Pregnancy-associated Plasma protein-A mRNA Expression Research Articles

Pregnancy-associated plasma protein-A modulates the anabolic effects of parathyroid hormone in mouse bone

Intermittent parathyroid hormone (PTH) is a potent anabolic therapy for bone, and several studies have implicated local insulin-like growth factor (IGF) signaling in mediating this effect. The IGF system is complex and includes ligands and receptors, as well as IGF binding proteins (IGFBPs) and IGFBP proteases. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease expressed by osteoblasts in vitro that has been shown to enhance local IGF action through cleavage of inhibitory IGFBP-4. This study was set up to test two specific hypotheses: 1) Intermittent PTH treatment increases the expression of IGF-I, IGFBP-4 and PAPP-A in bone in vivo, thereby increasing local IGF activity. 2) In the absence of PAPP-A, local IGF activity and the anabolic effects of PTH on bone are reduced. Wild-type (WT) and PAPP-A knock-out (KO) mice were treated with 80μg/kg human PTH 1-34 or vehicle by subcutaneous injection five days per week for six weeks. IGF-I, IGFBP-4 and PAPP-A mRNA expression in bone were significantly increased in response to PTH treatment. PTH treatment of WT mice, but not PAPP-A KO mice, significantly increased expression of an IGF-responsive gene. Bone mineral density (BMD), as measured by DEXA, was significantly decreased in femurs of PAPP-A KO compared to WT mice with PTH treatment. Volumetric BMD, as measured by pQCT, was significantly decreased in femoral midshaft (primarily cortical bone), but not metaphysis (primarily trabecular bone), of PAPP-A KO compared to WT mice with PTH treatment. These data suggest that stimulation of PAPP-A expression by intermittent PTH treatment contributes to PTH bone anabolism in mice.

Involvement of PAPP-A and IGFR1 in Cystic Ovarian Disease in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.

A novel neutralizing antibody targeting pregnancy-associated plasma protein-a inhibits ovarian cancer growth and ascites accumulation in patient mouse tumorgrafts.

The majority of ovarian cancer patients acquire resistance to standard platinum chemotherapy and novel therapies to reduce tumor burden and ascites accumulation are needed. Pregnancy-associated plasma protein-A (PAPP-A) plays a key role in promoting insulin-like growth factor (IGF) pathway activity, which directly correlates to ovarian cancer cell transformation, growth, and invasiveness. Herein, we evaluate PAPP-A expression in tumors and ascites of women with ovarian cancer, and determine the antitumor efficacy of a neutralizing monoclonal PAPP-A antibody (mAb-PA) in ovarian cancer using primary patient ovarian tumorgrafts ("Ovatars"). PAPP-A mRNA expression in patient ovarian tumors correlated with poor outcome and was validated as a prognostic surrogate in Ovatar tumors. Following confirmation of mAb-PA bioavailability and target efficacy in vivo, the antitumor efficacy of mAb-PA in multiple Ovatar tumor models was examined and the response was found to depend on PAPP-A expression. Strikingly, the addition of mAb-PA to standard platinum chemotherapy effectively sensitized platinum-resistant Ovatar tumors. PAPP-A protein in ascites was also assessed in a large cohort of patients and very high levels were evident across the entire sample set. Therefore, we evaluated targeted PAPP-A inhibition as a novel approach to managing ovarian ascites, and found that mAb-PA inhibited the development, attenuated the progression, and induced the regression of Ovatar ascites. Together, these data indicate PAPP-A as a potential palliative and adjunct therapeutic target for women with ovarian cancer.

