p50-NF-kappaB complexes partially compensate for the absence of RelB: severely increased pathology in p50(-/-)relB(-/-) double-knockout mice.

RelB-deficient mice (relB(-/-)) have a complex phenotype including multiorgan inflammation and hematopoietic abnormalities. To examine whether other NF-kappaB/Rel family members are required for the development of this phenotype or have a compensatory role, we have initiated a program to generate double-mutant mice that are deficient in more than one family member. Here we report the phenotypic changes in relB(-/-) mice that also lack the p50 subunit of NF-kappaB (p50(-/-)). The inflammatory phenotype of p50(-/-)relB(-/-) double-mutant mice was markedly increased in both severity and extent of organ involvement, leading to premature death within three to four weeks after birth. Double-knockout mice also had strongly increased myeloid hyperplasia and thymic atrophy. Moreover, B cell development was impaired and, in contrast to relB(-/-) single knockouts, B cells were absent from inflammatory infiltrates. Both p50(-/-) and heterozygous relB(-/+) animals are disease-free. In the absence of the p50, however, relB(-/+) mice (p50(-/-)relB(-/+)) had a mild inflammatory phenotype and moderate myeloid hyperplasia. Neither elevated mRNA levels of other family members, nor increased kappaB-binding activities of NF-kappaB/Rel complexes could be detected in single- or double-mutant mice compared to control animals. These results indicate that the lack of RelB is, in part, compensated by other p50-containing complexes and that the "classical" p50-RelA-NF-kappaB activity is not required for the development of the inflammatory phenotype.

Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses.

The nfkb2 gene is a member of the Rel/NF-kappa B family of transcription factors. COOH-terminal deletions and rearrangements of this gene have been associated with the development of human cutaneous T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas. To further investigate the function of NF-kappa B2, we have generated mutant mice carrying a germline mutation of the nfkb2 gene by homologous recombination. NF-kappa B2-deficient mice showed a marked reduction in the B cell compartment in spleen, bone marrow, and lymph nodes. Moreover, spleen and lymph nodes of mutant mice presented an altered architecture, characterized by diffuse, irregular B cell areas and the absence of discrete perifollicular marginal and mantle zones; the formation of secondary germinal centers in spleen was also impaired. Proliferation of NF-kappa B2-deficient B cells was moderately reduced in response to lipopolysaccharide, anti-IgD-dextran, and CD40, but maturation and immunoglobulin switching were normal. However, nfkb2 (-/-) animals presented a deficient immunological response to T cell-dependent and -independent antigens. These findings indicate an important role of NF-kappa B2 in the maintenance of the peripheral B cell population, humoral responses, and normal spleen architecture.

Neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine N51/KC in oligodendrocytes.

Chemokines (pro-inflammatory chemoattractant cytokines) are expressed in pathological conditions of the central nervous system (CNS). Previous studies suggested that the CNS is relatively resistant to leukocyte diapedesis after chemokine injection, leaving their functional role unresolved. The CNS function of N51/KC, a neutrophil-selective chemokine, was addressed by expressing N51/KC under control of the myelin basic protein (MBP) promoter in transgenic (tg) mice (MBP-N51/KC mice). CNS-specific N51/KC expression produced remarkable neutrophil infiltration into perivascular, meningeal, and parenchymal sites, demonstrating that this chemokine exerts the multiple functions in vivo required to recruit leukocytes into the CNS. MBP-N5 1/KC mice represent an incisive model for the molecular dissection of neutrophil entry into the CNS. Unexpectedly, MBP-N51/KC mice developed a neurological syndrome of pronounced postural instability and rigidity at high frequency beginning at 40 days of age, well after peak chemokine expression. 68/182 mice in one tg fine were found dead before one year of age, with prominent neurological symptoms premortem in 26 (38%). Florid microglial activation and blood-brain barrier disruption without dysmyelination were the major neuropathological alterations. Late-onset neurological symptoms in MBP-N51/KC mice may indicate unanticipated consequences of CNS chemokine expression.

Expression of constitutively active IkappaB beta in T cells of transgenic mice: persistent NF-kappaB activity is required for T-cell immune responses.

