Intrauterine Growth Restriction in Humans Is Associated with Abnormalities in Placental Insulin-Like Growth Factor Signaling
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内分泌学杂志 2005年第3期
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology and Metabolic Diseases (L.L., S.P., G.B., A.N., C.M., A.L., R.G., F.G.), and Department of Obstetrics and Gynecology, Clinica Ostetrica e Ginecologica II (A.V., M.S., L.S.), University of Bari, I-70124 Bari, Italy; and Department of Obstetrics and Gynecology, University of Foggia (P.G.), I-71100 Foggia, Italy
Address all correspondence and requests for reprints to: Dr. Francesco Giorgino, Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology and Metabolic Diseases, University of Bari, Piazza Giulio Cesare 11, I-70124 Bari, Italy. E-mail: f.giorgino@endo.uniba.it.
Abstract
The IGFs promote the growth and development of the feto-placental unit during gestation, and impairment of their placental actions may result in altered intrauterine growth of the fetus. In this study, proteins involved in IGF signaling were investigated in human placentas from pregnancies complicated by intrauterine growth restriction (IUGR) compared with those from normal pregnancies. IUGR placentas exhibited 33% reduction in the protein content of IGF-I receptors, but no changes in insulin receptor protein levels. In addition, insulin receptor substrate-2 (IRS-2) protein levels were reduced in IUGR placentas, with no changes in IRS-1 or Shc protein content, and this was associated with a parallel decrease in IRS-2-associated phosphatidyl inositol 3-kinase. Akt protein expression was also reduced in IUGR, whereas phosphorylation of Akt and its substrate glycogen synthase kinase-3 was unchanged. Finally, in IUGR placentas there was impaired activation of multiple members of the MAPK family, because phosphorylation of p38 and c-Jun N-terminal kinase was reduced 70%. In conclusion, human placentas from pregnancies complicated by IUGR are characterized by decreased IGF-I receptor content, selective impairment of the IRS-2/ phosphatidyl inositol 3-kinase pathway, and reduced p38 and c-Jun N-terminal kinase activation. The observed abnormalities in IGF-I signaling may contribute to altered fetal growth and development in human IUGR.
Introduction
FETAL GROWTH IS under control of genetic, environmental, and nutritional factors. The term intrauterine growth retardation (IUGR) is usually assigned to a fetus that has not reached its growth potential (1). The growth-restricted fetus/newborn is therefore characterized by abnormal morphometric parameters at prenatal sonographic analysis and low birth weight for its gestational age at the time of delivery (2). In addition, fetal growth restriction is usually heralded by abnormalities of flow impedance through the umbilical artery or other fetal vessels by Doppler velocimetry (1). The association of sonographic indicators of reduced fetal growth, low birth weight, and abnormal umbilical artery flow identifies IUGR fetuses carrying a significantly increased risk of perinatal mortality (1) and metabolic abnormalities in adult life, i.e. obesity, arterial hypertension, hypercholesterolemia, cardiovascular disease, impaired glucose tolerance, and type 2 diabetes mellitus (3). Conversely, small fetuses with normal Doppler studies have the same outcome as appropriately grown fetuses (4) and probably represent constitutionally small for gestational age neonates (1). The development of IUGR is thought to be a consequence of maternal, fetal, or placental factors that ultimately result in impaired trophoblastic invasion of the maternal spiral arteries in early pregnancy and reduced placental perfusion (5). Therefore, placental failure is the background to fetal growth constraint.
The IGFs and their receptors are implicated in the regulation of protein turnover and exert potent mitogenic and differentiating effects on most cell types. IGF-I and IGF-II exert a key role in feto-placental growth throughout gestation (6). Growth-promoting effects of IGFs are mediated by the type I IGF receptor, comprised of two extracellular -subunits, containing hormone-binding sites, and two membrane-spanning ?-subunits, encoding an intracellular tyrosine kinase. Hormone binding activates the receptor kinase, leading to receptor autophosphorylation and tyrosine phosphorylation of multiple substrates, including the insulin receptor substrate (IRS) and Shc proteins (7). Through these initial tyrosine phosphorylation reactions, IGF-I signals are transduced to a complex network of intracellular lipid and serine/threonine kinases that are ultimately responsible for cell proliferation, modulation of tissue differentiation, and protection from apoptosis (7, 8).
Dysregulation of IGF-I actions due to abnormal expression of IGFs, structural defects in their receptors, or impairment in the postreceptor signaling machinery may contribute to abnormal fetal growth. Low maternal serum IGF-I levels have been related to poor placental function, as indicated by Doppler velocimetry studies of umbilical arteries, rather than to low birth weight (9). However, studies carried out in human placentas did not show conclusive results on the potential role of the IGF system in the pathogenesis of impaired fetal growth. Expression levels of both IGF-I and IGF-II were found to be increased in IUGR term placentas (10, 11), whereas immunohistochemistry studies showed a conserved distribution of IGF-I receptor protein in placentas associated with various degrees of IUGR compared with those of normally grown fetuses (12). IGF-I receptor mRNA levels measured by quantitative PCR appeared to be increased in IUGR placentas (10), whereas a recent study of experimentally induced IUGR in rats showed reduced IGF-I receptor levels, measured by evaluation of both mRNA levels with RT-PCR and total protein content by immunoblotting (13). Limited information is available on molecules involved in the IGF-I receptor signaling system in human placentas and their potential abnormalities in placentas from women delivering IUGR neonates.
In this study we investigated the IGF-I receptor signal transduction system in human placentas from control and IUGR pregnancies. Placental tissue was removed immediately after delivery and analyzed by immunoprecipitation and immunoblotting techniques to study multiple signaling molecules involved in IGF-I regulation of growth and differentiation. We show that IUGR placentas have marked reduction in the protein content of IGF-I receptors and defects in specific postreceptor signaling intermediates.
Materials and Methods
Patients
This was a prospective observational study of women with pregnancies complicated by IUGR. The study was approved by the local institutional review committee. IUGR pregnancies (n = 14) were defined as having a singleton fetus with an ultrasonographically diagnosed abdominal circumference smaller than –2 SD for gestational age in the second half of pregnancy (14) and no suspicion of fetal abnormality. Major identifiable causes of reduced fetal growth, including congenital anomalies, genetic syndromes, fetal infections, gross placental abnormalities, and maternal malnutrition, medical disorders, or substance abuse were excluded. Umbilical artery Doppler velocimetry studies were performed immediately after the ultrasonic diagnosis of growth restriction. Doppler measurements were expressed as the pulsatility index, and values exceeding +2 SD for gestational age (15) were required for inclusion in the study group. Impaired Doppler velocimetry as an indicator of poor fetal conditions guided the indication for cesarean delivery in IUGR pregnancies. Abnormal neonates were reassessed at birth, and a birth weight less than the fifth percentile for gestational age for the Italian population (16) was requested for inclusion in the study. Controls (n = 14) were women, recruited from the same antenatal clinic and during the same period, who consented to have ultrasound and Doppler studies. Indications for cesarean section in control women were failure to progress in labor, previous cesarean delivery or hysterotomy, fetal malpresentation, or patient’s choice. Criteria for inclusion were both abdominal circumference and pulsatility index between –2 and +2 SD, and birth weight between the 5th and the 95th percentiles for gestational age. Criteria for exclusion were the development of gestational hypertension, diabetes, or a reduced amount of amniotic fluid at term. The characteristics of the IUGR and control neonates who fulfilled the criteria for inclusion in the study are shown in Table 1. All women gave their informed consent to the study.
