Structure-function relationships of insulin-like growth factor binding protein 6 (IGFBP-6) and its chimeras.

Insulin-like growth factor binding protein 6 (IGFBP-6) is a high-affinity IGFBP with substantially greater affinity for insulin-like growth factor-II (IGF-II) than IGF-I. IGFBP-6(3) is a chimera which has a 20 amino acidC -terminal portion of IGFBP-6 switched with the homologous area of IGFBP-3, P3. Unlike IGFBP-4(3), in which the P3 region was exchanged for the homologous region of IGFBP-4 (P4), IGFBP-6(3) does not bind to endothelial cells. Double mutations were made with the P3 region exchanged as well as a second area differing from IGFBP-3 to form IGFBP-6(3)A and IGFBP-6(3)B, by replacing this area with the homologous region of IGFBP-3. Neither [(125)I]IGFBP-6(3)A nor IGFBP-6(3)B specifically bound to endothelial cells. However, each double mutant competed for [(125)I]IGFBP-3 binding to cultured cells. In the perfused heart, transendothelial transport of IGFBP-6 and IGFBP-6(3) was only 25% of similar transendothelial transport of perfused IGFBP-3. We conclude that chimeras of IGFBP-6 and IGFBP-3(6) clearly differ from IGFBP-4(3) in their ability to bind specifically to endothelial cells and in their capacity to undergo transendothelial transportation in the perfused heart.

IGFBP-3 binding to endothelial cells inhibits plasmin and thrombin proteolysis.

Insulin-like growth factor-binding protein (IGFBP)-3 contains a highly basic COOH-terminal heparin-binding region, the P3 region, which is thought to be important in the binding of IGFBP-3 to endothelial cells. IGFBP-3 and IGFBP-4, and their chimeras IGFBP-3(4) and IGFBP-4(3), were treated with plasmin and with thrombin, proteases known to cleave IGFBP-3. IGFBP-3 was highly susceptible to plasmin, whereas IGFBP-4 was less so. Substitution of the P3 region for the P4 region in IGFBP-4 (IGFBP-4(3)) increased the ability of the protease to digest IGFBP-4(3); substitution of the P4 region for the P3 region in IGFBP-3 (IGFBP-3(4)) decreased the digestion of IGFBP-3(4). When 125I-labeled IGFBP-3 or 125I-IGFBP-4(3) was first bound to vascular endothelial cells, subsequent proteolysis by either plasmin or thrombin was substantially inhibited. Proteolysis of 125I-IGFBP-3(4) was not inhibited in the presence of endothelial cells. The P3 peptide was cleaved by plasmin but not by thrombin. We conclude that the P3 region is central to proteolysis of IGFBP-3 by plasmin and thrombin, processes which were inhibited by association of IGFBP-3 with endothelial cells.

Distribution of chimeric IGF binding protein (IGFBP)-3 and IGFBP-4 in the rat heart: importance of C-terminal basic region.

IGF binding proteins-3 and -4, whether given in the perfused rat heart or given iv in the intact animal, cross the microvascular endothelium of the heart and distribute in subendothelial tissues. IGF binding protein-3, like IGF-I/II, localizes in cardiac muscle, with lesser concentrations in CT elements. In contrast, IGFBP-4 preferentially localizes in CT. In this study, chimeric IGF binding proteins were prepared in which a basic 20-amino-acid C-terminal region of IGF binding protein-3 was switched with the homologous region of IGF binding protein-4, and vice-versa, to create IGF binding protein-3(4) and IGF binding protein-4(3). Perfused IGF binding protein-3(4) behaved like IGF binding protein-4, localizing in connective tissue elements, whereas IGF binding protein-4(3) now localized in cardiac muscle at concentrations identical to perfused IGF binding protein-3. To determine whether these small mutations altered the affinity of the chimera for cells, the ability of (125)I-IGF binding protein-3(4) and (125)I-IGF binding protein-4(3) to bind to microvascular endothelial cells was determined and compared with IGF binding protein-3. IGF binding protein-3(4) retained 15% of the binding capacity of IGF binding protein-3, whereas IGF binding protein-4(3) bound to microvessel endothelial cells with higher affinity and greater total binding than that of IGF binding protein-3. We conclude that small changes in the C-terminal basic domain of IGF binding protein-3 and the corresponding region of IGF binding protein-4 can alter their affinity for cultured cells and influence their tissue distribution in the rat heart.

