 large T antigen (SV40LT). These OSE(tsT) cells proliferate at 34 degrees C (i.e., when SV40LT-positive), but not at 39 degrees C, a temperature at which the SV40LT is unstable (SV40LT-negative). Proteolysis of radiolabeled IGFBP-4 in conditioned media from OSE(tsT) lines was IGF-dependent and blocked by anti-PAPP-A antisera. Temperature shifts that eliminated stable SV40LT induced a 7-fold increase in PAPP-A mRNA and a 4-fold increase in protein. The converse experiment (shifting to SV40LT-positive conditions) resulted in decreased levels of PAPP-A mRNA but little change in PAPP-A protein. Nevertheless, there was a marked reduction in IGF-BP-4 proteolytic activity in medium of SV40LT-positive OSE-(tsT) cells. This decreased PAPP-A activity coincided with a nearly 20-fold increase in mRNA encoding a physiological inhibitor of PAPP-A, the precursor form of eosinophil Major Basic Protein (proMBP), and 4- to 5-fold increases in proMBP protein. Primary cultures of unmodified OSE expressed high levels of PAPP-A and undetectable proMBP, and therefore produced abundant IGFBP-4 protease activity. Short-term ovarian tumor cell cultures expressed variable levels of PAPP-A and high levels of proMBP, and consequently secreted little or no IGFBP-4 protease activity. The concurrent regulation of PAPP-A and its inhibitor, proMBP, suggests that IGFBP-4 proteolysis and local regulation of IGF availability may be altered in malignant ovarian epithelial cells.

Regulation of pregnancy-associated plasma protein-A expression in cultured human osteoblasts

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase secreted by cultured human osteoblasts that has been implicated in the regulation of local insulin-like growth factor (IGF) bioavailability during bone growth and remodeling. However, very little is known about the regulation of PAPP-A expression in bone. In this study, we determined the effect of systemic and local osteoregulatory factors on PAPP-A mRNA and protein expression in normal human osteoblasts (hOB cells). Treatment of hOB cells with particular peptide growth factors (basic fibroblast growth factor, epidermal growth factor), steroid hormones (dexamethasone, 1,25-dihydroxyvitamin D 3), and cytokines [interleukin-6 (IL-6), IL-13, oncostatin M] with known involvement in bone cell physiology had no significant effect on PAPP-A expression. Agents that increase intracellular cyclic AMP (forskolin, prostaglandin E 2) increased PAPP-A mRNA and protein expression ∼3-fold. Tumor necrosis factor α (TNFα), IL-1β, and IL-4 also increased PAPP-A expression 3- to 4-fold. Transforming growth factor β (TGFβ) was previously shown to stimulate PAPP-A expression in hOB cells. The effects of TGFβ, TNFα, and IL-1β were additive, whereas the effects of TGFβ and IL-4 were synergistic. In summary, TNFα, IL-1β, and IL-4 were identified as potent stimulators of PAPP-A expression in primary cultures of human osteoblasts. These findings suggest a mechanism whereby cytokines present in bone and bone marrow could augment IGF bioavailability during skeletal growth and remodeling.

Pregnancy-associated plasma protein a gene expression as a target of inflammatory cytokines.

Pregnancy-associated plasma protein A (PAPP-A) cleaves IGF-binding protein-4 (IGFBP-4) and appears to enhance local IGF bioavailability in response to injury. In this study we determined the effects of growth factors and cytokines involved in the healing process on PAPP-A expression in human dermal fibroblasts. There was no effect of platelet-derived growth factor, epidermal growth factor, or basic fibroblast growth factor on PAPP-A mRNA expression in these cells. However, treatment with the proinflammatory cytokines, TNFalpha and IL-1 beta, resulted in time- and dose-dependent increases in PAPP-A mRNA and protein expression (3- to 4-fold maximal effects), which were prevented by actinomycin D. On the other hand, interferon-gamma (IFN gamma) treatment markedly inhibited PAPP-A expression. IGFBP-4 proteolytic activity was increased 4-fold in medium from TNFalpha- and IL-1 beta-treated (1 nm) cells and decreased 40% in medium from IFN gamma-treated (1 nm) cells. IGF-I-stimulated [(3)H]thymidine incorporation was significantly enhanced by pretreatment with 1 nm TNFalpha, and this enhancement was blocked in the presence of protease-resistant IGFBP-4. In conclusion, PAPP-A expression is regulated by inflammatory cytokines in adult human fibroblasts, with functional consequences on IGFBP-4 protease activity and IGF-I bioavailability. These data provide a mechanism for the regulation of PAPP-A in response to injury and further implicate PAPP-A in the wound-healing processes.

