Proteolysis Of Insulin-like Growth Factor-binding Protein-3 Research Articles

Indirect targeting of IGF receptor signalingin vivoby substrate-selective inhibition of PAPP-A proteolytic activity

The insulin-like growth factor (IGF) signaling pathway is involved in certain human cancers, and the feasibility of directly targeting the IGF receptor has been actively investigated. However, recent evidence from clinical trials suggests that this approach can be problematic. We have developed an alternative strategy to indirectly inhibit the IGF signaling by targeting the metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). PAPP-A associated with the cell surface cleaves IGF binding protein-4 (IGFBP-4), when IGF is bound to IGFBP-4, and thereby increases IGF bioavailability for receptor activation in an autocrine/paracrine manner. We hypothesized that inhibition of PAPP-A would suppress excessive local IGF signaling in tissues where this is caused by increased PAPP-A proteolytic activity. To test this hypothesis, we developed an inhibitory monoclonal antibody, mAb 1/41, which targets a unique substrate-binding exosite of PAPP-A. This inhibitor selectively and specifically inhibits proteolytic cleavage of IGFBP-4 with an inhibitory constant (Ki) of 135 pM. In addition, it inhibited intracellular signaling of the IGF receptor (AKT phosphorylation) in monolayers of A549 cells, an IGF-responsive lung cancer-derived cell line found to express high levels of PAPP-A. We further showed that mAb 1/41 is effective towards PAPP-A bound to cell surfaces, and that it is capable of inhibiting PAPP-A activity in vivo. Using a murine xenograft model of A549 cells, we demonstrated that mAb 1/41 administered intraperitoneally significantly inhibited tumor growth. Analysis of xenograft tumor tissue recovered from treated mice showed penetration of mAb 1/41, reduced IGFBP-4 proteolysis, and reduced AKT phosphorylation. Our study provides proof of concept that IGF signaling can be selectively reduced by targeting a regulatory proteinase that functions extracellularly, upstream of the IGF receptor. PAPP-A targeting thus represents an alternative therapeutic strategy for inhibiting IGF receptor signaling.

The Impact of Neutrophil Proteinase 3 on IGFBP-3 Proteolysis in Obesity

Obesity is a complex disorder and is a major risk factor associated with the incidence of insulin resistance (IR), diabetes, cardiovascular disease (CVD) and other metabolic disorders. The endocrine paradigm suggests that visceral fat in obesity, consisting primarily of adipocytes, secretes various pro-inflammatory adipokines such as tumor necrosis factor (TNF), leptin, visfatin, resistin, and IL-6 creating a state of local inflammation further resulting in chronic systemic inflammation and accelerating the events leading to systemic IR, diabetes and metabolic syndrome. The insulin-like growth factor (IGF) system plays a major role in growth, development and maintenance of homeostasis in normal cells. IGF binding protein-3 (IGFBP-3), the major binding protein in circulation, has been shown to be associated with obesity, IR, type II diabetes mellitus (T2DM) and CVD. Recent studies have demonstrated the IGFBP-3-specific receptor (IGFBP-3R) is a novel protein mediating the anti-inflammatory function of IGFBP-3. IGFBP-3 inhibits adipokine-induced insulin resistance and early manifestations of atherosclerosis via inhibition of NF-κB signaling in adipocytes. Furthermore, decreases in total IGFBP-3 levels and increases in proteolyzed IGFBP-3 in circulation have been documented in obese populations compared to their normal counterparts further establishing a positive correlation between IGFBP-3 proteolysis and adiposity parameters as well as IR. Conversely, our recent studies have identified that neutrophil serine protease (NSP) PR3, an IGFBP-3 specific protease in obesity, is positively correlated with IGFBP-3 proteolysis, IR, body mass index, TNF and IL-8. These findings strongly suggest that obesity-induced activation of PR3 abrogates the anti-inflammatory, insulin-sensitizing IGFBP-3/IGFBP-3R cascade, resulting in IR and its progression to T2DM. The complete characterization of the underlying mechanism and functional significance of the PR3-IGFBP-3/IGFBP-3R cascade in obesity will foster identification of the diagnostic and therapeutic potential of PR3 inhibition in insulin resistance and its sequelae.