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Insulin-like growth factor (IGF)-binding protein-4 proteolytic degradation in bovine, equine, and porcine preovulatory follicles: regulation by IGFs and heparin-binding domain-containing peptides.

We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I enhanced IGFBP-4 degradation. In contrast, IGFBP-2 or -3 or monoclonal antibodies against IGF-I or -II dose-dependently inhibited IGFBP-4 degradation, and IGF-I or -II reversed this inhibition in a dose-dependent manner. Heparin-binding peptides derived from the C-terminal domain of IGFBP-3 or -5 inhibited IGFBP-4 degradation. Other heparin-binding peptides derived from CTGF, HIP, and vitronectin also inhibited IGFBP-4 degradation, except in porcine follicles. Finally, IGFBP-3 that was mutated in its HBD was less effective at inhibiting IGFBP-4 degradation. Thus, in bovine, porcine, and equine preovulatory follicles, IGFBP-4 proteolytic degradation both depends on IGFs and is inhibited by peptides containing HBD. Overall, these results suggest that during terminal development of follicles to the preovulatory stage in domestic animal species, the increase in IGF bioavailability might enhance IGFBP-4 degradation. In contrast, in atretic follicles, the decrease in IGF bioavailability, resulting partly from the increase in IGFBP-2 (sow, heifer, mare) and IGFBP-5 (heifer) expression would participate in the decrease of IGFBP-4 degradation. In bovine atretic follicles, IGFBP-5 would also strengthen the inhibition of IGFBP-4 degradation by direct interaction of its HBD with the protease. The involvement of other HBD-containing proteins in the modulation of intrafollicular proteases degrading IGFBP-4 remains to be investigated.

Mechanism of coronary vasodilation to insulin and insulin-like growth factor I is dependent on vessel size.

Insulin and insulin-like growth factor I (IGF-I) influence numerous metabolic and mitogenic processes; these hormones also have vasoactive properties. This study examined mechanisms involved in insulin- and IGF-I-induced dilation in canine conduit and microvascular coronary segments. Tension of coronary artery segments was measured after constriction with PGF(2alpha). Internal diameter of coronary microvessels (resting diameter = 112.6+/-10.1 microm) was measured after endothelin constriction. Vessels were incubated in control (Krebs) solution and were treated with N(omega)-nitro-L-arginine (L-NA), indomethacin, or K(+) channel inhibitors. After constriction, cumulative doses of insulin or IGF-I (0.1-100 ng/ml) were administered. In conduit arteries, insulin produced modest maximal relaxation (32 +/- 5%) compared with IGF-I (66+/-12%). Vasodilation was attenuated by nitric oxide synthase (NOS) and cyclooxygenase inhibition and was blocked with KCl constriction. Coronary microvascular relaxation to insulin and IGF-I was not altered by L-NA, indomethacin, tetraethylammonium chloride, glibenclamide, charybdotoxin, and apamin; however, tetrabutylammonium chloride attenuated the response. In conclusion, insulin and IGF-I cause vasodilation in canine coronary conduit arteries and microvessels. In conduit vessels, NOS/cyclooxygenase pathways are involved in the vasodilation. In microvessels, relaxation to insulin and IGF-I is not mediated by NOS/cyclooxygenase pathways but rather through K(+)-dependent mechanisms.

Insulin-like growth factor binding protein-4 proteolytic degradation in ovine preovulatory follicles: studies of underlying mechanisms.