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

IGFBP-4 is an inhibitor of IGF-I in bone. We show that TGF-beta regulates IGFBP-4 and enhances IGF-I-stimulated growth of cultured human bone cells through increased expression of an IGFBP-4 protease, PAPP-A. This effect of TGF-beta on IGF-I bioavailability may promote local bone formation. Insulin-like growth factor binding protein (IGFBP-4) proteolysis is implicated in the regulation of local insulin-like growth factor (IGF)-I bioavailability during bone remodeling. The IGFBP-4 protease secreted by normal adult human osteoblastic (hOB) cells in culture is a novel metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). We have recently identified an inhibitor of PAPP-A, the precursor form of major basic protein (proMBP). Very little is known about the molecular regulation of this IGFBP-4 protease system. In the present study, we determined the effect of transforming growth factor (TGF)-beta and IGF-II, the two most abundant growth factors in human bone, on PAPP-A and proMBP expression in primary cultures of hOB cells. Treatment with TGF-beta resulted in time- and dose-dependent increases in PAPP-A mRNA expression, with a maximal 12-fold increase after 24 h of stimulation with 10 ng/ml TGF-beta. Increased PAPP-A levels in hOB cell-conditioned medium paralleled PAPP-A gene expression. In addition, TGF-beta completely suppressed proMBP expression. Treatment of hOB cells with IGF-II had no effect on PAPP-A or proMBP gene expression. However, IGFBP-4 proteolysis in cell-free assay was dependent on IGF-II, and there was increased IGF-II-dependent IGFBP-4 protease activity in conditioned medium from hOB cells that were treated with TGF-beta. IGF-I stimulation of hOB cell proliferation was markedly enhanced by pretreatment with TGF-beta and [Leu27]IGF-II, and this enhancement was prevented with protease-resistant IGFBP-4. In summary, TGF-beta regulates IGFBP-4 proteolysis in hOB cells through increased expression of the protease, PAPP-A, and decreased expression of the inhibitor, proMBP. However, functional activation of the IGFBP-4 protease system is dependent on IGF-II, which acts at a post-translational level. These data support a model whereby local TGF-beta and IGF-II in the bone microenvironment coordinately amplify IGF-I bioavailability through controlled IGFBP-4 proteolysis, which may be a means to promote bone formation.

Expression of Pregnancy-associated Plasma Protein-A (PAPP-A) During Human Villous Trophoblast Differentiation In Vitro

Pregnancy-associated placental protein-A (PAPP-A), first isolated from maternal serum, has been identified as a metalloprotease cleaving insulin-like growth factor binding protein-4 (IGFBP-4). The source of PAPP-A during pregnancy is unclear. We therefore investigated PAPP-A expression during in vitro human villous cytotrophoblast cell (CT) differentiation into syncytiotrophoblast (ST). CT were isolated from normal first trimester, second trimester and term placentae (n=10) and cultured to form ST. PAPP-A mRNA was quantified by real-time PCR, and PAPP-A protein expression was studied by immunocytochemistry and TRACE technology with specific monoclonal antibodies. PAPP-A mRNA expression in total placental extracts increased during the course of pregnancy. PAPP-A protein was detected in the cytoplasm of both CT and ST. ST formation in vitro was associated with a 19-fold increase in PAPP-A mRNA expression and an 8-fold increase in PAPP-A secretion into the culture medium. No significant difference in PAPP-A production was observed between cultured cells isolated from early and term placentae.In conclusion, PAPP-A production in vitro, is associated to the differentiation of villous cytotrophoblast cells into syncytiotrophoblast, independantly of the age of gestation.

The regulated expression of the pregnancy-associated plasma protein-A in the rodent ovary: a proposed role in the development of dominant follicles and of corpora lutea.

Compelling evidence exists displaying that the intrafollicular IGF-I system constitutes an obligatory mediator of FSH action in the murine ovary. Within this system, the ovarian IGF binding protein-4-directed protease (IGFBP-4ase) may have a critical role. Human IGFBP-4ase has been proved identical to the previously well-characterized pregnancy-associated plasma protein-A (PAPP-A). This communication reports the cloning and sequencing of the mouse PAPP-A cDNA as well as its expression and cellular localization in the mouse ovary. PAPP-A mRNA was undetectable in ovaries of untreated immature 25-d-old mice. Treatment with PMSG led to a marked time-dependent increase in PAPP-A expression in well-defined subsets of granulosa cells and follicles. Specifically, PAPP-A expression was detectable exclusively in centrifugally residing membrana granulosa cells of antral follicles during a 3- to 36-h period post PMSG. PAPP-A expression then fell to nondetectable levels in dominant preovulatory follicles at 48 h post PMSG. Treatment of PMSG-primed mice with human CG caused a rapid reinduction of PAPP-A expression in granulosa cells of dominant follicles and was sustained at relatively high levels throughout the ovulation and luteinization. These results suggest a role for gonadotropin-stimulated PAPP-A gene expression in the physiologic processes of dominant follicle development, ovulation, and luteogenesis in the mammalian ovary. The early onset and extended duration of gonadotropin-dependent PAPP-A expression in granulosa cells may serve to degrade the antigonadotropin IGFBP-4. Accordingly, successful antral follicle development, ovulation, and corpus luteum formation may be contingent on an IGFBP-4-deplete/PAPP-A-replete circumstance, hence resulting in an IGF-I-replete intrafollicular microenvironment.