Skp2B Overexpression Alters a Prohibitin-p53 Axis and the Transcription of PAPP-A, the Protease of Insulin-Like Growth Factor Binding Protein 4

BackgroundWe previously reported that the degradation of prohibitin by the SCFSkp2B ubiquitin ligase results in a defect in the activity of p53. We also reported that MMTV-Skp2B transgenic mice develop mammary gland tumors that are characterized by an increased proteolytic cleavage of the insulin-like growth factor binding protein 4 (IGFBP-4), an inhibitor of IGF signaling. However, whether a link exists between a defect in p53 activity and proteolysis of IGFBP-4 was not established.Methods and ResultsWe analyzed the levels of pregnancy-associated plasma protein A (PAPP-A), the protease of IGFBP-4, in MMTV-Skp2B transgenic mice and found that PAPP-A levels are elevated. Further, we found a p53 binding site in intron 1 of the PAPP-A gene and that both wild type and mutant p53 bind to this site. However, binding of wild type p53 results in the transcriptional repression of PAPP-A, while binding of mutant p53 results in the transcriptional activation of PAPP-A. Since MMTV-Skp2B mice express wild type p53 and yet show elevated levels of PAPP-A, at first, these observations appeared contradictory. However, further analysis revealed that the defect in p53 activity in Skp2B overexpressing cells does not only abolish the activity of wild type of p53 but actually mimics that of mutant p53. Our results suggest that in absence of prohibitin, the half-life of p53 is increased and like mutant p53, the conformation of p53 is denatured.ConclusionsThese observations revealed a novel function of prohibitin as a chaperone of p53. Further, they suggest that binding of denatured p53 in intron 1 causes an enhancer effect and increases the transcription of PAPP-A. Therefore, these findings indicate that the defect in p53 function and the increased proteolysis of IGFBP-4, we had observed, represent two components of the same pathway, which contributes to the oncogenic function of Skp2B.

Activity of Human Pregnancy Insulin-Like Growth Factor Binding Protein-3: Determination by Reconstituting Recombinant Complexes

During pregnancy, IGF binding protein-3 (IGFBP-3) is completely proteolyzed to fragments with low affinities for IGFs but appears to transport IGFs normally in high-molecular-mass complexes. We previously reported that synthetic isolated amino- and carboxyl-terminal domains of IGFBP-3 cooperate to bind IGFs, and we investigated whether this is the mechanism whereby proteolyzed IGFBP-3 fragments bind IGFs normally in pregnancy serum. Two fragments of IGFBP-3 have been isolated from pregnancy serum, one with the same N-terminal sequence as intact IGFBP-3 (GASSG) and the other with an N-terminal sequence (160)KVDYE. Recombinant forms of these proteins, IGFBP-3(1-159) and IGFBP-3(160-264), have been synthesized and characterized, demonstrating that although the fragments individually have greatly reduced affinity for IGF complex formation, when combined they cooperate to form complexes with IGF with or without the acid-labile subunit, inhibit IGF transport across endothelial cell monolayers and inhibit IGF-I-induced IGF type I receptor phosphorylation. It is proposed that proteolysis of IGFBP-3 into two discrete complementary fragments does not significantly increase IGF bioavailability, consistent with previous findings that proteolyzed IGFBP-3 in pregnancy serum is functionally normal and circulates as part of the IGF ternary complexes.