TABLE 1. Clinical features of the neonates
Immunoprecipitation and immunoblotting
Tissue specimens were taken from the placental perifunicular area during cesarean sections. Placental samples were rapidly cut into small pieces (2 x 2 x 2 cm) and immediately frozen in liquid N2. The frozen placental tissue was powdered in a stainless steel mortar and pestle with liquid N2 and homogenized for 30 sec with an Ultra-Turrax (Janke & Kunkel GmbH and Co., IKA-Werk, Staufen, Germany) in ice-cold lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM phenylmethylsulfonylfluoride, 5 μg/ml leupeptin, 2 mM sodium orthovanadate, and 1% Nonidet P-40. The tissue homogenate was incubated for 45 min at 4 C with gentle stirring and then centrifuged at 100,000 x g for 60 min. The resulting supernatant was collected and assayed for protein concentration using the Bradford dye binding assay kit with BSA as a standard.
For immunoprecipitation studies, equal amounts of placental extracts (1 mg) were subjected to immunoprecipitation overnight at 4 C with anti-IRS-1 or anti-IRS-2 antibodies, as indicated. The resulting immune complexes were adsorbed to protein A-Sepharose beads for 2 h at 4 C, washed three times with lysis buffer, then eluted with Laemmli buffer for 5 min at 100 C. For immunoblotting studies, equal amounts of solubilized placental proteins were resolved by electrophoresis on 7% or 10% SDS-polyacrylamide gels, as appropriate, directly or after immunoprecipitation with the specific antibodies, as indicated. The resolved proteins were electrophoretically transferred to nitrocellulose or polyvinylidene difluoride membranes (Hybond-ECL, Amersham Biosciences, Arlington Heights, IL) using a transfer buffer containing 192 mM glycine, 20% (vol/vol) methanol, and 0.02% SDS. To reduce nonspecific binding, the membranes were incubated in TNA buffer [10 mM Tris-HCl (pH 7.8), 0.9% NaCl, and 0.01% sodium azide] supplemented with 5% BSA and 0.05% Nonidet P-40 at 37 C for 2 h or in PBS supplemented with 3% nonfat dry milk for 2 h at room temperature, as appropriate, and then incubated overnight at 4 C with the indicated antibodies. The proteins were visualized by enhanced chemiluminescence using horseradish peroxidase-labeled antirabbit or antimouse IgG (Amersham Biosciences) and quantified by densitometric analysis using Optilab image analysis software (Graftek SA, Mirmande, France) or Quantity One image analysis software (Bio-Rad Laboratories, Inc., Hercules, CA).
Antibodies
Polyclonal anti-IGF-I receptor ?-subunit, polyclonal antiinsulin receptor ?-subunit, and monoclonal anti-phosphotyrosine antibodies (PY99) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-phospho-Shc (Tyr317), anti-Akt, anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-phospho-p42/44 MAPK [extracellular signal-regulated kinase (Erk-1/2); Thr202/Tyr204], and anti-phospho-glycogen synthase kinase-3 (GSK-3)/? (Ser9/Ser21) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal anti-Shc antibodies were purchased from Upstate Biotechnology, Inc. (Saranac Lake, NY). Polyclonal antibodies against the p85 subunit of phosphatidyl inositol 3-kinase (PI 3-kinase), IRS-1, IRS-2, or GSK-3 were purchased from Upstate Biotechnology, Inc. Anti-MAPK (Erk-1/2) antibodies were obtained from Zymed Laboratories (San Francisco, CA). Polyclonal anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-stress-activated protein kinase/c-Jun N-terminal kinase (JNK), and anti-phospho-stress-activated protein kinase/JNK (Thr183/Tyr185) were obtained from Cell Signaling Technology, Inc.
Statistical analyses
All data are expressed as the mean ± SE. Statistical analyses were performed by t tests.
Results
IGF-I receptor
To evaluate the expression levels of placental IGF-I receptors, tissue samples were analyzed by direct immunoblotting with anti-IGF-I receptor antibodies. As shown in Fig. 1A, a single band of approximately 105 kDa, corresponding to the IGF-I receptor ?-subunit, was detected. The total amount of IGF-I receptor protein was reduced 33% in IUGR compared with control placentas (Fig. 1A; P < 0.05). By contrast, the protein content of the insulin receptor, as assessed by reprobing the same filters with antiinsulin receptor antibodies, was not different in IUGR and control placentas (Fig. 1B; P = 0.383). To evaluate the phosphorylation state of IGF-I receptors, placental protein extracts were analyzed by sequential immunoprecipitation with antibodies against the IGF-I receptor ?-subunit and immunoblotting with anti-phosphotyrosine antibodies. However, tyrosine phosphorylation of IGF-I receptors could not be detected in either control or IUGR placentas (data not shown).
FIG. 1. IGF-I receptor and insulin receptor protein contents in human placentas. Equal amounts of solubilized proteins from control () and IUGR () placentas were resolved by 7% SDS-PAGE, as described in Materials and Methods. IGF-I receptor protein content (A) was detected by immunoblotting with anti-IGF-I receptor antibodies. The protein content of the insulin receptor in placental samples (B) was measured by immunoblotting with specific antibodies. The bar graphs in A and B show the quantitation of IGF-I receptor and insulin receptor protein contents in all samples (n = 14 for controls and IUGR, respectively). IGF-I R, IGF-I receptor; Ins R, insulin receptor. *, P < 0.05 vs. controls.
IRS proteins and PI 3-kinase
To investigate postreceptor IGF-I signaling proteins, the expression levels of IRS-1 and IRS-2, the major IGF-I receptor substrates, were next evaluated by immunoprecipitation and immunoblotting with anti-IRS-1 or anti-IRS-2 antibodies, respectively. The total levels of IRS-1 and IRS-2 proteins were differently regulated by IUGR. IRS-1 levels were not different in IUGR and control placentas (Fig. 2A; P = 0.553 vs. control). By contrast, IRS-2 levels were reduced 35% in IUGR compared with control placentas (Fig. 2B; P < 0.05 vs. control). Analysis of IRS-1 and IRS-2 immunoprecipitates with anti-phosphotyrosine antibodies showed minimal, if any, tyrosine phosphorylation of either substrate (data not shown).
FIG. 2. IRS-1 and IRS-2 protein contents and associated p85 protein in human placenta. Solubilized proteins from control () and IUGR () placentas were subjected to immunoprecipitation with anti-IRS-1 or anti-IRS-2 antibodies, as indicated. The resulting immune complexes were resolved by 7% SDS-PAGE and analyzed by immunoblotting with anti-IRS-1, anti-IRS-2, or anti-p85 antibodies, as indicated. Each panel shows a representative experiment, on the left, and the quantitation of results from all samples, on the right. A, IRS-1 protein content; B, IRS-2 protein content; C, IRS-1-associated p85; D, IRS-2-associated p85. *, P < 0.05 vs. controls.