LBA1 - A Novel Neutralizing Antibody Targeting PAPP-A Inhibits Tumor Growth and Ascites in Primary Patient Ovarian Tumorgrafts (Ovatars)

ABSTRACT The majority of ovarian cancer patients acquire resistance to standard platinum chemotherapy and novel therapies to reduce tumor burden and ascites accumulation are needed. Pregnancy-associated plasma protein-A (PAPP-A) plays a key role in promoting insulin-like growth factor (IGF) pathway activity, which directly correlates to ovarian cancer cell transformation, growth and invasiveness. Herein, we evaluate PAPP-A expression in tumors and ascites of women with ovarian cancer, and determine the anti-tumor efficacy of a neutralizing monoclonal PAPP-A antibody (mAb-PA) in ovarian cancer using primary patient ovarian tumorgrafts (“Ovatars”). PAPP-A mRNA expression in patient ovarian tumors correlated with poor outcome and was validated as a prognostic surrogate in Ovatar tumors. Following confirmation of mAb-PA bioavailability and target efficacy in vivo, the anti-tumor efficacy of mAb-PA in multiple Ovatar tumor models was examined and the response was found to depend on PAPP-A expression. Strikingly, the addition of mAb-PA to standard platinum chemotherapy effectively sensitized platinum-resistant Ovatar tumors. PAPP-A protein in ascites was also assessed in a large cohort of patients and very high levels were evident across the entire sample set. Therefore, we evaluated targeted PAPP-A inhibition as a novel approach to managing ovarian ascites, and found that mAb-PA inhibited the development, attenuated the progression and induced the regression of Ovatar ascites. Together, these data indicate PAPP-A as a potential palliative and adjunct therapeutic target for women with ovarian cancer.

Tissue-specific changes in pregnancy associated plasma protein-A expression with age in mice

Pregnancy-associated plasma protein-A (PAPP-A) is a novel zinc metalloproteinase that functions in many systems outside of pregnancy. Data in both humans and mice suggest a role for PAPP-A in aging and age-related diseases. However, our knowledge of tissue-specific PAPP-A expression and possible changes in this expression with age is limited. Thus, the aim of this study was to determine PAPP-A mRNA expression in multiple tissues with age in both male and female mice using real-time PCR. These included the heart, liver, kidney, bone, fat, skeletal muscle, gonads, brain, thymus and spleen. In young mice, PAPP-A mRNA was expressed at relatively high levels in all tissues examined except for liver. The only difference in expression between males and females was seen in the kidney, subcutaneous fat and gonads. The highest PAPP-A mRNA expression levels were found in visceral fat and these were 10-fold higher than in subcutaneous fat. PAPP-A expression significantly increased with age in kidney, brain and gonads. PAPP-A expression significantly deceased with age in bone and skeletal muscle. In the thymus, PAPP-A mRNA showed a biphasic response with age. There were no age-related changes in PAPP-A expression seen in any of the other tissues examined. Expression of IGFBP-5 mRNA, a marker of insulin-like growth factor-I (IGF-I) bioactivity known to be regulated by PAPP-A, paralleled the changes in PAPP-A expression with age in kidney, bone, skeletal muscle and thymus. Thus, tissue-specific PAPP-A expression in mice is differentially affected during aging, and may regulate local IGF-I bioactivity in certain tissues.

Preferential expression of PAPPA in human preadipocytes from omental fat

Fat distribution differs between individuals, and those with visceral fat predominance develop metabolic profiles that increase the risk of adverse cardiovascular events. This is due, in part, to the proinflammatory state associated with visceral obesity as well as depot-specific adipogenesis. The IGF system is important in adipose tissue development and metabolic function. Pregnancy-associated plasma protein A (PAPPA) is a novel zinc metalloproteinase that regulates local IGF availability. The first aim of this study was to characterize PAPPA mRNA and protein expression in primary cultures of human preadipocytes isolated from omental, mesenteric, and subcutaneous depots. PAPPA expression was significantly increased in omental preadipocytes compared with mesenteric and subcutaneous preadipocytes. The second aim of this study was to investigate the factors regulating PAPPA expression, focusing on proinflammatory cytokines and resveratrol that have been shown to have negative and positive effects, respectively, on metabolism and diet-induced obesity. Treatment of cultured primary human preadipocytes with tumor necrosis factor α and interleukin 1β led to significant increases in PAPPA expression. Activated pathways mediating cytokine-induced PAPPA expression include the nuclear factor κB pathway and the MAPK family, particularly c-Jun NH2-terminal kinase and p38 MAPK. Resveratrol, a polyphenolic compound with beneficial cardiometabolic effects, significantly downregulated PAPPA expression under basal and stimulated conditions. Effects of resveratrol on PAPPA appeared to be mediated through pathways independent of silent mating type information regulation 2 homolog 1 (SIRT1) and AMP kinase activation. Depot-specific PAPPA expression in human preadipocytes may contribute to a depot-specific function.

