Adrenomedullin and adrenomedullin binding protein-1 protect endothelium-dependent vascular relaxation in sepsis

Downregulation of vascular endothelial constitutive nitric oxide synthase (ecNOS) contributes to the vascular hyporesponsiveness in sepsis. Although coadministration of the potent vasodilatory peptide adrenomedulin (AM) and the newly discovered AM binding protein (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis, it remains unknown whether AM/AMBP-1 prevents endothelial cell dysfunction. To investigate this possibility, we subjected adult male rats to sepsis by cecal ligation and puncture (CLP), with or without subsequent intravenous administration of the combination of AM (12 microg/kg) and AMBP-1 (40 microg/kg). Thoracic aortae were harvested 20 h after CLP (i.e., the late stage of sepsis) and endothelium-dependent vascular relaxation was determined by the addition of acetylcholine (ACh) in an organ bath system. In addition, ecNOS gene and protein expression was assessed by RT-PCR and immunohistochemistry, respectively. The results indicate that ACh-induced (i.e., endothelium-dependent) vascular relaxation was significantly reduced 20 h after CLP. Administration of AM/AMBP-1 prevented the reduction of vascular relaxation. In addition, ecNOS gene expression in aortic and pulmonary tissues was downregulated 20 h after CLP and AM/AMBP-1 attenuated such a reduction. Moreover, the decreased ecNOS staining in thoracic aortae of septic animals was prevented by the treatment with AM/AMBP-1. These results, taken together, indicate that AM/AMBP-1 preserves ecNOS and prevents reduced endothelium-dependent vascular relaxation (i.e., endothelial cell dysfunction) in sepsis. In light of our recent finding that AM/AMBP-1 improves organ function and reduces mortality in sepsis, it is most likely that the protective effect of these compounds on ecNOS is a mechanism responsible for the salutary effect of AM/AMBP-1 in sepsis.     

Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-alpha from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-alpha release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-alpha production from RAW cells by 38-63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-alpha release by 14-22% and 13-22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-alpha production (by 24-35%). Moreover, the upregulated TNF-alpha mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-alpha production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-alpha production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-alpha mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-alpha expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-alpha may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.     

Purification and characterization of human adrenomedullin binding protein-1

We recently discovered that the vascular responsiveness to adrenomedullin (AM), a potent vasoactive peptide, decreased during sepsis and hemorrhage in the rat and was markedly improved by its novel binding protein (AMBP-1). Moreover, AM/AMBP-1 appears to be one of the leading candidates for further development to treat sepsis and hemorrhage. However, the extremely high cost of commercial AMBP-1 limits the development of human AM and AMBP-1 as therapeutic agents. The purpose of this study was to isolate and purify AMBP-1 from normal human serum and test its stability and biological activity under in vitro and in vivo conditions. AMBP-1 was isolated and purified from normal human serum with a yield of about 3.0 mg per 100 mL and purity of >99%. The purified AMBP-1 has a AM-binding capacity similar to that of the commercial AMBP-1. Human AM and human AMBP-1 in combination significantly inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production from macrophages. The biological activity of the purified human AMBP-1 was well preserved when stored at 45 degrees C for 5 d in solution or at 100 degrees C for 1 h in powder. Moreover, administration of AM and purified AMBP-1 to hemorrhaged rats attenuated tissue injury and neutrophil accumulation. Purified AMBP-1 in combination with AM also suppressed the hemorrhage-induced rise in serum cytokines TNF-alpha and IL-6. Thus, we have successfully purified biologically active AMBP-1 from human normal serum and demonstrated the stability of purified human AMBP-1. This technique will enable us to further develop human AM/AMBP-1 as a novel treatment for safe and effective therapy of patients with hemorrhagic shock, sepsis, and ischemic injury.     