The enzyme PI 3-kinase is recruited to IRS proteins through binding of the two SH2 domains contained in its 85-kDa regulatory subunit to specific phosphotyrosine residues in IRS-1 and IRS-2 (17). To assess whether association of the IRS proteins with p85, the regulatory subunit of PI 3-kinase, could be detected in human placenta, IRS-1 and IRS-2 immunoprecipitates were analyzed by immunoblotting with anti-p85 antibodies. The amount of p85 protein associated with IRS-1 was similar in control and IUGR placentas (Fig. 2C), whereas IRS-2-associated p85 appeared to be significantly reduced in the IUGR group (Fig. 2D; P < 0.05 vs. control), in agreement with the observed decrease in total IRS-2 protein content (Fig. 2B). No differences in the total p85 protein levels were observed in the IUGR and control placentas (Fig. 3A).
FIG. 3. p85, Akt and GSK-3 in human placenta. Solubilized proteins from control () and IUGR () placentas were resolved by 10% SDS-PAGE and analyzed by immunoblotting with specific antibodies, as indicated. Each panel shows a representative experiment, on the left, and the quantitation of results from all samples, on the right. A, p85 protein content; B, total protein content (top) and Ser473 phosphorylation (bottom) of Akt; C, total protein content (top) and Ser9/Ser21 phosphorylation (bottom) of GSK-3/?. *, P < 0.05 vs. controls.
Akt and GSK-3
The expression levels of Akt, a serine/threonine kinase that lies downstream of PI 3-kinase in the IGF-I signaling pathway, were investigated next. The total Akt protein level was significantly decreased in IUGR placentas (73% of control; P < 0.05; Fig. 3B). However, Akt activation, measured by immunoblotting with antibodies to phosphorylated Ser473 in the Akt protein, was not different in control and IUGR placentas (P = 0.737; Fig. 3B). Phosphorylation of Akt at Thr308 could not be demonstrated by immunoblotting with phospho-specific antibodies (data not shown). The total protein and phosphorylation levels of GSK-3, a direct substrate of Akt, were not different in control and IUGR placentas (Fig. 3C).
To assess whether the differences in total protein contents of specific signaling proteins in IUGR compared with those in control placentas were related to the different gestational ages of the two groups (Table 1), further analyses were carried out in control (n = 8) and IUGR (n = 7) placentas that were matched for gestational age (36.5 ± 1.1 vs. 35.7 ± 1.1 wk; P = 0.63; Table 2). The total protein content of IGF-I receptors was reduced 38% in these IUGR placentas compared with the related controls (P < 0.05), whereas insulin receptor expression was not altered (P = 0.56; Table 2). Moreover, IRS-1 protein levels were not different (P = 0.63), whereas IRS-2 and Akt protein levels were 45% and 30% lower in IUGR compared with control placentas, respectively (P < 0.05; Table 2). These changes were similar to those observed in the total IUGR group (Figs. 1–3). No significant differences in protein levels of IGF-I receptors, insulin receptors, IRS-1, IRS-2, or Akt were detected in these 35-wk-old IUGR placentas compared with the remaining IUGR samples with lower (i.e. 28.5 wk; P < 0.05) gestational age (data not shown). Together, these findings demonstrate that lower expression of specific signaling proteins in IUGR placentas was not related to gestational age.
TABLE 2. Characteristics of control and IUGR subgroups matched for gestational age
Shc and the MAPK family
The Shc proteins (i.e. p66Shc, p52Shc, and p46Shc) are substrates for the IGF-I receptor tyrosine kinase and are largely responsible for activation of the Erk-1/2 MAPK cascade, which is involved in growth and differentiation responses in mammalian cells (18). To examine potential defects of the Shc/Erk pathway in the placenta, expression levels of the Shc proteins were studied by immunoblotting with specific antibodies. Equal amounts of p66Shc, p52Shc, and p46Shc proteins were found in control and IUGR placental samples (Fig. 4A). Tyrosine phosphorylation of the Shc proteins, evaluated by immunoblotting with phospho-specific Shc antibodies, could not be demonstrated in either control or IUGR placental tissues (data not shown). Total expression levels of Erk-1 and Erk-2 MAPKs were reduced in IUGR compared with control placentas (Fig. 4B; P < 0.05). However, phosphorylation of Erk-1 and Erk-2 were not significantly different in IUGR samples (Fig. 4B).
FIG. 4. Shc and Erk-1/2 in human placenta. For Shc analyses, solubilized proteins from control () and IUGR () placentas were resolved by 10% SDS-PAGE and analyzed by immunoblotting with anti-Shc antibodies, as described in Materials and Methods. Erk-1/2 protein content and phosphorylation were determined by immunoblotting with the indicated antibodies. Each panel shows representative immunoblots, on the left, and the quantitation of results from all samples, on the right. A, Total protein content of Shc with quantitation of the p52Shc isoform; B, total protein content (top) and phosphorylation (bottom) of Erk-1 and Erk-2. *, P < 0.05 vs. controls.
The p38 MAPK and JNK belong to the MAPK family of intracellular signal transducers involved in mammalian growth by integrating a variety of environmental stimuli (19, 20). To verify whether abnormal intrauterine development is associated with altered expression and activation of these MAPKs in the placenta, protein content and phosphorylation of p38 and JNK MAPKs were analyzed. No differences were found in p38 protein content between the IUGR and control samples (Fig. 5A; P = 0.771). By contrast, the level of p38 phosphorylation was markedly reduced in IUGR compared with control placentas (Fig. 5A; P < 0.05). The protein content of JNK also did not differ in the two groups (Fig. 5B; P = 0.304), but the phosphorylation levels of both JNK-1 and JNK-2 were reduced 70% in IUGR (Fig. 5B; P < 0.05 vs. control). Therefore, phosphorylation of both p38 and JNK was markedly reduced in IUGR placentas, and this was not explained by changes in the total protein contents of these two kinases.
FIG. 5. Protein content and phosphorylation of p38 and JNK MAPKs in human placenta. Tissue lysates from control and IUGR placentas were analyzed by direct immunoblotting with the indicated antibodies, as described in Materials and Methods. Each panel shows representative immunoblots, on the left, and the quantitation of multiple experiments on all samples, on the right. A, Total protein (top) and phosphorylation (bottom) of p38. B, Total protein content (top) and phosphorylation (bottom) of JNK. *, P < 0.05 vs. controls.
Discussion
In this study we investigated the IGF-I signal transduction system in placentas of growth-restricted human fetuses. The experimental procedure used, i.e. sampling of placental tissues immediately after delivery and subsequent protein analysis using biochemical methods, allowed us to investigate the expression levels of various placental signaling proteins and their activation state in vivo during late gestation. Specific signaling events, such as tyrosine phosphorylation of the IGF-I receptor, IRS, and Shc proteins, were below the sensitivity of the immunoblotting techniques employed, whereas others, including formation of IRS-2/p85 signaling complexes and phosphorylation of Akt kinase and MAPK, could be measured. We demonstrate that the placentas of IUGR fetuses are characterized by multiple and specific abnormalities of the IGF-I signal transduction system, including reduced protein content of the IGF-I receptor, of its major substrate IRS-2, and of the serine/threonine kinases Akt and Erk-1/2. In addition, IUGR placentas feature a marked impairment of the coordinated activation of the MAPKs, with reduced p38 and JNK phosphorylation.