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

Pregnancy‐induced changes in insulin‐like growth factor I (IGF‐I), insulin‐like growth factor binding protein 3 (IGFBP‐3), and acid‐labile subunit (ALS) in patients with growth hormone (GH) deficiency and excess

Under most circumstances with altered growth hormone (GH) secretion, the changes of insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 3 (IGFBP-3), and acid-labile subunit (ALS) are in parallel. The aim of the present study was to compare the effects of pregnancy in a hypopituitary patient with those of pregnancy in an acromegalic patient on IGF-I, IGFBP-3, and ALS. IGF-I and ALS were low before pregnancy in the hypopituitary patient under glucocorticoid and thyroxine treatment. Gonadotropin treatment allowed her to become pregnant; IGF-I and ALS levels rose in the second half of pregnancy and fell again after delivery. IGF-I concentrations were elevated in the patient with persistent acromegaly before and dropped into the normal range during the first half of pregnancy. In the second half of pregnancy and following delivery, IGF-I levels increased again. IGFBP-3 levels (as assessed by immunoblot analysis as well as by 125I-IGF II ligand blotting) decreased markedly during pregnancy in both patients, suggesting that the placenta rather than pituitary GH regulates IGFBP-3 proteolysis in human pregnancy. The increase of IGF-I (and ALS) during the second half of pregnancy in the individual with pituitary GH deficiency may be attributed to placental GH. The fall of IGF-I (and ALS) into the normal range in the acromegalic patient during the first trimester of pregnancy may be related to decreased production or decreased half-life of these proteins. Our data suggest that measures to continuously replace GH or to suppress GH secretion during pregnancy in patients with GH deficiency or excess, respectively, may not be warranted.

Insulin-like growth factor, insulin-like growth factor–binding protein-4, and pregnancy-associated plasma protein-A gene expression in human granulosa cell tumors

The insulin-like growth factor (IGF) system plays an important role in folliculogenesis. It is also thought to contribute to the pathogenesis of many cancers, including those of the ovarian epithelium. In the human follicle, the predominant IGF is IGF-II and its actions are modulated by insulin-like growth factor-binding protein-4 (IGFBP-4), the IGFBP-4 protease, and the pregnancy-associated plasma protein-A (PAPP-A). These peptide components are synthesized by the granulosa cells of the developing follicle. The aim of this study was to characterize the expression of these components of the IGF system in granulosa cell tumors (GCT) of the ovary. IGF-I, IGF-II, IGFBP-4, and PAPP-A gene expression was determined in a panel of GCT and compared to the levels in normal ovary and in epithelial ovarian tumors. Although both the IGF-I and IGF-II genes were expressed in the GCT, the levels were lower than in the other tissue groups. IGFBP-4 expression was also low in the GCT, whereas PAPP-A gene expression was highest in the GCT. These findings were unexpected given the prominent role this signaling system plays in normal granulosa cells. In conclusion, these observations suggest that the IGF system may have a limited role in the pathogenesis of GCT with PAPP-A subserving a function other than IGFBP-4 proteolysis.

Dexamethasone administration attenuates the inhibitory effect of lipopolysaccharide on IGF-I and IGF-binding protein-3 in adult rats

The aim of this study was to investigate whether glucocorticoid administration had a beneficial effect on serum concentrations of insulin-like growth factor I (IGF-I) and on IGF-binding protein 3 (IGFBP-3) in rats injected with lipopolysaccharide (LPS). Adult male rats were injected with LPS or saline and pretreated with dexamethasone or saline. Dexamethasone administration decreased growth hormone (GH) receptor and IGF-I mRNA levels in the liver of control rats. LPS decreased GH receptor and IGF-I gene expression in the liver of saline-treated rats but not in the liver of dexamethasone-pretreated rats. In the kidney, GH receptor mRNA levels were not modified by dexamethasone or LPS treatment. However, LPS decreased renal IGF-I gene expression and dexamethasone pretreatment prevented this decrease. Serum concentrations of IGF-I were decreased by LPS, and dexamethasone pretreatment attenuated this effect. The gene expression of IGFBP-3 in the liver and kidney and its circulating levels were decreased by LPS. In control rats dexamethasone increased circulating IGFBP-3 and its gene expression in the liver, and decreased the proteolysis of this protein. Dexamethasone pretreatment attenuated the LPS-induced decrease in IGFBP-3 gene expression in the liver and prevented the LPS-induced decrease in IGFBP-3 gene expression in the kidney. Moreover, dexamethasone pretreatment attenuated the LPS-induced decrease in serum concentrations of IGFBP-3 and decreased the LPS-induced IGFBP-3 proteolysis in serum. In conclusion, dexamethasone pretreatment partially attenuates the inhibitory effect of LPS on serum IGF-I by blocking the decrease of its gene expression in the kidney as well as by attenuating the decrease in serum concentrations of IGFBP-3.