The transcription factor NF-kappaB is normally sequestered in the cytoplasm by members of the IkappaB family, including IkappaB alpha, IkappaB beta, and the recently cloned IkappaB epsilon. Upon cellular activation, these inhibitors are rapidly phosphorylated on two amino-terminal serines, ubiquitinated, and degraded by the 26S proteasome, releasing a functional NF-kappaB. To determine the importance of IkappaB beta in NF-kappaB regulation in T cells, we generated transgenic mice expressing a constitutively active IkappaB beta mutant (mIkappaB beta) under the control of the lck promoter. The transgene contains the two critical N-terminal serine residues mutated to alanines and therefore no longer susceptible to degradation upon cell activation. mIkappaB beta is unable to totally displace IkappaB alpha from RelA-containing complexes, thus allowing a transient activation of NF-kappaB upon T-cell stimulation. However, mIkappaB beta completely blocks NF-kappaB activity after IkappaB alpha degradation. In addition, as a consequence of this inhibition, ikba expression is down regulated, along with that of other NF-kappaB-regulated genes. These transgenic mice have a significant reduction in the peripheral T-cell population, especially CD8+ cells. The remaining T cells have impaired proliferation in response to phorbol 12-myristate 13-acetate plus phytohemagglutinin or calcium ionophore but not to anti-CD3/anti-CD28 costimulation. As a result of these alterations, transgenic animals present defects in immune responses such as delayed-type hypersensitivity and the generation of specific antibodies against T-cell-dependent antigens. These results show that in nonstimulated T cells, IkappaB beta cannot efficiently displace IkappaB alpha bound to RelA-containing complexes and that persistent NF-kappaB activity is required for proper T-cell responses in vivo.

Expression of FosB during mouse development: normal development of FosB knockout mice.

FosB, one of the members of the Fos family, is rapidly induced in many cell types upon stimulation and has a stimulatory effect on the proliferation of cultured cells. To understand the tissue distribution of FosB, we have studied its expression pattern by immunohistochemistry in newborn and late embryonic stage mice. These results show that FosB is widely expressed with the highest levels of expression observed in both bony and cartilagenous regions of developing bone. FosB is also detected within whisker follicles, liver, and epidermal tissue. To study the role of FosB in mammalian development we generated embryonic stem (ES) cells, mice and mouse embryo fibroblasts (MEFs) that are deficient for FosB. FosB -/- mice are born at a normal frequency, are fertile and present no obvious phenotypic or histologic abnormalities. FosB-deficient ES cells and MEFs proliferate and enter the S phase normally and we do not find upregulation of other fos family genes to compensate for the lack of FosB. However, we do find that the induction of two AP-1 containing genes is reduced after stimulation of FosB-deficient cells, demonstrating that FosB does indeed play a functional role in transcriptional regulation.

Glucokinase regulatory protein may interact with glucokinase in the hepatocyte nucleus.

Glucokinase (GK) plays a central role in the sensing of glucose in pancreatic beta-cells and parenchymal cells of the liver. Glucokinase regulatory protein is a physiological inhibitor of GK in the liver. To understand the role of the interaction of these two proteins in glucose sensing, we carried out a series of experiments to localize the protein in the liver cell. The regulatory protein was found to be present mainly in the nucleus of the cell under a variety of conditions that mimicked the glucose status of the fed and fasted state. GK was localized in the nucleus when the cells were exposed to low glucose concentrations. At higher glucose concentrations or in the presence of low concentrations of fructose, GK translocated to the cytoplasm. The effect of fructose was more robust and rapid than the effect of high glucose concentrations. Furthermore, the effect of fructose and high glucose on the translocation of GK from the nucleus could be partially reversed by glucagon. This unusual localization and behavior suggests a role for GK and its regulatory protein in hepatic energy metabolism that may be broader than glucose phosphorylation.

Conservation of an insulin response unit between mouse and human glucose-6-phosphatase catalytic subunit gene promoters: transcription factor FKHR binds the insulin response sequence.

Because overexpression of the glucose-6-phosphatase catalytic subunit (G-6-Pase) in both type 1 and type 2 diabetes may contribute to the characteristic increased rate of hepatic glucose production, we have investigated whether the insulin response unit (IRU) identified in the mouse G-6-Pase promoter is conserved in the human promoter. A series of human G-6-Pase-chloramphenicol acetyltransferase (CAT) fusion genes was transiently transfected into human HepG2 hepatoma cells, and the effect of insulin on basal CAT expression was analyzed. The results suggest that the IRU identified in the mouse promoter is conserved in the human promoter, but that an upstream multimerized insulin response sequence (IRS) motif that is only found in the human promoter appears to be functionally inactive. The G-6-Pase IRU comprises two distinct promoter regions, designated A and B. Region B contains an IRS, whereas region A acts as an accessory element to enhance the effect of insulin, mediated through region B, on basal G-6-Pase gene transcription. We have previously shown that the accessory factor binding region A is hepatocyte nuclear factor-1, and we show here that the forkhead protein FKHR is a candidate for the insulin-responsive transcription factor binding region B.

Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.

Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-gamma (IFN-gamma) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-gamma and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung.

Regulation of insulin-like growth factor binding protein 4 by a specific insulin-like growth factor binding protein 4 proteinase in normal human osteoblast-like cells: implications in bone cell physiology.

Insulin-like growth factor binding protein 4 (IGFBP-4) is secreted by normal human osteoblast-like cells (hOB) and is a potent inhibitor of insulin-like growth factor (IGF) action in vitro. In previous studies, IGF treatment of hOB in culture led to markedly reduced medium levels of IGFBP-4 as detected by western ligand blotting. In the present study, incubation of hOB-conditioned medium (hOB-CM) with IGF under cell-free conditions resulted in a similar loss of IGFBP-4. Both IGF-I and IGF-II were capable of inducing a decrease in IGFBP-4; however, IGF-II was more effective. When the six characterized IGFBP were added to hOB-CM, only IGFBP-4 disappeared in response to IGF-II addition. This IGF-regulated loss of IGFBP-4 was inhibited by metalloproteinase inhibitors and appeared to be due to a proteinase that cleaved IGFBP-4 in 18 and 14 kD fragments identified by western immunoblotting. Conditioned media from eight of eight different donor hOB lines tested exhibited IGFBP-4 proteinase activity. To assess the biologic consequences of IGF-II-induced IGFBP-4 proteolysis, we treated hOB with IGF-II for 5 h, which decreased medium IGF-BP-4 by 70%, and then measured IGF-I and insulin stimulation of [3H]thymidine incorporation. IGF-II itself was not mitogenic and had no effect on insulin-stimulated [3H]thymidine incorporation. However, pretreatment of cultured hOB with IGF-II enhanced IGF-I-stimulated [3H]thymidine incorporation threefold.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor (IGF) binding protein-3 potentiation of IGF action is mediated through the phosphatidylinositol-3-kinase pathway and is associated with alteration in protein kinase B/AKT sensitivity.

Cell-association and processing of insulin-like growth factor binding protein-3 (IGFBP-3) by cultured bovine fibroblasts results in markedly enhanced type I IGF receptor signaling at a step distal to ligand binding. The purpose of the present study was to determine the intracellular mediators of IGFBP-3's potentiating effect. Preincubation of cultured bovine fibroblasts with 50 nM IGFBP-3 had no effect alone, but enhanced by 3- to 4-fold IGF-I-stimulated 3H-aminoisobutryric acid (AIB) uptake. IGFBP-3-induced potentiation was specifically prevented if an inhibitor of phosphatidylinositol 3 (PI3)-kinase activation (LY294002), but not an inhibitor of mitogen-activated protein kinase activation (PD98059), was present during the preincubation period. IGFBP-3 did not directly activate the downstream effector of PI3-kinase, protein kinase B (PKB)/Akt. However, the sensitivity of PKB/Akt to activation by IGF-I was increased by 2- to 4-fold with IGFBP-3 pretreatment. This increased sensitivity was accompanied by altered mobility of PKB/Akt on SDS-polyacrylamide gels, suggestive of a diminished phosphorylation state. Consistent with this, okadaic acid, a potent serine/threonine phosphatase inhibitor, was able to block the potentiation effect of IGFBP-3 and prevent the altered mobility of the PKB/Akt molecule in response to IGFBP-3 treatment. PKB/Akt immunoprecipitated from IGFBP-3-pretreated cells was no longer recognized by an antibody specific for phosphorylated threonine followed by proline. These data indicate that IGFBP-3 modulates type I IGF receptor signaling through an effect on PI-3-kinase pathway substrates and suggest a novel mechanism of dephosphorylation whereby PKB/Akt is transformed into a more sensitive substrate of type I IGF receptor signaling.

Alterations in insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis in transformed osteoblastic cells.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106-01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide-sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity.

Conservation of a growth hormone-responsive promoter element in the human and mouse acid-labile subunit genes.