Multiple experimental settings have demonstrated a pivotal role of the IGF-I receptor/IRS/Akt pathway in controlling intrauterine growth in mammals. Targeted disruption of the IGF-I or IGF-I receptor genes is associated with marked IUGR in mice (21, 22), and partial deletion of the IGF-I gene has been reported to be responsible for IUGR and postnatal growth failure in humans (23). Knockout experiments in mice, in which gene expression of IRS-I or IRS-2 was abrogated in all tissues, also showed growth defects (24, 25, 26). Finally, Akt1-deficient mice showed a 20% reduction in body weight at birth compared with wild-type littermates (27). Thus, the observed reductions in IGF-I receptor, IRS-2, and Akt protein contents in placentas associated with IUGR may contribute to impaired fetal growth in humans. The IUGR placenta may be viewed as an IGF-resistant tissue, in which the IGF-I signaling potential is blunted due to subnormal expression levels of key signaling molecules. The impaired growth-promoting action of IGF-I on the feto-placental unit may thus explain the reduced neonatal and placental weight at birth (Table 1). Relevant to this concept, GH or IGF-I administration in experimental models of IUGR was associated with increased circulating levels of IGF-I, but failure to stimulate fetal growth and placental or fetal organ weight (28, 29).
In IUGR placentas, IRS-2 protein levels were markedly reduced, whereas those of IRS-1 were unchanged (Fig. 2, A and B). This finding was associated with decreased association of p85, the regulatory subunit of the enzyme PI 3-kinase, with IRS-2. Differential regulation of IRS-1 and IRS-2 has been observed in experimental and human diseases. IRS-1 is reduced, whereas IRS-2 is unchanged, in skeletal muscle and myocardium of streptozotocin-diabetic rats (30, 31) and in adipocytes from type 2 diabetic patients (32). Increased expression of IRS-2, with no change in IRS-1, has been described in rat insulinoma cells and human pancreatic cancer tissue compared with that in normal ?-cells (33, 34). Moreover, different physiological functions for IRS-1 and IRS-2 have been proposed in various cellular contexts, including L6 myoblasts (35), brown adipocytes (36), exercised skeletal muscle (37), and transgenic animals with selective ablation of the IRS-1 or IRS-2 gene (24, 38, 39). Although the specific roles of IRS-1 and IRS-2 in human placental cells have not been defined, the selective down-regulation of IRS-2 and its association with p85 in the IUGR placentas may play important roles in the impaired development of the feto-placental unit.
A low birth weight has reportedly been associated with impaired insulin secretion and insulin resistance in later life (40, 41). This can reflect both adaptation to disadvantageous environmental factors (i.e. malnutrition in utero) and genetically determined defects in specific signaling proteins. Interestingly, IRS-2 has been proposed as a major determinant of pancreatic ?-cell growth and insulin secretion (26, 42), and mice rendered null for IRS-2 develop ?-cell failure and various alterations resembling type 2 diabetes in humans (39). Even though it is not known whether IRS-2 levels are reduced in ?-cells from growth-restricted newborns, we show that placentas of IUGR fetuses exhibit a defect in IRS-2 expression and signaling. It is tempting to speculate that the impaired placental expression of IRS-2 may represent the molecular hallmark linking IUGR with the subsequent development of metabolic diseases in adult life (3). In an experimental model of IUGR, impaired activation of IRS-2 in the liver preceded the onset of insulin resistance and metabolic abnormalities (43).
Placental cells proliferate, differentiate, and eventually undergo apoptosis under the control of multiple growth factors (44). Under physiological conditions, apoptosis occurs during early pregnancy (at wk 5–7) and in the third trimester in human placentas (45, 46). However, pregnancies complicated by IUGR have reportedly been associated with increased apoptotic rates in placental tissue (47). Moreover, apoptosis was recently shown to be increased in cultured cytotrophoblasts from human IUGR placentas (48). Also in this study the amount of oligonucleosomes in cytosolic extracts, which correlates with early apoptosis, was found to be higher, although not significantly, in IUGR than in control placentas (Laviola, L., and F. Giorgino, unpublished observations). Proposed biological consequences of augmented apoptosis in the placenta include depletion of the syncytiotrophoblast layer, leading to impairment of materno-fetal exchange functions (49), and degeneration of placental villi, leading to reduced placental perfusion and oxygenation (50). Increased placental apoptosis in human placentas associated with IUGR may be the consequence of increased p53 expression (51) and reduced Bcl-2 protein levels (52). We suggest that it could also be the result of insufficient survival signals through the IGF-I receptor/IRS-2 pathway. Relevant to this concept, extensive apoptosis in preimplantation embryos is associated with down-regulation of the IGF-I receptor (53). It is important to note that the insulin receptor content was not altered in the IUGR placentas, as also shown by Reid and co-workers (13) in an experimental model of fetal growth restriction.
Coordinate activation of the MAPKs is required for protection from apoptosis and appropriate regulation of cell proliferation in response to growth factors and cytokines (20, 54, 55). Therefore, reduced expression and/or blunted activation of these intracellular enzymes are expected to impair development of the feto-placental unit. Indeed, embryos deficient in Erk-2 lack mesoderm differentiation and placental angiogenesis (56). In addition, a mutant mouse line with disruption of the mek1 gene, the upstream activator of Erk-1/2, was characterized by reduced placental vascularization, leading to the premature death of the mek1–/– embryos (57). Targeted disruption of the p38 MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. In particular, p38 mutant placentas display impaired vascularization and insufficient oxygen and nutrient transport as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis (58, 59). Finally, compound mutations of the jnk1 and jnk2 genes cause early embryonic death associated with impaired morphogenesis, decreased apoptosis in the hindbrain, and increased apoptosis in the forebrain (60). In primary human trophoblast, specific activation of JNK in response to placental growth factor protects from serum withdrawal-induced apoptosis (61). Reduced activation of JNK has also been observed in placental tissue from women with preeclampsia (62), which features a defective vascular development of the feto-placental unit, similar to IUGR pregnancies (5). Interestingly, mice with targeted disruption of the junB gene, a member of the activating protein-1 transcription factor complex, which lies downstream and is activated by JNK, show severe growth retardation in utero, embryonic lethality, and defective development of the feto-maternal vascularization (63). Together, these findings strongly support the hypothesis that the impairment in the integrated activation of the MAPKs observed in IUGR placentas may play an important role in altering placental angiogenesis, ultimately leading to reduced fetal growth.
Even though all deliveries occurred within the third trimester, the gestational age of the study group was significantly lower than that of the control group. To rule out that this could affect the expression levels of distinct signaling proteins, we studied selected control and IUGR subgroups, matched for gestational age. Analysis of the placental proteins in these subgroups revealed the same pattern of abnormalities detected in the larger groups, i.e. reduced expression of IGF-I receptors, IRS-2, and Akt, and no change in insulin receptor or IRS-1 protein levels, respectively. This suggests that the molecular mechanisms underlying the growth restriction in humans involve differences in IGF-I signaling independently of the stage of fetal development.