Lack of insulin-like growth factor binding protein-3 protease activation by venous cannulation

Disruption of the endothelium activates thrombogenic and fibrinolytic enzymes that cleave insulin-like growth factor binding protein-3 (IGFBP-3) in vitro. The aim of the present human study was to determine whether blood sampling, i.e., venous stasis and cannulation increase IGFBP-3 proteolysis before and/or after surgery by activating these enzymes. Serum samples obtained immediately after cannulation were compared with samples obtained from a previously inserted venous catheter. Cannulation did not increase serum IGFBP-3 proteolytic activity pre- and post-operatively, as determined by in vitro degradation of 125I-IGFBP-3. Furthermore, there was no effect on in vivo IGFBP-3 fragmentation assessed by western immunoblot. In addition, a standardized venous stasis did not affect IGFBP-3 proteolytic activity or fragmentation. Comparison of IGFBP-3 proteolytic activity before and after surgery demonstrated a significant post-operative increase. However, this could not be demonstrated immediately after the initial cannulation, due to a large individual variation at this time-point before surgery.

Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester.

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP-PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a approximately 470 kDa PAPP-A form (PAPP-A470) to a approximately 400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470 to the less-active PAPP-A400, could account for the TPA-induced suppression of PAPP-A activity.

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.

Matrix metalloproteinase-7 facilitates insulin-like growth factor bioavailability through its proteinase activity on insulin-like growth factor binding protein 3.

Matrix metalloproteinase-7 (MMP-7) secreted by cancer cells has been implicated classically in the basement membrane destruction associated with tumor cell invasion and metastasis. Recent epidemiologic studies have established a correlation between high levels of circulating insulin-like growth factor (IGF) and low levels of IGF binding protein 3 (IGFBP-3), and relative risk of developing colon, breast, prostate, and lung cancer, which are known to produce MMP-7. In this study, IGFBP-3 was assessed as a candidate for the physiologic substrate of MMP-7. MMP-7 proteolysis generated four major fragments (26 kDa, 17 kDa, 15.5 kDa, and 15.5 kDa), and two cleavage sites were identified: one at the site of hydrolysis of the K(144)-I(145) peptide bond and one at the R(95)-L(96) peptide bond. The former site is different from the previously reported site of cleavage of IGFBP-3 by other proteases. Addition of IGFBP-3 inhibited IGF-I-mediated IGF type 1 receptor (IGF-IR) phosphorylation and activation of the downstream molecule Akt in BALB/c 3T3 fibroblasts overexpressing human IGF-IR (3T3-IGF-IR) and in two human colon cancer cell lines (COLO201 and HT29). Coincubation of the IGF-I/IGFBP-3 complex with MMP-7 restored IGF-I-mediated IGF-IR phosphorylation and activation of Akt in these cell lines. The IGF-I signal recovered by MMP-7 protected against apoptosis induced by anoikis in 3T3-IGF-IR cells. These results indicate that MMP-7 proteolysis of IGFBP-3 plays a crucial role in regulating IGF-I bioavailability, thereby promoting cell survival. This mechanism may contribute to the tumorigenesis of MMP-7-producing IGF-IR-expressing tumors in the primary site and to organ-specific metastasis in a paracrine manner.

Effect of inducible nitric oxide synthase inhibition by aminoguanidine on insulin-like growth factor binding protein-3 in adjuvant-induced arthritic rats

This study was designed to investigate whether nitric oxide (NO) mediates changes in insulin-like growth factor binding protein-3 (IGFBP-3) levels in rats with adjuvant-induced arthritis. Male Wistar rats were injected with complete Freund's adjuvant, and 20 days afterwards arthritic and control rats were injected daily with an inhibitor of inducible NO synthase (iNOS), aminoguanidine, or vehicle for 8 days. The increase in serum levels of IGFBP-3 induced by arthritis was exacerbated by aminoguanidine treatment. Arthritis increased IGFBP-3 mRNA levels in the kidney but not in the liver. The inhibition of iNOS did not modify IGFBP-3 gene expression in the kidney or in the liver in arthritic rats. However, the inhibitory effect of arthritis on the proteolysis of IGFBP-3 in serum was potentiated by aminoguanidine administration. These results indicate that arthritis increases serum IGFBP-3 by increasing its synthesis in the kidney and decreasing its proteolysis in serum and that these effects are not mediated by NO.