During extrauterine life, insulin-like growth factors (IGFs) circulate in a ternary serum complex with one IGF-binding protein-3 (IGFBP-3) or IGFBP-5 protein and with a single acid-labile subunit (ALS). GH increases levels of this ternary complex; in mice, this effect is achieved in part by the ability of GH to stimulate mouse ALS (mALS) transcription through an interferon-gamma-activated sequence-like element (GLE) in the mALS promoter. To begin studying how GH regulates human ALS (hALS) gene expression, we cloned the hALS gene and found that it spans approximately 3.3 kb of DNA at chromosomal region 16p13.3. The hALS gene has two exons separated by a 1235-bp intron, which is found at the identical site in rat and mouse ALS genes. Sequence analysis reveals that the hALS 5'-flanking sequence is homologous to the mALS promoter, and that the GH-responsive GLE in the mALS promoter is conserved in both sequence and location in the hALS gene. The region spanning from -755 to -4 bp 5' to the hALS ATG translation start codon directs expression of a luciferase reporter gene in primary rat hepatocytes, and GH increases reporter expression in the presence of the native, but not a mutant, GLE in the hALS promoter. These data suggest that GH stimulates hALS and mALS gene expression by a similar mechanism, which involves at least in part a conserved GLE in the ALS promoter.

Hepatic nuclear factor 3 and high mobility group I/Y proteins bind the insulin response element of the insulin-like growth factor-binding protein-1 promoter.

The insulin response element (IRE) of the human insulin-like growth factor-binding protein-1 (IGFBP-1) promoter contains a palindrome of the T(A/G)TTT sequence crucial to hormonal regulation of many genes. In initial studies of how this IRE participates in hormonal regulation, the electromobility shift assay was used under a variety of conditions to identify IRE-binding proteins. An exhaustive search identified five proteins that specifically bind this IRE; purified proteins were used to show that all five are related to either the high mobility group I/Y (HMGI/Y) or hepatic nuclear factor 3 (HNF3) protein families. Further studies used purified HNF3 and HMGI proteins to show: 1) eah protects the IGFBP-1 IRE from deoxyribonuclease I (DNaseI) digestion; and 2) HNF3 but not HMGI/Y binds to the related phosphoenolpyruvate carboxykinase and Apo CIII IREs. A series of IRE mutants with variable responsiveness to insulin were used to show that the presence of a TGTTT sequence in the mutants did parallel, but HMGI/Y and HNF3 binding to the mutants did not parallel, the ability of the mutants to confer the inhibitory effect of insulin. In contrast, HNF3 binding to these IRE mutants roughly correlates with response of the mutants to glucocorticoids. The way by which HNF3 and/or other as yet unidentified IRE-binding proteins confer insulin inhibition to IGFBP-1 transcription and the role of HMGI/Y in IRE function have yet to be established.

FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter.

The insulin response element (IRE) in the IGFBP-1 promoter, and in other gene promoters, contains a T(A/G)TTT motif essential for insulin inhibition of transcription. Studies presented here test whether FKHR may be the transcription factor that confers insulin inhibition through this IRE motif. Immunoblots using antiserum to the synthetic peptide FKHR413-430, RNase protection, and Northerns blots show that FKHR is expressed in HEP G2 human hepatoma cells. Southwestern blots, electromobility shift assays, and DNase I protection assays show that Escherichia coli-expressed GST-FKHR binds specifically to IREs from the IGFBP-1, PEPCK and TAT genes; however, unlike HNF3beta, another protein proposed to be the insulin regulated factor, GST-FKHR does not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE. When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR expression induced a 5-fold increase in activity of the native IGFBP-1 promoter but no increase in activity of promoter constructs containing insulin unresponsive IRE mutants. These data suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription.

Phenotypic manifestations of insulin-like growth factor-binding protein-3 overexpression in transgenic mice.