In conclusion, we have demonstrated that abnormalities in expression and/or activation of specific IGF-I receptor signaling molecules and multiple members of the MAPK family occur in human placentas of IUGR fetuses at delivery. These molecular defects may be responsible for impaired IGF-I responsiveness and appropriate development of the feto-placental unit, leading to dysregulation of fetal growth in humans.
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Address all correspondence and requests for reprints to: Dr. Francesco Giorgino, Department of Emergency and Organ Transplantation, Section of Internal Medicine, Endocrinology and Metabolic Diseases, University of Bari, Piazza Giulio Cesare 11, I-70124 Bari, Italy. E-mail: f.giorgino@endo.uniba.it.
Abstract
The IGFs promote the growth and development of the feto-placental unit during gestation, and impairment of their placental actions may result in altered intrauterine growth of the fetus. In this study, proteins involved in IGF signaling were investigated in human placentas from pregnancies complicated by intrauterine growth restriction (IUGR) compared with those from normal pregnancies. IUGR placentas exhibited 33% reduction in the protein content of IGF-I receptors, but no changes in insulin receptor protein levels. In addition, insulin receptor substrate-2 (IRS-2) protein levels were reduced in IUGR placentas, with no changes in IRS-1 or Shc protein content, and this was associated with a parallel decrease in IRS-2-associated phosphatidyl inositol 3-kinase. Akt protein expression was also reduced in IUGR, whereas phosphorylation of Akt and its substrate glycogen synthase kinase-3 was unchanged. Finally, in IUGR placentas there was impaired activation of multiple members of the MAPK family, because phosphorylation of p38 and c-Jun N-terminal kinase was reduced 70%. In conclusion, human placentas from pregnancies complicated by IUGR are characterized by decreased IGF-I receptor content, selective impairment of the IRS-2/ phosphatidyl inositol 3-kinase pathway, and reduced p38 and c-Jun N-terminal kinase activation. The observed abnormalities in IGF-I signaling may contribute to altered fetal growth and development in human IUGR.
Introduction
FETAL GROWTH IS under control of genetic, environmental, and nutritional factors. The term intrauterine growth retardation (IUGR) is usually assigned to a fetus that has not reached its growth potential (1). The growth-restricted fetus/newborn is therefore characterized by abnormal morphometric parameters at prenatal sonographic analysis and low birth weight for its gestational age at the time of delivery (2). In addition, fetal growth restriction is usually heralded by abnormalities of flow impedance through the umbilical artery or other fetal vessels by Doppler velocimetry (1). The association of sonographic indicators of reduced fetal growth, low birth weight, and abnormal umbilical artery flow identifies IUGR fetuses carrying a significantly increased risk of perinatal mortality (1) and metabolic abnormalities in adult life, i.e. obesity, arterial hypertension, hypercholesterolemia, cardiovascular disease, impaired glucose tolerance, and type 2 diabetes mellitus (3). Conversely, small fetuses with normal Doppler studies have the same outcome as appropriately grown fetuses (4) and probably represent constitutionally small for gestational age neonates (1). The development of IUGR is thought to be a consequence of maternal, fetal, or placental factors that ultimately result in impaired trophoblastic invasion of the maternal spiral arteries in early pregnancy and reduced placental perfusion (5). Therefore, placental failure is the background to fetal growth constraint.
The IGFs and their receptors are implicated in the regulation of protein turnover and exert potent mitogenic and differentiating effects on most cell types. IGF-I and IGF-II exert a key role in feto-placental growth throughout gestation (6). Growth-promoting effects of IGFs are mediated by the type I IGF receptor, comprised of two extracellular -subunits, containing hormone-binding sites, and two membrane-spanning ?-subunits, encoding an intracellular tyrosine kinase. Hormone binding activates the receptor kinase, leading to receptor autophosphorylation and tyrosine phosphorylation of multiple substrates, including the insulin receptor substrate (IRS) and Shc proteins (7). Through these initial tyrosine phosphorylation reactions, IGF-I signals are transduced to a complex network of intracellular lipid and serine/threonine kinases that are ultimately responsible for cell proliferation, modulation of tissue differentiation, and protection from apoptosis (7, 8).
Dysregulation of IGF-I actions due to abnormal expression of IGFs, structural defects in their receptors, or impairment in the postreceptor signaling machinery may contribute to abnormal fetal growth. Low maternal serum IGF-I levels have been related to poor placental function, as indicated by Doppler velocimetry studies of umbilical arteries, rather than to low birth weight (9). However, studies carried out in human placentas did not show conclusive results on the potential role of the IGF system in the pathogenesis of impaired fetal growth. Expression levels of both IGF-I and IGF-II were found to be increased in IUGR term placentas (10, 11), whereas immunohistochemistry studies showed a conserved distribution of IGF-I receptor protein in placentas associated with various degrees of IUGR compared with those of normally grown fetuses (12). IGF-I receptor mRNA levels measured by quantitative PCR appeared to be increased in IUGR placentas (10), whereas a recent study of experimentally induced IUGR in rats showed reduced IGF-I receptor levels, measured by evaluation of both mRNA levels with RT-PCR and total protein content by immunoblotting (13). Limited information is available on molecules involved in the IGF-I receptor signaling system in human placentas and their potential abnormalities in placentas from women delivering IUGR neonates.
In this study we investigated the IGF-I receptor signal transduction system in human placentas from control and IUGR pregnancies. Placental tissue was removed immediately after delivery and analyzed by immunoprecipitation and immunoblotting techniques to study multiple signaling molecules involved in IGF-I regulation of growth and differentiation. We show that IUGR placentas have marked reduction in the protein content of IGF-I receptors and defects in specific postreceptor signaling intermediates.
Materials and Methods
Patients
This was a prospective observational study of women with pregnancies complicated by IUGR. The study was approved by the local institutional review committee. IUGR pregnancies (n = 14) were defined as having a singleton fetus with an ultrasonographically diagnosed abdominal circumference smaller than –2 SD for gestational age in the second half of pregnancy (14) and no suspicion of fetal abnormality. Major identifiable causes of reduced fetal growth, including congenital anomalies, genetic syndromes, fetal infections, gross placental abnormalities, and maternal malnutrition, medical disorders, or substance abuse were excluded. Umbilical artery Doppler velocimetry studies were performed immediately after the ultrasonic diagnosis of growth restriction. Doppler measurements were expressed as the pulsatility index, and values exceeding +2 SD for gestational age (15) were required for inclusion in the study group. Impaired Doppler velocimetry as an indicator of poor fetal conditions guided the indication for cesarean delivery in IUGR pregnancies. Abnormal neonates were reassessed at birth, and a birth weight less than the fifth percentile for gestational age for the Italian population (16) was requested for inclusion in the study. Controls (n = 14) were women, recruited from the same antenatal clinic and during the same period, who consented to have ultrasound and Doppler studies. Indications for cesarean section in control women were failure to progress in labor, previous cesarean delivery or hysterotomy, fetal malpresentation, or patient’s choice. Criteria for inclusion were both abdominal circumference and pulsatility index between –2 and +2 SD, and birth weight between the 5th and the 95th percentiles for gestational age. Criteria for exclusion were the development of gestational hypertension, diabetes, or a reduced amount of amniotic fluid at term. The characteristics of the IUGR and control neonates who fulfilled the criteria for inclusion in the study are shown in Table 1. All women gave their informed consent to the study.