Acute phase modulation of systemic insulin-like growth factor-1 and its binding proteins after major burn injuries.

To provide a detailed, sequential analysis of insulin-like growth factor-1 and its binding proteins in adults during the acute phase after a major burn injury. Descriptive, repeated measurements for quantitation and characterization of insulin-like growth factor-1 and its binding proteins in adult burn survivors. Burn center in a university hospital. A total of 17 severely burned (>15% total body-surface area burned) adult patients. Venous blood was collected twice a day for 10 days and centrifuged, and the sera were stored at -80 degrees C until analysis. A series of 340 serum samples were analyzed by radioimmunoassay to determine the circulating concentration of insulin-like growth factor-1 and its major binding proteins (insulin-like growth factor-binding protein), by Western ligand blotting. To better understand the changes seen in systemic insulin-like growth factor-binding protein-3 levels by Western ligand blotting, a proteolysis assay was performed. Insulin-like growth factor-1 levels were reduced from day 0 and correlated with insulin-like growth factor-binding protein-1 and -2 (p <.01), but not insulin-like growth factor-binding protein-3 and -4. Insulin-like growth factor-binding protein-3 was decreased relative to normal on day 0, declined further until day 3, and began recovering by day 6, but returned to only 35% of normal by day 10. Insulin-like growth factor-binding protein-1 and -2 were increased relative to normal and remained increased throughout the 10-day period. Insulin-like growth factor-binding protein-4 concentrations, however, were similar to normal at day 1 but gradually increased over time. Burn serum incubated with recombinant human glycosylated iodine-125 insulin-like growth factor-binding protein-3 did not demonstrate any proteolysis, although proteolysis of nonglycosylated iodine-125 insulin-like growth factor-binding protein-3 reached levels of approximately 40%. Insulin-like growth factor-binding protein-3 proteolysis does not seem to be the mechanism by which systemic insulin-like growth factor-1 levels are reduced in major burn survivors. In vitro proteolysis of recombinant human glycosylated and nonglycosylated iodine-125 insulin-like growth factor-binding protein-3 does not reflect the in vivo situation in major burn survivors.

Low Levels of the 150-kD Insulin-Like Growth Factor Binding Protein 3 Ternary Complex in Patients with Anorexia nervosa: Effect of Partial Weight Recovery

Background/Aim: In healthy adults, serum insulin-like growth factor I (IGF), IGF-binding protein 3 (IGFBP-3), and acid-labile subunit (ALS) form a 150-kD ternary complex under the control of growth hormone (GH). Circulating IGF-I half-life, bioavailability, and endocrine actions depend on the ternary complex formation. Despite GH hypersecretion, serum IGF-I, IGFBP-3, and ALS levels have all been reported to be low in patients with anorexia nervosa (AN), while the degree of ternary complex formation in AN is unknown. Methods: Serum ALS and 150-kD ternary complex formation were measured in 6 women with AN at the time of diagnosis and after partial weight recovery and in 6 healthy age-matched women serving as controls. Results: Patients with AN had low levels of ALS and IGFBP-3 contained in the 150-kD ternary complex and in the non-150-kD fraction. Following partial weight recovery, the 150-kD IGFBP-3 ternary complex was fully normalized, despite only partial normalization of serum GH and IGF-I levels. Patients with AN did not present with IGFBP-3 proteolysis different from controls. Conclusion: The present data indicate a pivotal role of the nutritional status in the regulation of each of the three components of the 150-kDa ternary IGFBP-3 complex and in the formation of the complex itself.