The regulation of insulin-like growth factor binding protein (IGFBP)-4 proteolytic degradation by insulin-like growth factors (IGFs) has been largely studied in vitro, but not in vivo. The aim of this study was to investigate the involvement of IGFs, IGFBP-2, IGFBP-3, and IGFBP-3 proteolytic fragments in the regulation of IGFBP-4 proteolytic activity in ovine ovarian follicles. Follicular fluid from preovulatory follicles contains proteolytic activity degrading exogenous IGFBP-4. The addition of an excess of IGF-I enhanced IGFBP-4 proteolytic degradation, whereas addition of IGFBP-2 or -3 or monoclonal antibodies against IGF-I and -II dose dependently inhibited IGFBP-4 proteolytic degradation. IGF-I and IGF-II, but not LongR3-IGF-I, reversed this inhibition in a dose-dependent manner. C-terminal, but not N-terminal, proteolytic fragments derived from IGFBP-3 (aa 161-264), as well as heparin-binding domain-containing peptides derived from the C-terminal domain of IGFBP-3 and -5 also induced the inhibition of IGFBP-4 proteolytic degradation. Other heparin-binding domain-containing peptides derived from the connective tissue growth factor (CTGF) and from proteins not related to IGFBP, heparan/heparin interacting protein (HIP) and vitronectin, but not from p36 subunit of annexin II tetramer, inhibited IGFBP-4 degradation. Furthermore, IGFBP-3, mutated on its heparin-binding domain, was not able to inhibit IGFBP-4 proteolytic degradation. So, in ovine preovulatory follicles, IGFBP-4 proteolytic degradation both 1) depends on IGFs, and 2) is inhibited by IGFBP-3 via its C-terminal heparin-binding domain as well as by heparin-binding domain containing peptides. These data suggest that in early atretic follicles, the increase in IGFBP-2 participates in the decrease in IGFBP-4 degradation. In late atretic follicles, the increase in the levels of C-terminal IGFBP-3 proteolytic fragments, generated by IGFBP-3 degradation, as well as the increase in IGFBP-5 expression would strengthen the inhibition of IGFBP-4 degradation. This inhibition might be partly mediated by direct interaction of IGFBP-4 proteinase(s) and heparin-binding domain within the C-terminal region from IGFBP-3 and -5.

Isolation and characterization of plasmin-generated bioactive fragments of IGFBP-3.

Insulin-like growth factor-binding protein-3 (IGFBP-3) was digested with plasmin, and the proteolytic fragments were isolated by HPLC and tested for bioactivity as measured by stimulation of glucose uptake in microvessel endothelial cells. Two of the pooled fractions of the digest stimulated glucose uptake. The major bioactive pool, at an estimated protein concentration <50 ng/ml, stimulated glucose uptake to 150% of control with greater stimulation and 220% of control at approximately 250 ng/ml. Two fragments were present in the bioactive fraction, the dominant one migrating at approximately 20,000 and the other at approximately 8,000. Both fragments bound 125I-labeled insulin-like growth factor and [3H]heparin. NH2-terminal amino acid analysis of the bioactive peak yielded two sequences. One, representing the majority of the material, had an NH2-terminal sequence identical to IGFBP-3; the second fragment began at amino acid 202 of IGFBP-3. In contrast to the bioactive fragments, intact IGFBP-3, at concentrations up to 130 microgram/ml, had no bioactivity. These findings demonstrate that IGFBP-3 can be degraded into fragments that have potent bioactivities that are not present in the intact IGFBP-3 molecule.

Transcriptional and posttranscriptional regulation of insulin-like growth factor binding protein-3 by cyclic adenosine 3',5'-monophosphate: messenger RNA stabilization is accompanied by decreased binding of a 42-kDa protein to a uridine-rich domain in the 3'-untranslated region.

The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased approximately 3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3'-untranslated region (3'-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.

Connective tissue growth factor (IGFBP-rP2) expression and regulation in cultured bovine endothelial cells.

