Insulin-like growth factor-binding protein-3 (IGFBP-3) blocks the effects of asthma by negatively regulating NF-κB signaling through IGFBP-3R-mediated activation of caspases.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a multifunctional protein known for modulating mitogenic and metabolic actions of IGFs as well as exerting a variety of biological actions not involving IGFs. Here, we show that IGFBP-3 blocks specific physiological consequences of asthma in an IGF-independent manner in vitro and in vivo. IGFBP-3 treatment effectively reduced all physiological manifestations of asthma examined in vivo (airway hyper-responsiveness, cellular and pathological changes in bronchoalveolar lavage fluid and lung tissue, and expression of numerous proinflammatory molecules). These unique IGFBP-3 effects were further confirmed in IGFBP-3-transgenic mice, thus strengthening the notion of IGFBP-3 actions within the respiratory system. Using human epithelial cells, we demonstrated the following: 1) IGFBP-3 blocks TNF-α-induced expression of proinflammatory molecules; 2) IGFBP-3 attenuates the TNF-α-induced migratory response of eosinophils; and 3) IGFBP-3 negatively regulates TNF-α-induced expression of the key NF-κB regulatory molecules IκBα and p65-NF-κB at the post-translational level. We identified that IGFBP-3 degrades IκBα and p65-NF-κB proteins through IGFBP-3 receptor (IGFBP-3R)-mediated activation of caspases thereby inhibiting TNF-α-induced activation of NF-κB signaling cascades. This unique IGFBP-3/IGFBP-3R action was further confirmed by demonstrating complete inhibition of IGFBP-3 action in the presence of caspase inhibitors as well as IGFBP-3R siRNAs. Non-IGF-binding IGFBP-3 mutants further proved the IGF-independent action of IGFBP-3. Our findings indicate that IGFBP-3 inhibits airway inflammation and hyper-responsiveness via an IGF-independent mechanism that involves activation of IGFBP-3R signaling and cross-talk with NF-κB signaling. The IGFBP-3/IGFBP-3R system therefore plays a pivotal role in the pathogenesis of asthma and can serve as a newly identified potential therapeutic target for this debilitating disease.

Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein.

Transglutaminase 2 (TG2, tissue transglutaminase) is a multifunctional protein involved in cross-linking a variety of proteins, including retinoblastoma protein (Rb). Here we show that Rb is also a substrate for the recently identified serine/threonine kinase activity of TG2 and that TG2 phosphorylates Rb at the critically important Ser780 residue. Furthermore, phosphorylation of Rb by TG2 destabilizes the Rb.E2F1 complex. TG2 phosphorylation of Rb was abrogated by high Ca2+ concentrations, whereas TG2 transamidating activity was inhibited by ATP. TG2 was itself phosphorylated by protein kinase A (PKA). Phosphorylation of TG2 by PKA attenuated its transamidating activity and enhanced its kinase activity. Activation of PKA in mouse embryonic fibroblasts (MEF) with dibutyryl-cAMP enhanced phosphorylation of both TG2 and Rb by a process that was inhibited by the PKA inhibitor H89. Treatment with dibutyryl-cAMP enhanced Rb phosphorylation in MEFtg2+/+ cells but not in MEFtg2-/- cells. These data indicate that Rb is a substrate for TG2 kinase activity and suggest that phosphorylation of Rb, which results from activation of PKA in fibroblasts, is indirect and requires TG2 kinase activity.

Ethnicity, insulin resistance, and inflammatory adipokines in women at high and low risk for vascular disease.

OBJECTIVE: We sought to compare the relationship between body composition, insulin resistance, and inflammatory adipokines in Aboriginal Canadian women, who are at high risk of vascular disease, with white women. RESEARCH DESIGN AND METHODS: A subgroup of the First Nations Bone Health Study population, consisting of 131 Aboriginal women and 132 matched white women, was utilized. Body composition was determined by whole-body dual X-ray absorptiometry, and blood analytes were measured after an overnight fast. RESULTS: After excluding individuals with diabetes, A1C, BMI, percent trunk fat, and homeostasis model assessment of insulin resistance (HOMA-IR) were greater in First Nation women compared with white women, whereas adiponectin, retinol binding protein (RBP)4, and insulin-like growth factor binding protein-1 (IGFBP-1) were lower. First Nation women had more trunk fat for any given level of total fat than white women. There were no differences in resistin, leptin, tumor necrosis factor (TNF)-alpha, or C-reactive protein (CRP) levels between First Nation and white women. Insulin resistance correlated with leptin and inversely with adiponectin levels in both First Nation and white women. There were weak correlations between insulin resistance and TNF-alpha, interleukin-6, and CRP, but these were not significant after correction for body fat. No correlation was found between RBP4 and insulin resistance. ANCOVA revealed a higher HOMA-IR adjusted for total body fat in First Nation women than in white women (P = 0.015) but not HOMA-IR adjusted for trunk fat (P > 0.2). CONCLUSIONS: First Nation women are more insulin resistant than white women, and this is explained by trunk fat but not total fat. Despite the increased insulin resistance, inflammatory adipokines are not significantly increased in First Nation women compared with white women.