Altered bone metabolism in children infected with human immunodeficiency virus.

Data on bone homoeostasis of children infected with human immunodeficiency virus (HIV), at the time of the gain in bone mass, are very rare. To determine possible alterations in bone metabolism, 13 prepubertal vertically HIV-infected children were studied. Viral load, CD4 count, interleukin-6 (IL-6), growth hormone, insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), acid-labile subunit (ALS), IGFBP-3 proteolysis, osteocalcin in blood and N-terminal telopeptide of type I collagen in urine were determined. Lumbar spine bone mineral density was examined by dual-energy X-ray absorptiometry. Low osteocalcin levels were found in all patients. Low IGF-I was found in only six children, who had low CD4 count and high IL-6 levels, with normal levels of IGFBP-3 and ALS, absent IGFBP-3 proteolysis and decreased bone mineral density, irrespective of viral load or growth. Low serum osteocalcin levels appear to be an initial warning sign of possible altered bone metabolism in HIV-infected children. However, only when the immune system becomes more seriously compromised is bone loss measurable by bone densitometry.

Effect of intense exercise on inflammatory cytokines and growth mediators in adolescent boys.

Exercise can enhance growth and development in children, but recent investigations have revealed an intriguing paradox. Namely, the early (4-5 weeks) response to training programs in children lead to a catabolic, growth hormone (GH)-resistant state rather than the expected anabolic activation of the GH-->insulin-like growth factor-I (IGF-I) axis. This paradox led us to hypothesize that single bouts of exercise in children could stimulate proinflammatory cytokines known to inhibit directly anabolic activity of the GH-->IGF-1 axis (interleukin [IL]-6, IL-1beta, and tumor necrosis factor-alpha [TNF-alpha]). Eleven healthy high school-age boys, age 14 to 18.5 years, performed a single, typical, 1.5-hour wrestling practice session. Blood was sampled before and after the session. We found significant decreases in anabolic mediators: total IGF-I (-11.2 +/- 2.3%), bound IGF-I (-11.2 +/- 2.4%), and insulin (-42 +/- 10%. However, there was no change in unbound IGF-I. Remarkable increases were found in proinflammatory cytokines IL-6 (795 +/- 156%), TNF-alpha (30 +/- 12%), and IL-1beta (286 +/- 129%) and in IGF-binding protein-1 (835 +/- 234%), which itself is stimulated by inflammatory cytokines and is known to inhibit IGF-I. Evidence for compensatory mechanisms to counter the antianabolic inflammatory response to acute exercise were also noted: IL-1ra increased (80 +/- 20%) and IGF-binding protein-3 proteolysis (which can maintain unbound, biologically active IGF-I despite losses in total IGF-I) increased significantly (101 +/- 39%) as well. These data demonstrate that an intense exercise bout in male adolescents leads to reductions in anabolic mediators and profound increases in inflammatory cytokines. This might explain the development of what seems to be a paradoxical catabolic state in the initial phases of exercise training programs.

Targeted Expression of a Protease-resistant IGFBP-4 Mutant in Smooth Muscle of Transgenic Mice Results in IGFBP-4 Stabilization and Smooth Muscle Hypotrophy

The insulin-like growth factor-binding protein 4 (IGFBP-4), the most abundant IGF-binding protein produced by rodent smooth muscle cells (SMC), is degraded by specific protease(s) potentially releasing IGF-I for local bioactivity. IGFBP-4 protease(s) recognizes basic residues within the midregion of the molecule. We constructed a mutant IGFBP-4 with the cleavage domain substitution 119-KHMAKVRDRSKMK-133 to 119-AAMAAVADASAMA-133. Myc-tagged native and IGFBP-4.7A retained equivalent IGF-I binding affinity. Whereas native IGFBP-4 was cleaved by SMC-conditioned medium, IGFBP-4.7A was completely resistant to proteolysis. To explore the function of the protease-resistant IGFBP-4 in vivo, expression of the mutant and native proteins was targeted to SMC of transgenic mice by means of a smooth muscle alpha-actin promoter. Transgene expression was confined to SMC-rich tissues in all lines. Bladder and aortic immunoreactive IGFBP-4/transgene mRNA ratios in SMP8-BP4.7A mice were increased by 2- to 4-fold relative to SMP8-BP4 mice, indicating that the IGFBP-4.7A protein was stabilized in vivo. SMP8-BP4.7A mice had lower aortic, bladder, and stomach weight and intestinal length relative to SMP8-BP4 counterparts matched for protein expression by Western blotting. Thus, IGFBP-4.7A results in greater growth inhibition than equivalent levels of native IGFBP-4 in vivo, demonstrating a role for IGFBP-4 proteolysis in the regulation of IGF-I action.