Human adrenomedullin combined with human adrenomedullin binding protein-1 is protective in gut ischemia and reperfusion injury in the rat

Previous studies have demonstrated that co-administration of rat adrenomedullin (AM) and human AM binding protein-1 (AMBP-1) has various beneficial effects following adverse circulatory conditions. In order to reduce rat proteins to elicit possible immune responses in humans, we determined the effect of human AM combined with human AMBP-1 after intestinal ischemia and reperfusion (I/R). Intestinal ischemia was induced in the rat by occluding the superior mesenteric artery for 90 min. At 60 min after the beginning of reperfusion, human AM/AMBP-1 at 3 different dosages was administered intravenously over 30 min. At 240 min after the treatment, blood and tissue samples were harvested and measured for pro-inflammatory cytokines (i.e., TNF-alpha and IL-6), myeloperoxidase activities in the gut and lungs, and cleaved caspase-3 expression in the lungs, as well as serum levels of hepatic enzymes and lactate. In additional groups of animals, a 10-day survival study was conducted. Results showed that administration of human AM/AMBP-1 reduced pro-inflammatory cytokines, attenuated organ injury, and improved the survival rate in a seemingly dose-response fashion. Co-administration of the highest dose of human AM/AMBP-1 in this study had the optimal therapeutic effect in the rat model of intestinal I/R.     

Pro-inflammatory cytokines from Kupffer cells downregulate hepatocyte expression of adrenomedullin binding protein-1

Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late hypodynamic phase. Adrenomedullin (AM), a vasodilatory peptide, inhibits this transition from the early phase to the late phase. Adrenomedullin binding protein-1 (AMBP-1) enhances AM-mediated activities. The decrease of AMBP-1 levels in late sepsis reduces the vascular response to AM and produces the hypodynamic phase. Studies have indicated that the administration of LPS downregulates AMBP-1 production in the liver. Since hepatocytes are the primary source of AMBP-1 biosynthesis in the liver, we employed a co-culture strategy using hepatocyte and Kupffer cells to determine whether LPS directly or by increasing pro-inflammatory cytokines from Kupffer cells downregulates AMBP-1 production. Hepatocytes and Kupffer cells isolated from rats were co-cultured and treated with LPS for 24 h. LPS significantly attenuated AMBP-1 protein expression in a dose-dependent manner. Since AMBP-1 is basically a secretory protein, cell supernatants from co-culture cells treated with LPS were examined for AMBP-1 protein levels. LPS treatment caused a dose related decrease in AMBP-1 protein secretion. Similarly, LPS treatment produced a significant decrease in AMBP-1 protein expression in hepatocytes and Kupffer cells cultured using transwell inserts. LPS had no direct effect on AMBP-1 levels in cultured hepatocytes or Kupffer cells alone. To confirm that the observed effects in co-culture were due to the cytokines released from Kupffer cells, hepatocytes were treated with IL-1beta or TNF-alpha for 24 h and AMBP-1 expression was examined. The results indicated that both cytokines significantly inhibited AMBP-1 protein levels. Thus, pro-inflammatory cytokines released from Kupffer cells are responsible for downregulation of AMBP-1.     

L-Name modulates responses to adrenomedullin in the hindquarters vascular bed of the rat

Responses to synthetic human adrenomedullin (ADM), a novel hypotensive peptide recently discovered in human pheochromocytoma cells, and calcitonin gene-related peptide (CGRP), a structurally related peptide, were investigated in the hintquarters vascular bed of the rat. Under conditions of controlled hintquarters blood flow, intraarterial injections of ADM (0.01–0.3 nmol) and of CGRP (0.03–0.3 nmol) caused dose-related decreases in hindquarters perfusion pressure and decreases in systemic arterial pressure. Following administration of the nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME), hindquarters vasodilator and systemic depressor responses to ADM were significantly decreased, whereas L-NAME did not significantly decrease the vasodilator response to CGRP in either the hindquarters or systemic vascular beds. Following administration of the cyclooxygenase inhibitor, meclofenamate, vasodilator responses to ADM and to CGRP were not significantly decreased. When the relative vasodilator activity of the two peptides was compared on a nmol basis, responses to ADM were similar to responses with CGRP in the hindquarters vascular bed, whereas ADM was 30–100 fold less potent than CGRP in decreasing systemic arterial pressure. The present data demonstrate that ADM has significant vasodilator activity in the hindquarters vascular bed of the rat, that hindquarters vasodilator and systemic vasodepressor responses to ADM, but not to CGRP, are dependent upon the release of nitric oxide from the endothelium.     

Cell-cell bond modulates vascular smooth muscle cell responsiveness to Angiotensin II

Cell attachment is provided by cell-matrix and cell-cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell-cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of β-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that β-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell-cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.     