Media from large vessel endothelial cells (pulmonary artery, aorta) contained intact connective tissue growth factor (CTGF) and a dominant 19-kDa band. N-terminal analysis of the 19-kDa band showed sequence corresponding to CTGF amino acid 181-190, suggesting that the 19-kDa band represented a proteolytic fragment of CTGF. Intact CTGF was increased by cAMP but not by transforming growth factor-beta (TGFbeta). CTGF messenger RNA (mRNA) was not changed by cAMP nor TGFbeta. In two microvessel endothelial cells, mRNA was found at low levels by PCR and Northern analysis, but no CTGF protein was seen on Western analysis. In the microvessel cells, TGFbeta increased and cAMP did not change CTGF mRNA levels, with neither TGFbeta nor cAMP increasing CTGF protein. The discordance between protein and mRNA levels in large vessel and microvessel endothelial cells was mostly explained by the effects of cAMP and TGFbeta on media proteolytic activity; in large vessel cells, cAMP inhibited degradation of CTGF, whereas in microvessel cells, TGFbeta and cAMP stimulated proteolytic activity against CTGF. We conclude that in large vessel endothelial cells, cAMP increased intact CTGF protein by inhibiting degradation of CTGF, whereas TGFbeta stimulated neither CTGF mRNA nor protein; in microvessel cells, TGFbeta increased CTGF mRNA, while both TGFbeta and cAMP stimulated CTGF degradation.

Infused IGF-I/IGFBP-3 complex causes glomerular localization of IGF-I in the rat kidney.

Insulin-like growth factor I (IGF-I) increases renal blood flow, glomerular filtration rate (GFR), and proximal tubule reabsorption of phosphate in humans and rodents. The biological effects of IGF-I are likely to be influenced by cellular localization of IGF-I within the kidney. We therefore tested whether the renal localization of infused IGF-I could be altered if given with selected IGF-binding proteins (IGFBPs). Rats were treated with intravenous injections of 125I-labeled IGF-I, 125I-IGFBP-3, or 125I-IGFBP-4 alone or with complexes of 125I-IGF-I and IGFBP-3 or IGFBP-4. The cellular localization of IGF and the IGFBP within the kidney was then determined. 125I-IGF-I, 125I-IGFBP-4, and 125I-IGF-I/IGFBP-4 complexes were found almost exclusively in vacuolar structures (endosomes) of proximal renal tubules. In contrast, about one-third of renal 125I-IGFBP-3 and 125I-IGF-I/IGFBP-3 was localized to glomeruli. When 125I-IGF-I was given alone, 3% was found in glomeruli and 89% in proximal tubules. When given as 125I-IGF-I/IGFBP-3, 29% was in glomeruli and 65% in proximal tubules. We conclude that the cellular localization of IGF-I within the kidney can be directed to glomerular elements if the IGF-I is given with IGFBP-3.

Regulations of IGF binding proteins in human aorta vascular smooth muscle cells by cAMP, dexamethasone and IGF-I.

Human vascular smooth muscle cells produce IGFBP-3, IGFBP-4, IGFBP-6 and proteases specific for IGFBP-3 and IGFBP-4. This study evaluated the regulation of IGFBPs in human aorta smooth muscle cells by cyclic AMP, dexamethasone and IGF-I. cAMP decreased IGFBP-3, increased IGFBP-4 and increased IGFBP-6. Dexamethasone decreased IGFBP-3, slightly increased IGFBP-4 and increased IGFBP-6. IGF-I increased IGFBP-3 and IGFBP-6 while decreasing IGFBP-4. Co-incubation with IGF-I and dexamethasone or cAMP increased media IGFBP-3, despite a decrease in IGFBP-3 mRNA, due to the dominant effect of IGF-I-induced dissociation of cell surface-bound IGFBP-3. In cells incubated with cAMP and IGF-I, media IGFBP-4 was decreased, despite increased IGFBP-4 mRNA, in this case secondary to the dominant effect of IGF-I-stimulated IGFBP-4 protease. These findings suggest that cAMP, dexamethasone and IGF-I regulate IGFBP production in human aorta smooth muscle cells via a complex interplay of changes in transcription, protease activation and dissociation of cell surface-bound IGFBPs.