In cell culture systems insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can both enhance and inhibit IGF-I action. To investigate the biological role of IGFBP-3 in vivo, transgenic (Tg) mice that constitutively overexpress the human IGFBP-3 complementary DNA (cDNA) driven by the mouse phosphoglycerate kinase I (PGK) and the cytomegalovirus (CMV) promoters were examined. Serum levels of human IGFBP-3 in CMVBP-3 and PGKBP-3 Tg mice were 4.7 and 5.8 microgram/ml, respectively and total IGFBP-3 was increased 4.9- and 7.7-fold compared with that in wild-type (Wt) mice. In PGKBP-3 Tg mice the levels of transgene expression were similar in all tissues. Although CMVBP-3 mice demonstrated similar levels of expression of the transgene as PGKBP-3 mice in most tissues, markedly elevated expression was apparent in the kidney and heart. The transgene-derived IGFBP-3 circulated as a 150-kDa ternary complex, and serum IGF-I levels were elevated 1.9- to 2.8-fold in Tg mice compared with Wt mice. A significant reduction in birth weight of approximately 10% and a modest reduction in litter size were apparent in both Tg strains. Early postnatal growth, as assessed by both body weight and length, was significantly reduced in Tg mice compared with Wt mice. This was more marked in PGKBP-3 than in CMVBP-3 mice, who demonstrated a propensity to adiposity after weaning. The relative organ weights of brain and kidney were reduced in both Tg strains, whereas liver size and epididymal fat were significantly increased in CMVBP-3, but not PGKBP-3, mice. Our data indicate that overexpression of IGFBP-3 is associated with modest intrauterine and postnatal growth retardation despite elevated circulating IGF-I levels.

Overexpression of the acid-labile subunit of the IGF ternary complex in transgenic mice.

The ternary complex, composed of IGF-I or IGF-II, IGF-binding protein-3, and the acid-labile subunit, is responsible for transport of the majority of the IGF-I and IGF-II present in the circulation. Acid-labile subunit is developmentally and hormonally regulated, suggesting an important, although unclear, role in regulating the availability and action of the IGFs. To investigate the biological role of acid-labile subunit, we generated transgenic mice, which constitutively overexpress a human acid-labile subunit cDNA driven by the cytomegalovirus promoter. Two independent transgenic strains, CMVALS-1 and CMVALS-2, with mean serum levels of human acid-labile subunit of 19.3 +/- 4.2 and 20.2 +/- 3.2 microg/ml respectively, were characterized. Total acid-labile subunit, endogenous plus transgene derived, was measured by Western blotting and was found to be significantly increased in transgenic compared with wild-type mice (1.51 +/- 0.02-fold; P < 0.001). There were no significant differences in serum IGF-binding protein-3 or IGF-I levels between transgenic and wild-type mice. Similar chromatographic elution patterns were observed when sera from transgenic and wild-type mice were preincubated with [(125)I]IGF-I, indicating that acid-labile subunit overexpression had no measurable effect on compartmentalization of IGF-I in the circulation. Transgene-derived human acid-labile subunit mRNA was detected in 17-d-old embryos and all adult mouse tissues examined. A significant reduction in litter size was also observed in each of the acid-labile subunit transgenic mouse strains. This reduction in litter size was due to a maternal effect, as it was apparent when transgenic female mice were crossed with wild-type male mice, but not when male transgenic mice were crossed with female wild-type mice. The transgenic mice were phenotypically normal at birth, but demonstrated a significant reduction in postnatal body weight gain, particularly during the first 3 wk of life. Over the first 3 months of life, average body weights were significantly reduced by 5.3 +/- 0.6%, 4.2 +/- 0.6%, 8.1 +/- 0.9%, and 5.6 +/- 0.8%, compared with those in wild-type mice, for male and female CMVALS-1 mice and male and female CMVALS-2 mice, respectively. Double transgenic mice, generated by crossing acid-labile subunit transgenic mice with transgenic mice that overexpress IGF-binding protein-3, demonstrated a significantly more marked reduction in body weight gain than acid-labile subunit transgenic mice. These data demonstrate that overexpression of acid-labile subunit has significant effects on postnatal growth and reproduction. As there is little measurable alteration in the circulating components of the IGF system, these effects are most likely to be mediated via disturbances in tissue IGF availability.

Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia.