Prohibitin binds to C3 and enhances complement activation.

Prohibitin (PHB1) is a multifunction protein that is released in lipid droplets from adipocytes and possibly other cells and is detectable in the circulation. We used crosslinking, immunoprecipitation and proteomic analysis to investigate binding partners for circulating PHB1. Crosslinking of PHB1 to serum resulted in two complexes of approximately 150 and 100 kDa, which contained both PHB1 and fragments of C3. The binding of PHB1 to C3 was confirmed using a solid phase assay where the dissociation constant was approximately 90 fmol/l. PHB1, but not the closely related PHB2, was able to enhance complement activation and induce lysis of sensitized sheep erythrocytes when added with normal serum but not with C3-deficient serum. The ability of PHB1 to bind to, and activate C3 suggests that PHB1 may have a previously unrecognized role in innate immunity.

Variations in parametrial white adipose tissue mass during the mouse estrous cycle: relationship with the expression of peroxisome proliferator-activated receptor-gamma and retinoic acid receptor-alpha.

Estrogen and progestin participate in the regulation of adipose tissue metabolism, and peroxisome proliferator-activated receptor-gamma (PPARgamma) and retinoic acid receptor-alpha (RXRalpha) are absolutely required for adipose tissue development. The present study is to investigate the changes in parametrial fat mass and expression of PPARgamma and RXRalpha during estrous cycle in mice. Parametrial white adipose tissues (WAT), inter-scapula brown adipose tissues, and uteri from female mice were weighed. Blood samples were collected for the measurement of 17 beta-estradiol and progesterone levels. An RNase protection assay and Western blot analysis were used to compare the expression of PPARgamma and RXRalpha in adipose tissue. The mass of parametrial WAT in diestrus was significantly higher compared with estrus. However, there is no significant difference on the mass of brown adipose tissues during estrous cycle. The expression of PPARgamma in WAT in diestrus was significantly higher than that in estrus. The expression of RXRalpha during estrous cycle was unchanged in both white and brown adipose tissues. In conclusion, the variation in parametrial WAT mass during the mouse estrous cycle correlates with changes in the expression of PPARgamma in WAT.

Plasma adipokines and body composition in response to modest dietary manipulations in the mouse.

OBJECTIVE: The relationship between adipokine levels and body composition has not been carefully examined. Most studies in humans are cross-sectional, and the few studies in mice have been restricted to a comparison of control animals with markedly obese, insulin-resistant mice. Our objective was to study changes in adipokine levels and body composition in response to modest dietary intervention. RESEARCH METHODS AND PROCEDURES: Plasma resistin, adiponectin, and leptin levels were examined in mice fed ad libitum, a 75% restricted diet, or a diet supplemented with 10% sucrose. Body composition was determined by whole-body DXA. RESULTS: The percentage body fat was reduced in mice subjected to the restricted diet and increased in mice supplemented with 10% dextrose. Adipokine levels were not different in either of these groups compared with the control mice. A significant inverse correlation was observed between resistin levels and total body fat, whereas there was no significant correlation between body fat and adiponectin levels. Positive correlations were observed between leptin levels and percentage body fat, total body fat, and abdominal fat. Leptin levels correlated with plasma glucose, but multivariate analysis revealed that this correlation was the result of a strong positive correlation between leptin and insulin levels. There were no correlations between glycemia and resistin or glycemia and adiponectin levels, and no correlation was observed between any of the adipokine levels and bone mineral content or density. DISCUSSION: These data suggest that in the mouse, modest dietary perturbations have little effect on resistin and adiponectin levels despite significant effects on glycemia, insulin levels, and bone parameters.

Phosphorylation of transglutaminase 2 by PKA at Ser216 creates 14-3-3 binding sites.