Bovine insulin-like growth factor binding protein-3: organization of the chromosomal gene and functional analysis of its promoter.

Insulin-like growth factor binding protein-3 (IGFBP-3), the major IGFBP in the circulation, is synthesized by the vascular endothelium in vivo and has been shown to be an important modulator of the physiological effects of IGF. IGFBP-3 is regulated by a number of growth factors/cytokines to which the vascular endothelium is exposed, including IGF-I stimulation and TGF-beta1 inhibition of IGFBP-3 in cultured endothelial cells. To understand the mechanisms of transcriptional regulation of IGFBP-3, we have cloned the bovine IGFBP-3 gene and begun the functional analysis of its promoter. Southern analysis indicated a single copy gene. The gene spanned approximately 10 kb and was divided into five exons, the fifth containing the 3' untranslated region. The transcription start site was 137 bp upstream of the initiation codon and a TATA box was located 26 bp 5' to this CAP site. No CAAT box was present but a GC rich sequence element, containing two overlapping putative AP-2 binding elements, was located 5' to the TATA box. Transient transfection studies with a series of 5' truncated luciferase reporter constructs were conducted in primary cultures of bovine aorta endothelial cells. Results of the transfection studies indicated that 1) nearly 80% of the maximal basal promoter activity was retained within the first 130 bp of the 5' flanking sequence; 2) this region responded to IGF-I, despite lacking the TTF-1/TTF-2 (thyroid specific transcription factors) binding elements that are required for IGF-I stimulation of thyroglobulin synthesis. These binding elements have also been suggested to be involved in IGF-I regulation of IGFBP-3 transcription, thus, implying the existence of novel cis-acting elements that mediate the IGF-I stimulation of bovine endothelial cell IGFBP-3 mRNA synthesis; 3) deletion of the GC rich sequence element resulted in a 60% reduction in basal promoter activity as well as loss of the IGF-1 stimulatory effect; 4) the TGF-beta1 mediated inhibition of IGFBP-3 transcription required sequence element(s) beyond 1.5 kb of its promoter.

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle.

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.

Structure-function relationships in the heparin-binding C-terminal region of insulin-like growth factor binding protein-3.

IGFBP-3 contains a carboxyterminal basic region which, when present as an isolated 18 amino acid peptide (P3), binds heparin, associates with cultured endothelial cells and stimulates glucose uptake. The P3 molecule has now been modified relative to charge, amino acid sequence and size to determine structure-function relationships relative to four properties of P3: affinity for heparin; inhibition of IGFBP-3 binding; stimulation of glucose uptake; and displacement of bFGF from the extracellular matrix of endothelial cells. Results indicate: (1) the presence or absence of heparin binding was concordant with the presence/absence of the other three properties; (2) the number of basic amino acids was an important, if not limiting, factor for each property; (3) the order of potency of the basic amino acids was arginine = lysine > > histidine; (4) the unrelated, basic protein, protamine, mimics all properties of P3; and (5) the putative consensus heparin-binding sequence of P3 was not essential for any of the P3 activities.

Four- to twenty-four-hour uptake ratio: an index of rapid iodine-131 turnover in hyperthyroidism.

UNLABELLED: Rapid thyroidal iodine turnover may contribute to 131I therapy failure in patients with hyperthyroidism. The utility of a 4- to 24-hr 131I uptake ratio was evaluated as an index of thyroidal iodide retention in hyperthyroid patients. METHODS: In 433 hyperthyroid patients, the success of 131I therapy was correlated with the following factors: gender, pretreatment with antithyroid drugs, clinical diagnosis, magnitude of early and late thyroidal 131I uptake values, and the 4- to 24-hr 131I uptake ratio. RESULTS: Of the 433 patients, 362 patients (84%) had a successful outcome after a single therapeutic dose of 131I while 71 (16%) did not. Multiple linear regression analysis revealed that the highest statistically significant predictor of outcome was the 4- to 24-hr 131I uptake ratio (p-value < 0.001); all other factors showed a weaker association. An 131I uptake ratio of > 1 was found in 67 (15%) patients. Thirty-two of these 67 patients (48%) failed 131I therapy, whereas those patients with uptake ratios of < 1.0, only 39/366 (11%) failed 131I therapy. CONCLUSION: The 4- to 24-hr 131I thyroidal uptake ratio is a practical substitute for exact determination of the effective half-life. It identifies patients who are likely to have a rapid 131I turnover without the need for extended thyroid uptake measurements. An 131I uptake ratio of > or = 1 was found in 15% of hyperthyroid patients and was associated with a near 50% 131I therapy failure rate.