The imbalance between normal insulin-like growth factor-I (IGF-I) and markedly increased IGF binding protein (IGFBP) plasma levels plays a pathogenic role for growth retardation and catabolism in children with chronic renal failure. To investigate the mechanism of these alterations, experiments were performed in an experimental model of uremia in rats (5/6 nephrectomy) and in pair-fed and ad libitum-fed sham-operated controls Using a specific solution hybridization/RNase protection assay, we observed a marked reduction of hepatic IGF-I messenger RNA (mRNA) abundance at steady state in uremic animals (37 +/- 5% of control) compared both with pair-fed (65 +/- 10%) and ad libitum-fed controls (100 +/- 11%) (P < 0.001). Reduced IGF-I gene expression was clearly organ-specific; it was most pronounced in liver (significant vs., pair-fed controls) and lung and muscle tissue (significant vs., ad libitum-fed controls); no change was observed in kidney and heart tissue. To determine a potential mechanism of reduced hepatic IGF-I gene expression in uremia, the hepatic GH receptor gene expression in the same experimental animals was analyzed by specific solution hybridization/RNase protection assay. Uremic animals had a 20-30% reduction of hepatic GH receptor mRNA abundance compared with controls. Hepatic GHBP expression in uremia was decreased in parallel. Despite the reduction of hepatic IGF-I mRNA abundance, plasma IGF-I levels in uremia were not different from ad libitum-fed controls. This discrepancy is explained by an increased concentration of IGFBPs in uremic plasma. By RIA, plasma IGFBP-1 levels in uremia were increased 4-fold; by Western immunoblot, plasma IGFBP-2 levels were increased 7-fold and plasma IGFBP-4 levels were increased 2-fold compared with both control groups. Intact IGFBP-3 (M(r), approximately 48 kDa) and low molecular IGFBP-3 fragments were not significantly different among the three groups. By Northern blot analysis, hepatic IGFBP-1 mRNA levels in uremia were 2-fold higher than in controls. IGFBP-2 mRNA abundance in liver tissue was increased 4-fold, whereas in kidney there was a significant reduction of IGFBP-2 mRNA (30% of control). IGFBP-4 mRNA was increased by 50% in kidney but not in liver. Plasma insulin and corticosterone levels were not different among the groups. Our study shows that hepatic IGF-I gene expression was specifically reduced in uremia, partially as the consequence of a reduced hepatic GH receptor gene expression. One of the mechanisms contributing to increased IGFBP levels in uremia is increased hepatic gene expression of IGFBP-1 and IGFBP-2. The imbalance between reduced hepatic IGF-I production and increased hepatic IGFBP-1 and 2 production is likely to play a pathogenic role for catabolism and growth failure in CRF.

The insulin-like growth factor-binding protein-4 (IGFBP-4)-IGFBP-4 protease system in normal human osteoblast-like cells: regulation by transforming growth factor-beta.

Insulin-like growth factor-binding protein-4 (IGFBP-4) is secreted by normal human osteoblast-like (hOB) cells and acts as a potent inhibitor of IGF action. hOB cells also secrete a protease, which requires IGFs for activation and specifically cleaves IGFBP-4. To study the regulation of this IGFBP-4 protease, hOB cells from 26 different adult donors were cultured in serum-free medium for 24 h in the absence or presence of hormones and other factors known to regulate bone growth. hOB cell-conditioned medium (hOB-CM) was collected for measurement of IGFBP-4 protease activity in a cell-free assay. This assay involved incubation of hOB-CM (50 microL) without or with IGF-II at 37 C for 6 h. IGF-II-activated IGFBP-4 hydrolysis was assessed by Western ligand blotting and quantitated using laser densitometry. Conditioned medium from all hOB cells examined exhibited IGFBP-4 protease activity. PTH, GH, insulin, calcitonin, glucocorticoids, sex steroids, 1,25-dihydroxyvitamin D3, and epidermal growth factor had no significant effect on IGF-dependent IGFBP-4 protease activity. In comparison, hOB-CM from cells treated with transforming growth factor-beta (TGF beta) exhibited significantly augmented IGF-II-dependent proteolysis of endogenous IGFBP-4. Enhanced proteolysis of exogenous IGFBP-4 was also demonstrated: 1) 92% of recombinant human IGFBP-4 added to conditioned medium from TGF beta-treated hOB cells was hydrolyzed during the assay in the presence of IGF-II compared to 45% of recombinant human IGFBP-4 added to control hOB-CM; and 2) increased radiolabeled IGFBP-4 fragments were generated in conditioned medium from TGF beta-treated hOB cells compared with control hOB-CM in the presence of IGF-II. In addition to its effect on IGFBP-4 proteolysis, TGF beta treatment decreased IGFBP-4 messenger ribonucleic acid expression, as measured by Northern analysis. Our results indicate that the IGFBP-4-IGFBP-4 protease system in hOB cells can be controlled by two of the most abundant local growth factors for bone (IGF-II and TGF beta), with each acting via different mechanisms. Regulation of IGFBP-4 availability may play an important role in the modulation of bone cell responsiveness to IGFs.