Pulmonary clearance of adrenomedullin is reduced during the late stage of sepsis

Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Although upregulation of adrenomedullin (ADM), a novel potent vasodilatory peptide, plays an important role in producing cardiovascular responses during the progression of sepsis, it remains unknown whether the clearance of this peptide is altered under such conditions. To determine this, male adult rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. At 5 h (i.e., the hyperdynamic phase of sepsis) or 20 h (the hypodynamic phase) after CLP, the animals were injected with 125I-labeled ADM through the jugular vein. Blood and tissue samples (including the lungs, kidneys, gastrointestinal tract, pancreas, spleen, mesentery, liver, brain, skeletal muscle, heart, and skin) were harvested 30 min after the injection and the radioactivity was determined. The results indicate that there were no significant alterations in tissue [125I]ADM distribution at 5 h after CLP compared to shams. At 20 h after CLP, however, there was a significant decrease in radioactivity in the lungs. In contrast, a significant increase of radioactivity was observed in all other organs except the liver and kidneys. The pulmonary distribution of [125I]ADM was found to be far greater than in any other organs tested, irrespective of the effect of sepsis. In separate groups of animals, injection of [125I]ADM into the left ventricle resulted in a significant decrease in radioactivity in the lungs of both sham and septic animals at 20 h after surgery. These results suggest that the lungs are the primary site of ADM clearance, which is significantly diminished during the late stage of sepsis. The decreased clearance of ADM by the lungs may play an important role in maintaining the sustained levels of plasma ADM under such conditions.     

IL-1 receptor-associated kinase modulates host responsiveness to endotoxin

Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock. Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer. The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction. LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells. We demonstrate that LPS activates IRAK in murine macrophages. IRAK-deficient macrophages, in contrast, are resistant to LPS. Deletion of IRAK disrupts several endotoxin-triggered signaling cascades. Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS. These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection.     

Cholesterol modulates cellular TGF-beta responsiveness by altering TGF-beta binding to TGF-beta receptors

Transforming growth factor-beta (TGF-beta) responsiveness in cultured cells can be modulated by TGF-beta partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. The TbetaR-II/TbetaR-I binding ratio of TGF-beta on the cell surface has recently been found to be a signal that controls TGF-beta partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF-beta binding to TGF-beta receptors and TGF-beta responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol, alone or complexed in lipoproteins, decreases the TbetaR-II/TbetaR-I binding ratio of TGF-beta while treatment with cholesterol-lowering or cholesterol-depleting agents increases the TbetaR-II/TbetaR-I binding ratio of TGF-beta in all cell types studied. Among cholesterol derivatives and analogs examined, cholesterol is the most potent agent for decreasing the TbetaR-II/TbetaR-I binding ratio of TGF-beta. Cholesterol treatment increases accumulation of the TGF-beta receptors in lipid rafts/caveolae as determined by sucrose density gradient ultracentrifugation analysis of cell lysates. Cholesterol/LDL suppresses TGF-beta responsiveness and statins/beta-CD enhances it, as measured by the levels of P-Smad2 and PAI-1 expression in cells stimulated with TGF-beta. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE-null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels may contribute to the pathogenesis of certain diseases (e.g., atherosclerosis) by suppressing TGF-beta responsiveness.     

Amyloid precursor protein binding protein-1 modulates cell cycle progression in fetal neural stem cells

Amyloid precursor protein binding protein-1 (APP-BP1) binds to the carboxyl terminus of the amyloid precursor protein (APP) and serves as the bipartite activation enzyme for the ubiquitin-like protein, NEDD8. In the present study, we explored the physiological role of APP-BP1 in the cell cycle progression of fetal neural stem cells. Our results show that cell cycle progression of the cells is arrested at the G1 phase by depletion of APP-BP1, which results in a marked decrease in the proliferation of the cells. This action of APP-BP1 is antagonistically regulated by the interaction with APP. Consistent with the evidence that APP-BP1 function is critical for cell cycle progression, the amount of APP-BP1 varies depending upon cell cycle phase, with culminating expression at S-phase. Furthermore, our FRET experiment revealed that phosphorylation of APP at threonine 668, known to occur during the G2/M phase, is required for the interaction between APP and APP-BP1. We also found a moderate ubiquitous level of APP-BP1 mRNA in developing embryonic and early postnatal brains; however, APP-BP1 expression is reduced by P12, and only low levels of APP-BP1 were found in the adult brain. In the cerebral cortex of E16 rats, substantial expression of both APP-BP1 and APP mRNAs was observed in the ventricular zone. Collectively, these results indicate that APP-BP1 plays an important role in the cell cycle progression of fetal neural stem cells, through the interaction with APP, which is fostered by phosphorylation of threonine 668.     