Transglutaminase 2 (TG2) is a multifunctional ubiquitous enzyme which is present in various cellular compartments and is subject to phosphorylation by PKA. To better understand the relevance of PKA induced phosphorylation of TG2, we performed pull-down assays using phosphorylated biotinylated-TG2(209-223) peptides spanning PKA induced phosphorylation sites as a bait. Subsequent analysis of pull-down protein by SDS-PAGE and LC/MS identified 14-3-3epsilon as the binding partner for TG2 which was further confirmed by immunoblotting with 14-3-3 specific antiserum. In contrast, non-phosphorylated and/or phosphorylation site substituted peptides fail to pull-down 14-3-3. Furthermore, we demonstrate that 14-3-3 co-immunoprecipitated with TG2 antiserum after activation of PKA from mouse embryonic fibroblasts (MEF)(TG2+/+) cells but not from MEF(TG2-/-) cells. In summary, we provide convincing evidence that phosphorylation of TG2 by PKA creates binding site(s) for 14-3-3 both in vitro and in vivo.

The Prohibitins: emerging roles in diverse functions.

The prohibitins, Phb1 and Phb2 are highly conserved proteins in eukaryotic cells that are present in multiple cellular compartments. Initial investigations focused on the role of Phb1 as an inhibitor of cell proliferation hence the original name prohibitin. However both proteins appear to have a diverse range of functions and recent evidence suggests that the prohibitins have very similar but as yet only partially understood functions. In addition to their role as chaperone proteins in the mitochondria, and their ability to target to lipid rafts, their is now compelling evidence that both prohibitins are localized in the nucleus and can modulate transcriptional activity by interacting with various transcription factors, including the steroid hormone receptors, either directly or indirectly. In addition Phb1 and Phb2 are present in the circulation and can be internalized when added to cultured cells suggesting that the circulating prohibitins may have some regulatory role. This review presents some of the recent developments in prohibitin research and focuses on the similarities in the structure and function of these interesting proteins.

Canadian guidelines for the management of adult growth hormone deficiency.

PURPOSE: To develop guidelines for the management of growth hormone (GH) deficiency in Canadian adults to facilitate rational use of GH in appropriate indications. METHODS: The guidelines were developed by group of endocrinologists and an endocrine specialist nurse with an interest in neuroendocrine disorders, representing all regions of Canada and practicing in a variety of settings. A steering committee with broad expertise undertook a systematic review of the evidence relating to adult GHD and GH replacement. This evidence was reviewed by the whole group, and guidelines were developed using a consensus approach. RESULTS: The document addresses the causes and clinical characteristics of GHD in adults and reviews the evidence in support of GH replacement in this group. The authors provide recommendations for the identification of adult GHD, and guidelines for GH replacement in appropriate patients. Also, access to GH therapy across Canada is reviewed.

Insulin-like growth factor (IGF) binding protein-3 attenuates prostate tumor growth by IGF-dependent and IGF-independent mechanisms.

IGF binding protein (IGFBP)-3 inhibits cell growth and promotes apoptosis by sequestering free IGFs. In addition IGFBP-3 has IGF-independent, proapoptotic, antiproliferative effects on prostate cancer cells in vitro. Expression of the large T-antigen (Tag) under the long probasin promoter (LPB) in LPB-Tag mice results in prostate tumorigenesis. To investigate the IGF-dependent and IGF-independent effects of IGFBP-3 on prostate tumor growth, we crossed LPB-Tag mice with cytomegalovirus (CMVBP-3) and phosphoglycerate kinase (PGKBP-3) mice that overexpress IGFBP-3 under the cytomegalovirus promoter and the phosphoglycerate kinase promoter, respectively, and also I56G/L80G/L81G-mutant IGFBP-3 (PGKmBP-3) mice that express I56G/L80G/L81G-IGFBP-3, a mutant, that does not bind IGF-I but retains IGF-independent proapoptotic effects in vitro. Prostate tumor size and the steady-state level of p53 were attenuated in LPB-Tag/CMVBP-3 and LPB-Tag/PGKBP-3 mice, compared with LPB-Tag/wild-type (Wt) mice. A more marked effect was observed in LPB-Tag/CMVBP-3, compared with LPB-Tag/PGKBP-3, reflecting increased levels of transgene expression in CMVBP-3 prostate tissue. No attenuation of tumor growth was observed in LPB-Tag/PGKmBP-3 mice during the early tumor development, indicating that the inhibitory effects of IGFBP-3 were most likely IGF dependent during the initiation of tumorigenesis. At 15 wk of age, epidermal growth factor receptor expression was increased in LPB-Tag/Wt and LPB-Tag/PGKmBP-3 tissue, compared with LPB-Tag/PGKBP-3. IGF receptor was increased in all transgenic mice, but pAkt expression, a marker of downstream IGF-I action, was increased only in LPB-Tag/Wt and LPB-Tag/PGKmBP-3. After 15 wk of age, a marked reduction in tumor growth was apparent in LPB-Tag/PGKmBP-3 mice, indicating that the IGF-independent effects of IGFBP-3 may be important in inhibiting tumor progression.