TABLE 1. Clinical features of the neonates
Immunoprecipitation and immunoblotting
Tissue specimens were taken from the placental perifunicular area during cesarean sections. Placental samples were rapidly cut into small pieces (2 x 2 x 2 cm) and immediately frozen in liquid N2. The frozen placental tissue was powdered in a stainless steel mortar and pestle with liquid N2 and homogenized for 30 sec with an Ultra-Turrax (Janke & Kunkel GmbH and Co., IKA-Werk, Staufen, Germany) in ice-cold lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM phenylmethylsulfonylfluoride, 5 μg/ml leupeptin, 2 mM sodium orthovanadate, and 1% Nonidet P-40. The tissue homogenate was incubated for 45 min at 4 C with gentle stirring and then centrifuged at 100,000 x g for 60 min. The resulting supernatant was collected and assayed for protein concentration using the Bradford dye binding assay kit with BSA as a standard.
For immunoprecipitation studies, equal amounts of placental extracts (1 mg) were subjected to immunoprecipitation overnight at 4 C with anti-IRS-1 or anti-IRS-2 antibodies, as indicated. The resulting immune complexes were adsorbed to protein A-Sepharose beads for 2 h at 4 C, washed three times with lysis buffer, then eluted with Laemmli buffer for 5 min at 100 C. For immunoblotting studies, equal amounts of solubilized placental proteins were resolved by electrophoresis on 7% or 10% SDS-polyacrylamide gels, as appropriate, directly or after immunoprecipitation with the specific antibodies, as indicated. The resolved proteins were electrophoretically transferred to nitrocellulose or polyvinylidene difluoride membranes (Hybond-ECL, Amersham Biosciences, Arlington Heights, IL) using a transfer buffer containing 192 mM glycine, 20% (vol/vol) methanol, and 0.02% SDS. To reduce nonspecific binding, the membranes were incubated in TNA buffer [10 mM Tris-HCl (pH 7.8), 0.9% NaCl, and 0.01% sodium azide] supplemented with 5% BSA and 0.05% Nonidet P-40 at 37 C for 2 h or in PBS supplemented with 3% nonfat dry milk for 2 h at room temperature, as appropriate, and then incubated overnight at 4 C with the indicated antibodies. The proteins were visualized by enhanced chemiluminescence using horseradish peroxidase-labeled antirabbit or antimouse IgG (Amersham Biosciences) and quantified by densitometric analysis using Optilab image analysis software (Graftek SA, Mirmande, France) or Quantity One image analysis software (Bio-Rad Laboratories, Inc., Hercules, CA).
Antibodies
Polyclonal anti-IGF-I receptor ?-subunit, polyclonal antiinsulin receptor ?-subunit, and monoclonal anti-phosphotyrosine antibodies (PY99) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-phospho-Shc (Tyr317), anti-Akt, anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-phospho-p42/44 MAPK [extracellular signal-regulated kinase (Erk-1/2); Thr202/Tyr204], and anti-phospho-glycogen synthase kinase-3 (GSK-3)/? (Ser9/Ser21) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal anti-Shc antibodies were purchased from Upstate Biotechnology, Inc. (Saranac Lake, NY). Polyclonal antibodies against the p85 subunit of phosphatidyl inositol 3-kinase (PI 3-kinase), IRS-1, IRS-2, or GSK-3 were purchased from Upstate Biotechnology, Inc. Anti-MAPK (Erk-1/2) antibodies were obtained from Zymed Laboratories (San Francisco, CA). Polyclonal anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-stress-activated protein kinase/c-Jun N-terminal kinase (JNK), and anti-phospho-stress-activated protein kinase/JNK (Thr183/Tyr185) were obtained from Cell Signaling Technology, Inc.
Statistical analyses
All data are expressed as the mean ± SE. Statistical analyses were performed by t tests.
Results
IGF-I receptor
To evaluate the expression levels of placental IGF-I receptors, tissue samples were analyzed by direct immunoblotting with anti-IGF-I receptor antibodies. As shown in Fig. 1A, a single band of approximately 105 kDa, corresponding to the IGF-I receptor ?-subunit, was detected. The total amount of IGF-I receptor protein was reduced 33% in IUGR compared with control placentas (Fig. 1A; P < 0.05). By contrast, the protein content of the insulin receptor, as assessed by reprobing the same filters with antiinsulin receptor antibodies, was not different in IUGR and control placentas (Fig. 1B; P = 0.383). To evaluate the phosphorylation state of IGF-I receptors, placental protein extracts were analyzed by sequential immunoprecipitation with antibodies against the IGF-I receptor ?-subunit and immunoblotting with anti-phosphotyrosine antibodies. However, tyrosine phosphorylation of IGF-I receptors could not be detected in either control or IUGR placentas (data not shown).
FIG. 1. IGF-I receptor and insulin receptor protein contents in human placentas. Equal amounts of solubilized proteins from control () and IUGR () placentas were resolved by 7% SDS-PAGE, as described in Materials and Methods. IGF-I receptor protein content (A) was detected by immunoblotting with anti-IGF-I receptor antibodies. The protein content of the insulin receptor in placental samples (B) was measured by immunoblotting with specific antibodies. The bar graphs in A and B show the quantitation of IGF-I receptor and insulin receptor protein contents in all samples (n = 14 for controls and IUGR, respectively). IGF-I R, IGF-I receptor; Ins R, insulin receptor. *, P < 0.05 vs. controls.
IRS proteins and PI 3-kinase
To investigate postreceptor IGF-I signaling proteins, the expression levels of IRS-1 and IRS-2, the major IGF-I receptor substrates, were next evaluated by immunoprecipitation and immunoblotting with anti-IRS-1 or anti-IRS-2 antibodies, respectively. The total levels of IRS-1 and IRS-2 proteins were differently regulated by IUGR. IRS-1 levels were not different in IUGR and control placentas (Fig. 2A; P = 0.553 vs. control). By contrast, IRS-2 levels were reduced 35% in IUGR compared with control placentas (Fig. 2B; P < 0.05 vs. control). Analysis of IRS-1 and IRS-2 immunoprecipitates with anti-phosphotyrosine antibodies showed minimal, if any, tyrosine phosphorylation of either substrate (data not shown).
FIG. 2. IRS-1 and IRS-2 protein contents and associated p85 protein in human placenta. Solubilized proteins from control () and IUGR () placentas were subjected to immunoprecipitation with anti-IRS-1 or anti-IRS-2 antibodies, as indicated. The resulting immune complexes were resolved by 7% SDS-PAGE and analyzed by immunoblotting with anti-IRS-1, anti-IRS-2, or anti-p85 antibodies, as indicated. Each panel shows a representative experiment, on the left, and the quantitation of results from all samples, on the right. A, IRS-1 protein content; B, IRS-2 protein content; C, IRS-1-associated p85; D, IRS-2-associated p85. *, P < 0.05 vs. controls.