Adrenomedullin is upregulated in the heart and aorta during the early and late stages of sepsis

Although circulating levels of adrenomedullin (ADM), a newly reported vasodilatory peptide with 52 amino acid residues in the human and 50 amino acid residues in the rat, are elevated during the early and late stages of sepsis, ADM levels in cardiovascular tissues and its precise localization remain to be determined. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP), followed by administration of 3 ml/100 g b.wt. normal saline to these and sham-operated animals. The heart and thoracic aorta were harvested at 5 h (i.e. the early stage of sepsis) and 20 h (late sepsis) after CLP. Tissue levels of ADM were determined by radioimmunoassay. The localization of ADM in the left ventricle and thoracic aorta was examined by using immunohistochemistry and electron microscopy techniques. The results indicated that ADM levels in the heart and thoracic aorta increased significantly at 5 h after CLP and remained elevated at 20 h after the onset of sepsis. Immunohistochemistry findings showed that ADM immunoreaction products were localized in the cytoplasm of the cardiac myocytes and aortic endothelial cells. Using electron microscopy, ADM immunoreaction products were found in the cytoplasmic matrixes. The immunostainings were also associated with the outer membranes of mitochondria and vesicles of the myocytes as well as vascular endothelial cells. It appears that the cardiovascular tissues, among other organ systems, contribute to the increased levels of plasma ADM under those conditions. Since ADM is localized in different cell populations in the heart and the large blood vessel (i.e. myocytes versus vascular endothelial cells), this peptide may play a differential role in regulating cardiac and vascular functions during sepsis as an autocrine and/or paracrine mediator.     

Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility

In addition to their role in many vital cellular functions, arachidonic acid (AA) and its eicosanoid metabolites are involved in the pathogenesis of several diseases, including atherosclerosis and cancer. To understand the potential mechanisms by which these lipid molecules could influence the disease processes, particularly cardiovascular diseases, we studied AA's effects on vascular smooth muscle cell (VSMC) motility and the role of cAMP-response element binding protein-1 (CREB-1) in this process. AA exerted differential effects on VSMC motility; at lower doses, it stimulated motility, whereas at higher doses, it was inhibitory. AA-induced VSMC motility requires its conversion via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. AA stimulated the phosphorylation of extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) in a time-dependent manner, and blockade of these serine/threonine kinases significantly attenuated AA-induced VSMC motility. In addition, AA stimulated CREB-1 phosphorylation and activity in a manner that was also dependent on its metabolic conversion via the LOX and COX pathways and the activation of ERKs and p38MAPK but not JNKs. Furthermore, suppression of CREB-1 activation inhibited AA-induced VSMC motility. 15(S)-Hydroxyeicosatetraenoic acid and prostaglandin F2alpha, the 15-LOX and COX metabolites of AA, respectively, that are produced by VSMC at lower doses, were also found to stimulate motility in these cells. Together, these results suggest that AA induces VSMC motility by complex mechanisms involving its metabolism via the LOX and COX pathways as well as the ERK- and p38MAPK-dependent and JNK-independent activation of CREB-1.     

Adrenomedullin is a novel adipokine: adrenomedullin in adipocytes and adipose tissues

Adrenomedullin (AM) is a multifunctional regulatory peptide that is produced and secreted by various types of cells. The production and the secretion of AM have been demonstrated in cultured adipocytes and adipose tissues. Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide are strong stimulators for AM expression in adipocytes. Furthermore, AM expression in the adipose tissue is increased in obesity, and plasma concentrations of AM are increased in obese subjects. One possible (patho)physiological role of AM secreted by adipose tissue may be actions against complications of the metabolic syndrome characterized by obesity, type 2 diabetic mellitus and hypertension, via its antioxidant and potent vasodilator effects. These findings indicate that AM is a new member of the adipokine family.     