IGFBP-3 proteolysis by plasmin, thrombin, serum: heparin binding, IGF binding, and structure of fragments.

Insulin-like growth factor binding protein (IGFBP)-3 was exposed to plasmin, thrombin, and pregnancy serum, substances normally present at the endothelial surface in enriched concentrations. The NH2-termini of the proteolytic fragments were sequenced, and their ability to bind insulin-like growth factor (IGF) and heparin was assessed by ligand blotting. Plasmin generated at least five fragments, three beginning at the NH2-terminus of IGFBP-3 and two with NH2-termini corresponding to middle portions of IGFBP-3. The dominant fragment bound both IGF and heparin while NH2-terminal fragments bound only IGF. Thrombin generated three and serum five easily identified fragments; the dominant fragments, beginning at midportions of IGFBP-3, retained IGF and heparin affinity, whereas the remaining fragments had differential affinities for IGF and heparin. We suggest that such fragments, when generated at the endothelia surface, have the potential to alter regional vascular concentrations of IGF and thus influence both IGF and endothelial function.

Regulation of endothelial IGFBP-3 synthesis and secretion by IGF-I and TGF-beta.

We have examined the regulation of endothelial IGFBP-3 production by IGF-I and TGF-beta, two growth factors thought to play a major roles in the complications of diabetes mellitus. In addition, we developed a sensitive method for IGFBP-3 mRNA quantitation by adapting the fluorescent modification of the competitive PCR strategy. Our results using both Northern analysis and the fluorescent competitive PCR method indicate that: (1) IGFBP-3 mRNA is increased 2- to 10-fold by IGF-I and maximally reduced to 20% of control by TGF-beta; (2) the changes in mRNA levels correlate with the levels of IGFBP-3 protein secreted into the media by these cells; (3) the induction of IGFBP-3 mRNA and protein by IGF-I analogs was directly related to their ability to bind to the type I IGF receptor, reflecting an IGF-I receptor-mediated process; and (4) steady state IGFBP-3 mRNA levels did not change significantly after a 6 h incubation with actinomycin D in the presence or absence of the growth factors suggesting that the observed IGF-I/TGF-beta effects occur at the level of gene transcription rather than mRNA stability.

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.

Insulin-like growth factor binding protein (IGFBP)4 accounts for the connective tissue distribution of endothelial cell IGFBPs perfused through the isolated heart.

Insulin-like growth factor binding protein 4 (IGFBP4) was purified to homogeneity from conditioned media of bovine pulmonary artery endothelial cells and shown to have the N-terminal amino acid sequence DEAIHCPPCS, a sequence unique to IGFBP4. The IGFBP4 was separated into predominantly glycosylated and nonglycosylated fractions, with each fraction separately perfused through isolated, beating rat hearts. Both forms of IGFBP4 crossed the capillary boundary of the heart and distributed primarily in subendothelial connective tissue components with a connective tissue/cardiac muscle distribution ratio of 20:1 for the glycosylated fraction and 27:1 for the nonglycosylated fraction. Perfused IGFBP1, 2, 3, and IGF-I also crossed the capillary boundary but in contrast to IGFBP4, preferentially localized in cardiac muscle with a connective tissue/muscle ratio of approximately 1:3. We conclude that the connective tissue distribution previously reported for IGFBPs in conditioned media of pulmonary artery endothelial cells is due to IGFBP4.