The enzyme PI 3-kinase is recruited to IRS proteins through binding of the two SH2 domains contained in its 85-kDa regulatory subunit to specific phosphotyrosine residues in IRS-1 and IRS-2 (17). To assess whether association of the IRS proteins with p85, the regulatory subunit of PI 3-kinase, could be detected in human placenta, IRS-1 and IRS-2 immunoprecipitates were analyzed by immunoblotting with anti-p85 antibodies. The amount of p85 protein associated with IRS-1 was similar in control and IUGR placentas (Fig. 2C), whereas IRS-2-associated p85 appeared to be significantly reduced in the IUGR group (Fig. 2D; P < 0.05 vs. control), in agreement with the observed decrease in total IRS-2 protein content (Fig. 2B). No differences in the total p85 protein levels were observed in the IUGR and control placentas (Fig. 3A).
FIG. 3. p85, Akt and GSK-3 in human placenta. Solubilized proteins from control () and IUGR () placentas were resolved by 10% SDS-PAGE and analyzed by immunoblotting with specific antibodies, as indicated. Each panel shows a representative experiment, on the left, and the quantitation of results from all samples, on the right. A, p85 protein content; B, total protein content (top) and Ser473 phosphorylation (bottom) of Akt; C, total protein content (top) and Ser9/Ser21 phosphorylation (bottom) of GSK-3/?. *, P < 0.05 vs. controls.
Akt and GSK-3
The expression levels of Akt, a serine/threonine kinase that lies downstream of PI 3-kinase in the IGF-I signaling pathway, were investigated next. The total Akt protein level was significantly decreased in IUGR placentas (73% of control; P < 0.05; Fig. 3B). However, Akt activation, measured by immunoblotting with antibodies to phosphorylated Ser473 in the Akt protein, was not different in control and IUGR placentas (P = 0.737; Fig. 3B). Phosphorylation of Akt at Thr308 could not be demonstrated by immunoblotting with phospho-specific antibodies (data not shown). The total protein and phosphorylation levels of GSK-3, a direct substrate of Akt, were not different in control and IUGR placentas (Fig. 3C).
To assess whether the differences in total protein contents of specific signaling proteins in IUGR compared with those in control placentas were related to the different gestational ages of the two groups (Table 1), further analyses were carried out in control (n = 8) and IUGR (n = 7) placentas that were matched for gestational age (36.5 ± 1.1 vs. 35.7 ± 1.1 wk; P = 0.63; Table 2). The total protein content of IGF-I receptors was reduced 38% in these IUGR placentas compared with the related controls (P < 0.05), whereas insulin receptor expression was not altered (P = 0.56; Table 2). Moreover, IRS-1 protein levels were not different (P = 0.63), whereas IRS-2 and Akt protein levels were 45% and 30% lower in IUGR compared with control placentas, respectively (P < 0.05; Table 2). These changes were similar to those observed in the total IUGR group (Figs. 1–3). No significant differences in protein levels of IGF-I receptors, insulin receptors, IRS-1, IRS-2, or Akt were detected in these 35-wk-old IUGR placentas compared with the remaining IUGR samples with lower (i.e. 28.5 wk; P < 0.05) gestational age (data not shown). Together, these findings demonstrate that lower expression of specific signaling proteins in IUGR placentas was not related to gestational age.
TABLE 2. Characteristics of control and IUGR subgroups matched for gestational age
Shc and the MAPK family
The Shc proteins (i.e. p66Shc, p52Shc, and p46Shc) are substrates for the IGF-I receptor tyrosine kinase and are largely responsible for activation of the Erk-1/2 MAPK cascade, which is involved in growth and differentiation responses in mammalian cells (18). To examine potential defects of the Shc/Erk pathway in the placenta, expression levels of the Shc proteins were studied by immunoblotting with specific antibodies. Equal amounts of p66Shc, p52Shc, and p46Shc proteins were found in control and IUGR placental samples (Fig. 4A). Tyrosine phosphorylation of the Shc proteins, evaluated by immunoblotting with phospho-specific Shc antibodies, could not be demonstrated in either control or IUGR placental tissues (data not shown). Total expression levels of Erk-1 and Erk-2 MAPKs were reduced in IUGR compared with control placentas (Fig. 4B; P < 0.05). However, phosphorylation of Erk-1 and Erk-2 were not significantly different in IUGR samples (Fig. 4B).
FIG. 4. Shc and Erk-1/2 in human placenta. For Shc analyses, solubilized proteins from control () and IUGR () placentas were resolved by 10% SDS-PAGE and analyzed by immunoblotting with anti-Shc antibodies, as described in Materials and Methods. Erk-1/2 protein content and phosphorylation were determined by immunoblotting with the indicated antibodies. Each panel shows representative immunoblots, on the left, and the quantitation of results from all samples, on the right. A, Total protein content of Shc with quantitation of the p52Shc isoform; B, total protein content (top) and phosphorylation (bottom) of Erk-1 and Erk-2. *, P < 0.05 vs. controls.
The p38 MAPK and JNK belong to the MAPK family of intracellular signal transducers involved in mammalian growth by integrating a variety of environmental stimuli (19, 20). To verify whether abnormal intrauterine development is associated with altered expression and activation of these MAPKs in the placenta, protein content and phosphorylation of p38 and JNK MAPKs were analyzed. No differences were found in p38 protein content between the IUGR and control samples (Fig. 5A; P = 0.771). By contrast, the level of p38 phosphorylation was markedly reduced in IUGR compared with control placentas (Fig. 5A; P < 0.05). The protein content of JNK also did not differ in the two groups (Fig. 5B; P = 0.304), but the phosphorylation levels of both JNK-1 and JNK-2 were reduced 70% in IUGR (Fig. 5B; P < 0.05 vs. control). Therefore, phosphorylation of both p38 and JNK was markedly reduced in IUGR placentas, and this was not explained by changes in the total protein contents of these two kinases.
FIG. 5. Protein content and phosphorylation of p38 and JNK MAPKs in human placenta. Tissue lysates from control and IUGR placentas were analyzed by direct immunoblotting with the indicated antibodies, as described in Materials and Methods. Each panel shows representative immunoblots, on the left, and the quantitation of multiple experiments on all samples, on the right. A, Total protein (top) and phosphorylation (bottom) of p38. B, Total protein content (top) and phosphorylation (bottom) of JNK. *, P < 0.05 vs. controls.
Discussion
In this study we investigated the IGF-I signal transduction system in placentas of growth-restricted human fetuses. The experimental procedure used, i.e. sampling of placental tissues immediately after delivery and subsequent protein analysis using biochemical methods, allowed us to investigate the expression levels of various placental signaling proteins and their activation state in vivo during late gestation. Specific signaling events, such as tyrosine phosphorylation of the IGF-I receptor, IRS, and Shc proteins, were below the sensitivity of the immunoblotting techniques employed, whereas others, including formation of IRS-2/p85 signaling complexes and phosphorylation of Akt kinase and MAPK, could be measured. We demonstrate that the placentas of IUGR fetuses are characterized by multiple and specific abnormalities of the IGF-I signal transduction system, including reduced protein content of the IGF-I receptor, of its major substrate IRS-2, and of the serine/threonine kinases Akt and Erk-1/2. In addition, IUGR placentas feature a marked impairment of the coordinated activation of the MAPKs, with reduced p38 and JNK phosphorylation.