Effects of retinol binding protein-4 on vascular endothelial cells

The study was designed to investigate the effect of retinol binding protein (RBP)-4 on the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which mediate the effects of insulin in vascular endothelial cells. The effects of RBP4 on nitric oxide (NO) and insulin-stimulated endothelin-1 (ET-1) secretion and on phosphorylation (p) of Akt, endothelial NO synthetase (eNOS), and extracellular signal-regulated kinase (ERK)1/2 were investigated in bovine vascular aortic endothelial cells (BAECs). RBP4 showed an acute vasodilatatory effect on aortic rings of rats within a few minutes. In BAECs, RBP4-treatment for 5 min significantly increased NO production, but inhibited insulin-stimulated ET-1 secretion. RBP4-induced NO production was not inhibited by tetraacetoxymethylester (BAPTA-AM), an intracellular calcium chelator, but was completely abolished by wortmannin, a PI3K inhibitor. RBP4 significantly increased p-Akt and p-eNOS production, and significantly inhibited p-ERK1/2 production. Triciribine, an Akt inhibitor, and wortmannin significantly inhibited RBP4-induced p-Akt and p-eNOS production. Inhibition of Akt1 by small interfering RNA decreased p-eNOS production enhanced by RBP4 in human umbilical vein endothelial cells. In conclusion, RBP4 has a robust acute effect of enhancement of NO production via stimulation of part of the PI3K/Akt/eNOS pathway and inhibition of ERK1/2 phosphorylation and insulin-induced ET-1 secretion, probably in the MAPK pathway, which results in vasodilatation.     

Endurance training in dogs increases vascular responsiveness to an alpha 1-agonist

The effects of endurance training on vascular responsiveness to an alpha 1-agonist and the associated changes in baroreflex modulation of heart rate and vascular resistance were studied. Graded dosages of phenylephrine were given to eight treadmill-trained dogs and to eight untrained dogs; both groups were chronically instrumented and were sedated and resting when tested. These dosages were repeated after ganglionic blockade. Aortic pressure, cardiac output, central venous pressure, peripheral resistance, and heart rate were each averaged over 30 s before injection and 90 s after injection. The slope of the peripheral resistance-dose relationship was significantly increased in trained compared with untrained dogs in both the unblocked and blocked cases [unblocked: trained 0.89, untrained 0.47; blocked: trained 4.30, untrained 2.05 (mmHg.l-1.min)/(microgram.kg-1)]. The unblocked resistance slopes were reduced with respect to the blocked slopes by 77 (untrained) and 79% (trained). The slope of the heart rate-aortic pressure response was reduced, but not significantly, by endurance training. We conclude that 6 wk of endurance training in dogs resulted in a doubling of the vascular responsiveness to an alpha 1-agonist, with no significant change in the baroreflex regulation of resistance or heart rate.     

Calcium modulates the binding of high-mobility-group protein 1 to DNA

Binding of 45Ca2+ to nonhistone protein HMG1 was detected after fixation of the protein to nitrocellulose membrane. The same experiment with HMG1 peptides, derived from HMG1 by protease V8 digestion, allowed to identify the highly glutamic and aspartic C-terminal domain of HMG1 as a 45Ca2(+)-binding region. Measurements of 32P-labeled DNA retention on nitrocellulose filters revealed that in the absence of Ca2+, the affinity of HMG1 for linear DNA decreased upon an increase of pH from 7 to 8.4. However, when Ca2+ was included in the assay buffer, the affinity of HMG1 for DNA remained unchanged between pH 7 to 8.4 and was higher than in the absence of Ca2+. The effect of Ca2+ on HMG1 - DNA interaction was no longer observed upon removal of the C-terminal domain from HMG1.     

Interaction of insulin-like growth factor binding protein-3 with latent transforming growth factor-beta binding protein-1

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits the replication and promotes apoptosis in various cell lines in an IGF-independent manner. We utilized a yeast two-hybrid system to identify binding partners for IGFBP-3 in a mouse embryo cDNA library. A partial cDNA encoding mouse latent transforming growth factor beta (TGF-) binding protein-1 (LTBP-1) was identified. This cDNA encoded a mouse LTBP-1 mRNA fragment corresponding to amino acid residues 1160–1712. Analysis of C-terminal deleted mutants indicated that the IGFBP-3 interacting domain resides in the 552 residue C-terminal fragment encoding amino acids 831–1383. The interaction of IGFBP-3 with recombinant human LTBP-1 immobilized on nitrocellulose was also demonstrated. Neither binding of IGF-I to IGFBP-3 nor binding of latency associated protein (LAP) with LTBP-1 inhibited the interaction of IGFBP-3 with LTBP-1. Furthermore the large latent complex, ¹²⁵I-TGF-/LAP/LTBP-1 was able to bind to immobilized IGFBP-3. These data demonstrate that IGFBP-3 can bind to LTBP-1 and provide a potential mechanism whereby IGFBP-3 can interact with the TGF- system.