Prohibitin attenuates insulin-stimulated glucose and fatty acid oxidation in adipose tissue by inhibition of pyruvate carboxylase.

Prohibitin (PHB-1) is a highly conserved protein involved in mitochondrial biogenesis and function. It is secreted in lipid droplets from adipocytes and is present in the circulation. In adipose tissue it functions as a membrane receptor and can target binding partners to the mitochondria. Here we report that PHB-1 has a hitherto undescribed role as an inhibitor of pyruvate carboxylase (PC). As a consequence, it can modulate insulin-stimulated glucose and fatty acid oxidation. It had no effect on insulin-stimulated 2-deoxglucose uptake by isolated adipocytes but inhibited insulin-stimulated oxidation of [14C]glucose with a half-maximal concentration of approximately 4 nM. It also inhibited oleic acid oxidation in glucose-depleted adipocytes via depletion of oxaloacetate. In vitro experiments using broken-cell assays confirmed that PHB-1 inhibited PC. MALDI-TOF analysis of proteins identified by cross-linking of PHB-1 to adipocyte membranes indicated that PHB-1 is closely associated with PC and EH domain 2 (EHD2). On the basis of these data, we propose that PHB-1 is recycled between the extracellular space and the mitochondria by a mechanism involving lipid rafts and EHD2 and can modulate mitochondrial fuel metabolism by inhibition of PC.

Phosphorylation of histones by tissue transglutaminase.

Tissue transglutaminase 2 (TG2) has recently been shown to have intrinsic serine/threonine kinase activity. Since histones are known to be cross-linked by TG2, we investigated whether histones are also substrates for TG2 kinase activity. TG2 was able to phosphorylate H1, H2A, H2B, H3, and H4 histones in vitro. Using peptide substrates and phosphospecific antibodies we demonstrated that TG2 phosphorylated Ser(10) in H3 and that this phosphorylation was reduced by acetylation, whereas phosphorylation of Ser(10) by TG2 enhanced acetylation. Furthermore we demonstrated that exogenous TG2 phosphorylated H1 and H3 in nucleosome preparations. We examined the abundance of TG2 in DNA-associated proteins from MCF-7 cells treated with phorbol ester (TPA) and 17beta-estradiol (E2). TG2 abundance was significantly reduced in E2-treated cells and enhanced in TPA-treated cells. In summary we have demonstrated that TG2 is able to phosphorylate purified histone proteins, and H3 and H1 in chromatin preparations, and it is associated with chromatin in breast cancer cells. These studies suggest a novel role for TG2 in the regulation of chromatin structure and function.

The p53 oncoprotein is a substrate for tissue transglutaminase kinase activity.

Increased expression and activity of the ubiquitous enzyme, tissue transglutaminase (TG2), is consistently seen in a variety of models of apoptosis. The p53 oncoprotein is also involved in apoptosis. Here we investigated the interaction of TG2 with p53 and show that the p53 is a substrate for the recently identified serine/threonine kinase activity of TG2. Phosphospecific antibodies indicated that TG2 phosphorylated p53 at Ser(15) and Ser(20), residues that are critically important in the interaction of p53 with Mdm2. The TG2-induced phosphorylation was abrogated by high Ca(2+) concentrations and inhibited by cystamine, a known inhibitor of TG2 cross-linking activity. Furthermore, we demonstrate that TG2-induced phosphorylation of p53 reduces the ability of p53 to interact with Mdm2. Although TG2 cross-linking activity has been clearly implicated in apoptosis, our observations reported here suggest TG2 modification of p53 could be an additional mechanism whereby TG2 could facilitate apoptosis.

Insulin-like growth factor binding proteins in development.

IGFBPs regulate growth and development by regulating IGF transport to tissues and IGF bioavailability to IGF receptors at cell membrane level. IGFBP excess leads predominantly to inhibition of IGF action and growth retardation with impaired organogenesis. Absence of human and also mouse ALS leads to decreased IGF-I levels in circulation and causes mild growth retardation. Although IGFBP KO mice demonstrate relatively minor phenotypes, the possibility of compensatory mechanisms that mask the phenotypic manifestation of lack of individual binding proteins needs to be further investigated. Recent studies of hepatic regeneration in IGFBP-1 KO mice and also with mutant IGFBP-3 Tg mice provide some limited support for the existence of IGF-independent mechanism of action in vivo.