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.

PAPP-A in Indian Water Buffalo (Bubalus bubalis) Ovary: Phylogeny, Expression, Hormonal Regulation, and Sequence Characterization

The insulin-like growth factor binding proteins and pregnancy-associated plasma protein-A (PAPP-A) within the future dominant follicle is a critical part of selection of one follicle. This study was aimed to find out the expression and hormonal regulation of PAPP-A mRNAs in the Indian water buffalo ovary. The results showed stage-specific expression of PAPP-A in the whole follicles and postovulatory structures, corpus luteum and corpus albicans. Expression of PAPP-A mRNA was found to be up regulated in corpus luteum. The 341 nucleotide partial cDNA sequence of buffalo PAPP-A was determined. Sequence analyses have revealed that bubaline PAPP-A belongs to Zinc-metzincin superfamily (zinc-dependent metalloprotease) and pappalysin-like subfamily. It also confirmed the stimulatory effect of follicle-stimulating hormone on PAPP-A mRNA expression. The PAPP-A sequences showed more than 90 % homology with reported sequences of cattle, sheep, pig, and human. The study was an attempt in understanding local regulatory mechanism of follicular selection and development in buffalo ovary.

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.

CRP and TNF-α Induce PAPP-A Expression in Human Peripheral Blood Mononuclear Cells

Objective. The effects of C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α) on pregnancy-associated plasma protein-A (PAPP-A) expression in human peripheral blood mononuclear cells (PBMCs) require further investigation. Methods. The PAPP-A levels in culture supernatants, PAPP-A mRNA expression, and cellular PAPP-A expression were measured in human PBMCs isolated from fresh blood donations provided by 6 healthy volunteers (4 donations per volunteer). Analyses were conducted by ultrasensitive ELISA, western blotting, and RT-PCR following stimulation with CRP or TNF-α cytokines. Results. PAPP-A mRNA and protein levels after CRP stimulation peaked at 24 hours, whereas peak PAPP-A mRNA and protein levels were achieved after TNF-α stimulation at only 2 and 8 hours, respectively. These findings indicate the dose-dependent effect of CRP and TNF-α stimulation. Actinomycin D treatment completely prevented CRP and TNF-α induction of PAPP-A mRNA and protein expression. Additionally, nuclear factor- (NF-) κB inhibitor (BAY11-7082) potently inhibited both CRP and TNF-α stimulated PAPP-A mRNA and protein expression. Conclusions. Human PBMCs are capable of expressing PAPP-A in vitro, expression that may be regulated by CRP and TNF-α through the NF-κB pathway. This mechanism may play a significant role in the observed increase of serum PAPP-A levels in acute coronary syndrome (ACS).

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.

Insulin-like growth factor-I (IGF-I) system during follicle development in the bovine ovary: Relationship among IGF-I, type 1 IGF receptor (IGFR-1) and pregnancy-associated plasma protein-A (PAPP-A)

Insulin-like growth factor-I (IGF-I) system that is exerted mainly through the type 1 IGF receptor (IGFR-1) and releasing of free IGF-I is regulated by the proteases of IGF-binding proteins (IGFBPs), an important factor in follicle development of bovine ovary. The aims of the present study were to examine the mRNA expressions of IGF-I, IGFR-1 and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells and theca tissues during bovine follicular development and the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of these genes in cultured bovine granulosa cells. Follicles were classified into four groups such as small follicle (SF), estrogen inactive dominant follicle (EID), estrogen active dominant follicle (EAD) and preovulatory follicle (POF). The concentration of free IGF-I in follicular fluid of POF was significantly higher than those in EID, whereas the total IGF-I in follicular fluid did not change at all developmental stages. The expression of IGF-I mRNA was not detected in the granulosa cells at all at any developmental stages but the expression was detected in the theca tissues. The amount of IGFR-1 mRNA in granulosa cell showed the constant level at all developmental stages except EID. The expressions of IGFR-1 and PAPP-A in cultured bovine granulosa cells were stimulated with FSH but not with E2. The PAPP-A mRNA expression was stimulated by FSH in presence of 1 ng/ml E2. These results indicate that IGF-I in follicular fluid is mainly derived from the circulation and that FSH is an inducer for the expression of IGFR-1 and PAPP-A genes in granulosa cells. Therefore, we suggest that PAPP-A stimulated with FSH play a crucial role for IGF-I system in bovine follicular development.