Expression of recombinant murine pregnancy-associated plasma protein-A (PAPP-A) and a novel variant (PAPP-Ai) with differential proteolytic activity.

Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP-A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse. The overall sequence identity between murine and human PAPP-A is 91%, and murine PAPP-A contains sequence motifs previously described in the sequence of human PAPP-A. Through expression in mammalian cells, we show that murine PAPP-A and PAPP-Ai are active metalloproteinases, both capable of cleaving insulin-like growth factor binding protein (IGFBP)-4 and -5. Cleavage of IGFBP-4 is dramatically enhanced by the addition of IGF, whereas cleavage of IGFBP-5 is slightly inhibited by IGF, as previously established with human PAPP-A. Surprisingly, however, quantitative analyses demonstrate that the murine PAPP-Ai cleaves IGFBP-4 very slowly compared to PAPP-A, even though its ability to cleave IGFBP-5 is unaffected by the presence of the insert. By RT-PCR analysis, we find that both variants are expressed in several tissues. The level of mRNA in the murine placenta does not exceed the levels of other tissues analyzed. Furthermore, the IGFBP-4-proteolytic activity of murine pregnancy serum is not elevated. This is in striking contrast to the increase seen in human pregnancy serum, and the expression of PAPP-A in the human placenta, which exceeds other tissues at least 250-fold. Interestingly, the position of the insert of PAPP-Ai, within the proteolytic domain, lies in close proximity to the cysteine residue, which in human PAPP-A forms a disulfide bond with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP-A-proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP-Ai and the linkage to proMBP is not known. Importantly, these data support the development of the mouse as a model organism for the study of PAPP-A, which must take into account the differences between the mouse and the human.

Pregnancy-associated plasma protein A as a marker of acute coronary syndromes.

Circulating markers indicating the instability of atherosclerotic plaques could have diagnostic value in unstable angina or acute myocardial infarction. We evaluated pregnancy-associated plasma protein A (PAPP-A), a potentially proatherosclerotic metalloproteinase, as a marker of acute coronary syndromes. We examined the level of expression of PAPP-A in eight culprit unstable coronary plaques and four stable plaques from eight patients who had died suddenly of cardiac causes. We also measured circulating levels of PAPP-A, C-reactive protein, and insulin-like growth factor I (IGF-I) in 17 patients with acute myocardial infarction, 20 with unstable angina, 19 with stable angina, and 13 controls without atherosclerosis. PAPP-A was abundantly expressed in plaque cells and extracellular matrix of ruptured and eroded unstable plaques, but not in stable plaques. Circulating PAPP-A levels were significantly higher in patients with unstable angina or acute myocardial infarction than in patients with stable angina and controls (P<0.001). A PAPP-A threshold value of 10 mlU per liter identified patients who had acute coronary syndromes with a sensitivity of 89.2 percent and a specificity of 81.3 percent. PAPP-A levels correlated with levels of C-reactive protein and free IGF-I, but not with markers of myocardial injury (troponin I and the MB isoform of creatine kinase). PAPP-A is present in unstable plaques, and circulating levels are elevated in acute coronary syndromes; these increased levels may reflect the instability of atherosclerotic plaques. PAPP-A is a new candidate marker of unstable angina and acute myocardial infarction.

Insulin-like growth factor binding protein-4 protease produced by smooth muscle cells increases in the coronary artery after angioplasty.