Inhaled insulin improves glycemic control when substituted for or added to oral combination therapy in type 2 diabetes: a randomized, controlled trial.

BACKGROUND: Patients with type 2 diabetes who do not achieve glycemic control with oral agent therapy eventually require insulin. OBJECTIVE: To determine the effect on glycemic control of inhaled insulin alone or added to dual oral therapy (insulin secretagogue and sensitizer) after failure of dual oral therapy. DESIGN: Open-label, randomized, controlled trial. SETTING: 48 outpatient centers in the United States and Canada. PATIENTS: 309 patients with type 2 diabetes, no clinically significant respiratory disease, and hemoglobin A(1c) level of 8% to 11% who were receiving dual oral therapy. MEASUREMENTS: Primary end point was change in hemoglobin A(1c) level from baseline to 12 weeks. Secondary outcomes included hemoglobin A(1c) level less than 8% and less than 7%, hypoglycemia, weight, lipid levels, pulmonary function, insulin antibody binding, and adverse events. INTERVENTION: Inhaled insulin (Exubera; Pfizer Inc. [New York, New York], sanofi-aventis Group [Paris, France], and Nektar Therapeutics [San Carlos, California]), titrated to blood glucose, administered alone (n = 104) or added to dual oral agents (n = 103) versus oral therapy alone (n = 99). RESULTS: Reductions in hemoglobin A(1c) level were greater with inhaled insulin. Adjusted treatment group differences for inhaled insulin plus oral agents and inhaled insulin alone compared with continued oral agent therapy were -1.67 percentage points (95% CI, -1.90 to -1.44 percentage points; P < 0.001) and -1.18 percentage points (CI, -1.41 to -0.95 percentage point; P < 0.001), respectively. Hemoglobin A(1c) level less than 7% was achieved by 32% (inhaled insulin plus oral agents) and by 1% (oral agent therapy) of patients (adjusted odds ratio, 44.7 [CI, 6.0 to 335.2]). Hypoglycemia, mild weight gain, mild cough, and insulin antibodies were more frequent with inhaled insulin than with oral agent therapy alone. Pulmonary function was similar in all groups. LIMITATIONS: This study evaluated only patients with hemoglobin A1c levels of 8% to 11%, did not compare inhaled insulin with other insulins or oral therapy except a dual regimen of secretagogue and sensitizer, and lasted only 12 weeks. CONCLUSIONS: Inhaled insulin improved overall glycemic control and hemoglobin A1c level when added to or substituted for dual oral agent therapy with an insulin secretagogue and sensitizer. Consistent with other insulin therapies, hypoglycemia and mild weight gain occurred. Pulmonary function showed no between-group differences.

The effects of the insulin-like growth factor-I aptamer, NBI-31772, on glucose homeostasis in the mouse.

The majority of insulin-like growth factor-I (IGF-I) in the adult rodent circulation is bound to high affinity IGF binding proteins. We investigated the changes in IGF-I clearance, blood glucose and plasma insulin levels, and tissue 2-deoxyglucose uptake after intravenous administration of the IGF aptamer, NBI-31772, which selectively competes with IGF-I for binding to the IGFBPs, but has no effect at the IGF-I receptor. Clearance of 125I-IGF-I was significantly increased in NBI-31772-treated mice compared with vehicle-treated mice (t1/2 = 45.0 +/- 1.9 vs. 56.3 +/- 3.9 min, respectively; p = 0.021). However, NBI-31772 had no significant effect on glucose levels, and no insulin sparing effect was apparent neither under basal conditions nor during an intravenous glucose challenge. The decline in the specific activity after 3H-2-deoxyglucose administration was significantly less rapid in NBI-31772-treated mice compared with controls, suggesting that the IGF-I aptamer had an inhibitory effect on hepatic gluconeogenesis. In contrast, no insulin-like effect was apparent in other tissues examined. 3H-2-deoxyglucose accumulation was similar in all tissues analyzed, including skeletal muscle, which is thought to be particularly sensitive to IGF-I. These data suggest that the IGF-I aptamer affects clearance of radiolabeled IGF-I from the circulation, but has no marked effects on glucose nor insulin homeostasis. The search for hydrophilic IGF aptamers with longer duration of action that could be used in the treatment of diabetes may be rewarding.