Arthritis-induced increase in serum levels of IGF-binding protein-3 in rats is secondary to the decrease in its proteolytic activity.

Adjuvant-induced arthritis is a chronic inflammatory illness that induces a catabolic state, with a decrease in pituitary GH and hepatic IGF-I synthesis. We have previously observed an increase in serum IGF-binding protein-3 (IGFBP-3) in arthritic rats, and found that GH administration prevents the increase in circulating IGFBP-3 in arthritic rats. The aim of this work was therefore to study IGFBP-3 synthesis in the liver as well as its proteolysis in serum as the two possible causes of the increased circulating IGFBP-3 in arthritic rats. The effect of recombinant human GH (rhGH) administration was also analysed. Adult male Wistar rats were injected with complete Freund's adjuvant or vehicle, and 14 days later they were injected s.c. daily until day 22 after adjuvant injection with rhGH (3 IU/kg) or saline. Three hours after the last GH injection, all rats were killed by decapitation. Arthritis increased serum IGFBP-3 levels (P<0.01). The increase in serum IGFBP-3 levels in arthritic rats seems to be due to decreased proteolysis (P<0.01) rather than to an increased synthesis, since liver IGFBP-3 mRNA content was not modified by arthritis. GH administration to control rats resulted in an increase in both hepatic IGFBP-3 mRNA content and in serum IGFBP-3 levels in spite of the increase in IGFBP-3 proteolysis in serum. In arthritic rats, GH treatment did not modify liver IGFBP-3 synthesis, but it increased serum proteolysis of IGFBP-3, leading to a serum concentration of IGFBP-3 similar to that of control rats. Furthermore, there was a negative correlation between circulating IGFBP-3 and its proteolytic activity in the serum of adjuvant-induced arthritic rats. These data suggest that in chronic arthritis the increase in IGFBP-3 serum concentration is secondary to a decrease in proteolytic activity, rather than to an increase in hepatic IGFBP-3 gene expression.

Effect of IL-6 on IGF binding protein-3: a study in IL-6 transgenic mice and in patients with systemic juvenile idiopathic arthritis.

Stunted growth is a common complication of childhood diseases characterized by chronic inflammation or infections. We previously demonstrated that NSE/hIL-6 transgenic mice, overexpressing the inflammatory cytokine IL-6 since early phase of life, showed a marked growth defect associated with decreased IGF-I levels, suggesting that IL-6 is one of the factors involved in stunted growth complicating chronic inflammation in childhood. Here we show that NSE/hIL-6 mice have normal liver IGF-I production, decreased levels of IGF binding protein-3 (IGFBP-3) and increased serum IGFBP-3 proteolysis. Reduced IGFBP-3 levels results in a marked decrease in the circulating 150-kDa ternary complex, even in the presence of normally functional acid labile subunit. Pharmacokinetic studies showed that NSE/hIL-6 mice have accelerated IGF-I clearance. Patients with systemic juvenile idiopathic arthritis (s-JIA), a chronic inflammatory disease characterized by prominent IL-6 production and complicated by stunted growth associated with low IGF-I levels, have markedly decreased IGFBP-3 levels, increased serum IGFBP-3 proteolysis and normal acid labile subunit levels. Our data show that chronic overproduction of IL-6 causes decreased IGFBP-3 levels, resulting in a decreased association of IGF-I in the 150-kDa complex. Decreased levels of IGF-I appear to be secondary to increased clearance.