Insulin-like growth factor (IGF)-I stimulates vascular smooth muscle cell (VSMC) migration and proliferation, which are fundamental to neointimal hyperplasia in postangioplasty restenosis. IGF-I action is modulated by several high-affinity IGF binding proteins (IGFBPs). IGFBP-4 is the predominant IGFBP produced by VSMCs and is a potent inhibitor of IGF-I action. However, specific IGFBP-4 proteases can cleave IGFBP-4 and liberate active IGF-I. In this study, we document IGFBP-4 protease produced by human and porcine coronary artery VSMCs in culture as pregnancy-associated plasma protein-A (PAPP-A). This was shown by a distinctive IGFBP-4 cleavage pattern, specific inhibition of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition of PAPP-A by monoclonal antibodies. Furthermore, we found a 2-fold increase in IGFBP-4 protease activity in injured porcine VSMC cultures in vitro (P<0.05). We also evaluated IGFBP-4 protease/PAPP-A expression in vivo after coronary artery balloon injury. Twenty-five immature female pigs underwent coronary overstretch balloon injury, and vessels were examined at defined time points after the procedure. Abundant PAPP-A expression was observed in the cytoplasm of medial and neointimal cells 7, 14, and 28 days after angioplasty (P<0.01 vs control). The highest PAPP-A labeling indices were located in the neointima (36.1+/-2.1%) and the media (31.7+/-1.2%) 28 days after injury. Western blot analysis confirmed increased PAPP-A in injured vessels. PAPP-A, a regulator of IGF-I bioavailability through proteolysis of IGFBP-4, is thus expressed by VSMCs in vitro and in restenotic lesions in vivo. These results suggest a possible role for PAPP-A in neointimal hyperplasia.

Pregnancy-Associated Plasma Protein-A Gene Expression in Human Ovaries Is Restricted to Healthy Follicles and Corpora Lutea

Recently, Pregnancy-Associated Plasma Protein-A (PAPP-A) in human follicular fluid was identified as an insulin-like growth factor binding protein-4 protease (IGFBP-4ase). The ability of IGFBP-4ase to inactivate the FSH antagonist, IGFBP-4, has suggested a possible role for PAPP-A in regulating FSH action. Despite growing interest in this protease, the question of whether the PAPP-A gene is expressed in ovaries of normal cycling women is unknown. To fill this basic gap in our knowledge, we have identified the cellular sites of PAPP-A gene expression in normal human ovaries by in situ hybridization. PAPP-A mRNA was low or undetectable in preantral follicles, small (1–2 mm) healthy and atretic antral follicles, larger atretic antral follicles, surface epithelium, tunica albuginea and connective tissue cells. In contrast, an intense PAPP-A hybridization signal was evident in the healthy antral follicles examined from 5 mm to the preovulatory stage. In these follicles, the signal was restricted to the granulosa cells (GC). An intense signal for PAPP-A mRNA was also present in healthy corpora lutea (CL), being localized to a subset of large luteal cells. Collectively, these results provide the first evidence that the gene encoding PAPP-A is expressed in ovaries of normal cycling women and show that the gene is expressed almost exclusively in healthy GC and CL cells. The restricted pattern of PAPP-A expression in normal human ovaries suggests that PAPP-A may be a functional marker of the dominant follicle and its product, the CL. Although the physiological function of ovarian PAPP-A remains to be identified, we hypothesize it might play a role in controlling survival, growth, and/or differentiation of the dominant follicle and CL by inactivating the gonadotropin antagonist, IGFBP-4.

Pregnancy-associated plasma protein-A gene expression in human ovaries is restricted to healthy follicles and corpora lutea.

Recently, Pregnancy-Associated Plasma Protein-A (PAPP-A) in human follicular fluid was identified as an insulin-like growth factor binding protein-4 protease (IGFBP-4ase). The ability of IGFBP-4ase to inactivate the FSH antagonist, IGFBP-4, has suggested a possible role for PAPP-A in regulating FSH action. Despite growing interest in this protease, the question of whether the PAPP-A gene is expressed in ovaries of normal cycling women is unknown. To fill this basic gap in our knowledge, we have identified the cellular sites of PAPP-A gene expression in normal human ovaries by in situ hybridization. PAPP-A mRNA was low or undetectable in preantral follicles, small (1-2 mm) healthy and atretic antral follicles, larger atretic antral follicles, surface epithelium, tunica albuginea and connective tissue cells. In contrast, an intense PAPP-A hybridization signal was evident in the healthy antral follicles examined from 5 mm to the preovulatory stage. In these follicles, the signal was restricted to the granulosa cells (GC). An intense signal for PAPP-A mRNA was also present in healthy corpora lutea (CL), being localized to a subset of large luteal cells. Collectively, these results provide the first evidence that the gene encoding PAPP-A is expressed in ovaries of normal cycling women and show that the gene is expressed almost exclusively in healthy GC and CL cells. The restricted pattern of PAPP-A expression in normal human ovaries suggests that PAPP-A may be a functional marker of the dominant follicle and its product, the CL. Although the physiological function of ovarian PAPP-A remains to be identified, we hypothesize it might play a role in controlling survival, growth, and/or differentiation of the dominant follicle and CL by inactivating the gonadotropin antagonist, IGFBP-4.