Multiple experimental settings have demonstrated a pivotal role of the IGF-I receptor/IRS/Akt pathway in controlling intrauterine growth in mammals. Targeted disruption of the IGF-I or IGF-I receptor genes is associated with marked IUGR in mice (21, 22), and partial deletion of the IGF-I gene has been reported to be responsible for IUGR and postnatal growth failure in humans (23). Knockout experiments in mice, in which gene expression of IRS-I or IRS-2 was abrogated in all tissues, also showed growth defects (24, 25, 26). Finally, Akt1-deficient mice showed a 20% reduction in body weight at birth compared with wild-type littermates (27). Thus, the observed reductions in IGF-I receptor, IRS-2, and Akt protein contents in placentas associated with IUGR may contribute to impaired fetal growth in humans. The IUGR placenta may be viewed as an IGF-resistant tissue, in which the IGF-I signaling potential is blunted due to subnormal expression levels of key signaling molecules. The impaired growth-promoting action of IGF-I on the feto-placental unit may thus explain the reduced neonatal and placental weight at birth (Table 1). Relevant to this concept, GH or IGF-I administration in experimental models of IUGR was associated with increased circulating levels of IGF-I, but failure to stimulate fetal growth and placental or fetal organ weight (28, 29).
In IUGR placentas, IRS-2 protein levels were markedly reduced, whereas those of IRS-1 were unchanged (Fig. 2, A and B). This finding was associated with decreased association of p85, the regulatory subunit of the enzyme PI 3-kinase, with IRS-2. Differential regulation of IRS-1 and IRS-2 has been observed in experimental and human diseases. IRS-1 is reduced, whereas IRS-2 is unchanged, in skeletal muscle and myocardium of streptozotocin-diabetic rats (30, 31) and in adipocytes from type 2 diabetic patients (32). Increased expression of IRS-2, with no change in IRS-1, has been described in rat insulinoma cells and human pancreatic cancer tissue compared with that in normal ?-cells (33, 34). Moreover, different physiological functions for IRS-1 and IRS-2 have been proposed in various cellular contexts, including L6 myoblasts (35), brown adipocytes (36), exercised skeletal muscle (37), and transgenic animals with selective ablation of the IRS-1 or IRS-2 gene (24, 38, 39). Although the specific roles of IRS-1 and IRS-2 in human placental cells have not been defined, the selective down-regulation of IRS-2 and its association with p85 in the IUGR placentas may play important roles in the impaired development of the feto-placental unit.
A low birth weight has reportedly been associated with impaired insulin secretion and insulin resistance in later life (40, 41). This can reflect both adaptation to disadvantageous environmental factors (i.e. malnutrition in utero) and genetically determined defects in specific signaling proteins. Interestingly, IRS-2 has been proposed as a major determinant of pancreatic ?-cell growth and insulin secretion (26, 42), and mice rendered null for IRS-2 develop ?-cell failure and various alterations resembling type 2 diabetes in humans (39). Even though it is not known whether IRS-2 levels are reduced in ?-cells from growth-restricted newborns, we show that placentas of IUGR fetuses exhibit a defect in IRS-2 expression and signaling. It is tempting to speculate that the impaired placental expression of IRS-2 may represent the molecular hallmark linking IUGR with the subsequent development of metabolic diseases in adult life (3). In an experimental model of IUGR, impaired activation of IRS-2 in the liver preceded the onset of insulin resistance and metabolic abnormalities (43).
Placental cells proliferate, differentiate, and eventually undergo apoptosis under the control of multiple growth factors (44). Under physiological conditions, apoptosis occurs during early pregnancy (at wk 5–7) and in the third trimester in human placentas (45, 46). However, pregnancies complicated by IUGR have reportedly been associated with increased apoptotic rates in placental tissue (47). Moreover, apoptosis was recently shown to be increased in cultured cytotrophoblasts from human IUGR placentas (48). Also in this study the amount of oligonucleosomes in cytosolic extracts, which correlates with early apoptosis, was found to be higher, although not significantly, in IUGR than in control placentas (Laviola, L., and F. Giorgino, unpublished observations). Proposed biological consequences of augmented apoptosis in the placenta include depletion of the syncytiotrophoblast layer, leading to impairment of materno-fetal exchange functions (49), and degeneration of placental villi, leading to reduced placental perfusion and oxygenation (50). Increased placental apoptosis in human placentas associated with IUGR may be the consequence of increased p53 expression (51) and reduced Bcl-2 protein levels (52). We suggest that it could also be the result of insufficient survival signals through the IGF-I receptor/IRS-2 pathway. Relevant to this concept, extensive apoptosis in preimplantation embryos is associated with down-regulation of the IGF-I receptor (53). It is important to note that the insulin receptor content was not altered in the IUGR placentas, as also shown by Reid and co-workers (13) in an experimental model of fetal growth restriction.
Coordinate activation of the MAPKs is required for protection from apoptosis and appropriate regulation of cell proliferation in response to growth factors and cytokines (20, 54, 55). Therefore, reduced expression and/or blunted activation of these intracellular enzymes are expected to impair development of the feto-placental unit. Indeed, embryos deficient in Erk-2 lack mesoderm differentiation and placental angiogenesis (56). In addition, a mutant mouse line with disruption of the mek1 gene, the upstream activator of Erk-1/2, was characterized by reduced placental vascularization, leading to the premature death of the mek1–/– embryos (57). Targeted disruption of the p38 MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. In particular, p38 mutant placentas display impaired vascularization and insufficient oxygen and nutrient transport as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis (58, 59). Finally, compound mutations of the jnk1 and jnk2 genes cause early embryonic death associated with impaired morphogenesis, decreased apoptosis in the hindbrain, and increased apoptosis in the forebrain (60). In primary human trophoblast, specific activation of JNK in response to placental growth factor protects from serum withdrawal-induced apoptosis (61). Reduced activation of JNK has also been observed in placental tissue from women with preeclampsia (62), which features a defective vascular development of the feto-placental unit, similar to IUGR pregnancies (5). Interestingly, mice with targeted disruption of the junB gene, a member of the activating protein-1 transcription factor complex, which lies downstream and is activated by JNK, show severe growth retardation in utero, embryonic lethality, and defective development of the feto-maternal vascularization (63). Together, these findings strongly support the hypothesis that the impairment in the integrated activation of the MAPKs observed in IUGR placentas may play an important role in altering placental angiogenesis, ultimately leading to reduced fetal growth.
Even though all deliveries occurred within the third trimester, the gestational age of the study group was significantly lower than that of the control group. To rule out that this could affect the expression levels of distinct signaling proteins, we studied selected control and IUGR subgroups, matched for gestational age. Analysis of the placental proteins in these subgroups revealed the same pattern of abnormalities detected in the larger groups, i.e. reduced expression of IGF-I receptors, IRS-2, and Akt, and no change in insulin receptor or IRS-1 protein levels, respectively. This suggests that the molecular mechanisms underlying the growth restriction in humans involve differences in IGF-I signaling independently of the stage of fetal development.
In conclusion, we have demonstrated that abnormalities in expression and/or activation of specific IGF-I receptor signaling molecules and multiple members of the MAPK family occur in human placentas of IUGR fetuses at delivery. These molecular defects may be responsible for impaired IGF-I responsiveness and appropriate development of the feto-placental unit, leading to dysregulation of fetal growth in humans.
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