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.

Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows ( n = 16) were synchronized with two injections of prostaglandin (PGF2α) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2α. FFL from dominant follicles had lower concentrations of progesterone ( P < 0.08) and higher concentrations of estradiol ( P < 0.05), androstenedione ( P < 0.0001), estradiol:progesterone ratio ( P < 0.0001), free IGF-I ( P < 0.0001), and calculated percentage free IGF-I ( P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- ( P < 0.05), 2.4- ( P < 0.06), and 3.4-fold ( P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ ( P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater ( P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased ( P < 0.05) 62% to 92%, between 24 h and 48 h post-PGF2α. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.

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.

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.

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.

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.

Pregnancy-associated plasma protein-A is involved in insulin-like growth factor binding protein-2 (IGFBP-2) proteolytic degradation in bovine and porcine preovulatory follicles: identification of cleavage site and characterization of IGFBP-2 degradation.

In mammalian ovaries, terminal follicular growth is accompanied by a decrease in levels of intrafollicular insulin-like growth factor binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 is essentially due to an increase in proteolytic degradation by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. In contrast, the decrease in IGFBP-2 is partly due to a decrease in mRNA expression by follicular cells and also to an increase in IGFBP-2 proteolytic degradation, as previously shown in ewes and sows. In the present work we show that bovine and porcine preovulatory follicular fluid contains a proteolytic activity that degrades IGFBP-2. Bovine and porcine preovulatory follicular fluids contain undetectable levels of native IGFBP-2 as assessed by Western ligand blotting in comparison with the corresponding serum. In contrast, much higher levels of 23- and 12-kDa proteolytic fragments were found by immunoblotting in bovine and porcine preovulatory follicular fluid than in the corresponding serum. Moreover, bovine and porcine preovulatory follicular fluids were able to induce proteolytic degradation of exogenous IGFBP-2, and this degradation was enhanced by insulin-like growth factors. Intrafollicular IGFBP-2 proteolytic activity was surprisingly immunoneutralized in both species by a polyclonal antibody raised against human PAPP-A. In addition, recombinant human PAPP-A (rhPAPP-A) was able to cleave IGFBP-2 between Gln165 and Met166 in vitro, generating 23- and 12-kDa proteolytic fragments. IGFBP-2 was shown to be less sensitive than IGFBP-4 to cleavage by rhPAPP-A in vitro. As in follicular fluid, cleavage of IGFBP-2 by rhPAPP-A was dose-dependently enhanced by IGFs and inhibited by a peptide derived from the heparin-binding domain of IGFBP-5 (P5). Finally, Biacore analysis showed that P5 peptide-induced inhibition of IGFBP-2 cleavage was due to a direct interaction of P5 with PAPP-A rather than with IGFBP-2. Overall, these data show that in bovine and porcine preovulatory follicles, PAPP-A is responsible for IGF-dependent IGFBP-2 degradation. During follicular growth, the increase in IGFBP-2 cleavage by PAPP-A, as well as the decrease in IGFBP-2 expression, are responsible for the decrease in intact IGFBP-2 levels and the increase in IGF bioavailability. In atretic follicles, the increase and decrease in IGFBP-2 and PAPP-A mRNA expression, respectively, as well as the inhibition of PAPP-A activity by heparin-binding domains present in IGFBP-5 or other proteins, might participate in higher IGFBP-2 levels and a decrease in IGF bioavailability.