The effects of growth hormone status on circulating levels of vascular growth factors.

BACKGROUND: Vascular growth factors are important not only in angiogenesis but also for the maintenance of normal endothelial integrity and function. Elevated levels of vascular endothelial growth factor (VEGF), angiopoietin-2, hepatocyte growth factor (HGF), endostatin and angiogenin have been associated with endothelial dysfunction and atherosclerosis. Both acromegaly and growth hormone deficiency (GHD) are associated with endothelial dysfunction and changes in blood vessel morphology. AIM: To investigate the effect of GH status on the circulating levels of angiogenic factors. DESIGN: We measured the levels of six endothelial growth modulators, four angiogenic growth factors and two inhibitors of angiogenesis in 35 untreated acromegalics, 36 untreated GH-deficient subjects and 101 normal control subjects. Fifteen GH-deficient subjects were also studied before and 1 year after treatment with GH. RESULTS: Mean angiogenin concentrations were increased in acromegaly and decreased in GH-deficient subjects compared to control subjects. Endostatin levels showed a similar pattern although the elevated levels in acromegalic subjects did not achieve statistical significance. Angiogenin and endostatin levels both correlated significantly with IGF-I levels (R = 0.61, P < 0.001 and R = 0.22, P < 0.01, respectively). The relationship between angiogenin and IGF-I levels remained significant even after correction for gender, age, body mass index (BMI) and insulin resistance. There were no significant differences in the levels of HGF, VEGF, VEGF-C or angiopoietin-2 between the three groups. VEGF-D levels were elevated in both acromegalic and GH-deficient male subjects. A similar pattern was apparent in female subjects. After GH treatment, a significant reduction in VEGF-D levels and a significant rise in endostatin levels were observed in GH-deficient subjects. A nonsignificant increase in angiogenin levels was also observed. CONCLUSION: These data indicate that significant perturbations in the levels of vascular growth modulators are present in both acromegaly and GHD. While changes in endostatin and angiogenin levels appear to correlate with IGF-I levels, VEGF-D levels show similar perturbations in both acromegaly and GHD. Further studies are required to determine the relationship of the perturbations to endothelial dysfunction in these conditions.

Prohibitin: a potential target for new therapeutics.

Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function and protection against senescence. There is considerable controversy concerning the function of nuclear-localized PHB. PHB has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. PHB might also function as a cell-surface receptor for an as-yet unidentified ligand. Cell-associated PHB in the gastrointestinal tract has been implicated in protection against infection and inflammation and the induction of apoptosis in other tissues. The diverse array of functions of PHB, together with the emerging evidence that its function can be modulated specifically in certain tissues, suggest that targeting PHB would be a useful therapeutic approach for the treatment of variety of disease states, including inflammation, obesity and cancer.

Overexpression of gly56/gly80/gly81-mutant insulin-like growth factor-binding protein-3 in transgenic mice.

IGF-independent effects of IGF-binding protein-3 (IGFBP-3) have been demonstrated in vitro; however, the physiological significance of these effects in vivo is unclear. We generated two transgenic (Tg) mouse strains that overexpress a human Gly56/Gly80/Gly81-mutant IGFBP-3 cDNA. This mutant has a markedly reduced affinity for the IGFs, but retains the IGF-independent effects. Serum levels of mutant IGFBP-3 were 156 +/- 12 and 400 +/- 24 ng/ml in hemizygous mice of strains 5005 and 5012, respectively. When Tg and wild-type mice were compared, there was no reduction in birth weight, litter size, or postnatal growth. Despite differences in transgene expression in various tissues, relative organ weight was similar in Tg and wild-type mice, with exception of brain, where a modest reduction in brain weight was observed in the high-expressing 5012 lineage. There was also a significant reduction in proliferating cell nuclear antigen-staining cells observed in the periventricular region of the developing brain in embryonic d 18 Tg embryos. In the higher expressing 5012 Tg strain, IGF-I and murine IGFBP-3 levels, marker of GH action were increased. Furthermore, there was a positive correlation between mutant IGFBP-3 levels and IGF-I levels and between mutant IGFBP-3 levels and murine IGFBP-3 (P = 0.002 and P < 0.001, respectively). These data indicate that overexpression of mutant IGFBP-3 is not associated with growth retardation. The higher levels of IGF-I and murine IGFBP-3 in the 5012 Tg strain suggest that the growth inhibitory effect of mutant IGFBP-3 may be compensated for by other mechanisms.