Regulation of insulin-like growth factor (IGF)-binding protein-4 availability in normal human osteoblast-like cells: role of endogenous IGFs.

Insulin-like growth factor-binding protein-4 (IGFBP-4) is an important regulator of insulin-like growth factor-I (IGF-I) anabolic activity in bone. Although cultured human osteoblast-like (hOB) cells have been reported to secrete IGFBP-4, we could not detect IGFBP-4 protein in 8 of 27 individual donor-derived hOB-cell conditioned medium (hOB-CM) samples examined by Western ligand blotting. Nonetheless, this subset of hOB cells had normal IGFBP-4 messenger ribonucleic acid expression and protein secretion. Regulation of IGFBP-4 levels in hOB cultures appeared to occur extracellularly. hOB cells produce an IGFBP-4 proteinase that requires the presence of IGF for cleavage of the IGFBP-4 molecule into 2 fragments of approximately 18 and 14 kilodaltons. These fragments are not detected by Western ligand blotting. Our data indicate that elevated endogenous levels of IGF can activate IGFBP-4 proteolysis, because in hOB cultures lacking detectable IGFBP-4 protein 1) basal IGF messenger ribonucleic acid expression was increased; 2) IGF-II peptide levels were elevated; 3) IGF-neutralizing antibodies added to hOB-CM attenuated the proteolysis of exogenous IGFBP-4; and 4) recombinant human IGFBP-4 was proteolyzed into 2 immunoreactive fragments of approximately 18 and 14 kilodaltons during cell-free incubations in these hOB-CM without the addition of exogenous IGF. In conclusion, elevated IGF expression and secretion can contribute to enhanced proteolysis of endogenous and exogenous IGFBP-4 via a proteinase secreted by cultured hOB cells. Levels of endogenous IGF peptide may determine IGFBP-4 availability in the bone microenvironment and, thus, modulate the local cell response to IGF-I.

Kinetics of insulin-like growth factor (IGF) and IGF-binding protein responses to a single dose of growth hormone.

The in vivo physiological relationships among GH, the insulin-like growth factors (IGFs), and the IGF-binding proteins (IGFBPs) are not completely defined, and single random measurements of these serum proteins do not completely reveal their dynamic relationships. We report the kinetic responses of the IGFs and IGF-binding proteins to exogenous GH in 23 subjects with untreated GH deficiency [5 women and 18 men; age, 15.0 +/- 6.2 yr (+/- s.d.), height z-score = -4.4 +/- 2.2 (+/- s.d.); body mass index = 19.3 +/- 2.4 kg/m2]. After an overnight fast, subjects were given a sc dose of recombinant human GH (2.85 i.u./m2), and blood was sampled from an indwelling peripheral venous catheter 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, and 24 h after the injection. Subjects were then treated with recombinant human GH (2.85 i.u./m2.day); fasting samples were obtained at 3 months (n = 22), and timed sampling was repeated at 6 months (n = 21). Fasting levels of IGF-I, free IGF-I, IGF-II, IGFBP-3, and insulin increased significantly within 3 months of GH treatment, whereas IGFBP-1, IGFBP-2, and IGFBP-6 showed no change. In the timed sampling studies at 0 and 6 months, GH levels peaked 3 h after treatment; the degree of rise and the rate of decline were both greater at 6 months. IGF-I levels increased beginning at 4 h, continuing throughout the 24-h period at month 0, whereas a plateau was observed after 6-8 h during the 6-month study. Free IGF-I paralleled total IGF-I except during fasting, when it varied inversely with IGFBP-1. IGFBP-3 and IGF-II both showed late (> 20 h) responses to a dose of GH, whereas IGFBP-2 and IGFBP-6 showed minimal changes. IGFBP-1 varied inversely with insulin, which, in turn, varied with meal intake. Comparative studies in 2 subjects with GH receptor deficiency showed no response to exogenous GH. However, both IGFBP-1 and IGFBP-2 were several-fold elevated, and IGFBP-1 varied inversely with the low insulin levels. Our data are the first to examine multiple elements of the serum IGF system in response to GH in both GH-deficient and replete states. The relationships of the different response patterns provide insight into the physiology of this system and may guide future studies.