Activity-independent cell adhesion to tissue-type transglutaminase is mediated by alpha4beta1 integrin

Transglutaminases (TGases) are enzymes which catalyze cross-link formation between glutamine residues and lysine residues in substrate proteins. We have previously reported that one of the TGases, blood coagulation factor XIIIa (FXIIIa), is capable of mediating adhesion of various cells. In this paper, we report for the first time that tissue-type transglutaminase (TGc) also has cell adhesion activity. TGc-coated plastic surface promoted adhesion and spreading of cells in a TGc concentration-dependent manner. However, there are some obvious differences between cell adhesion mediated by TGc and FXIIIa. As was reported previously, the adhesion to FXIIIa is dependent on its TGase activity. In contrast, the TGc-mediated cell adhesion is independent of its TGase activity: 1) The modification of the active center cysteine with iodoacetamide blocked the enzyme activity without any effect on cell adhesion; 2) the addition of Mg2+ did not induce the enzyme activity, but it was as effective as Ca2+ for cell adhesion; 3) the addition of NH4+ inhibited the enzyme activity but did not affect the cell adhesion significantly. The integrins involved in these cell adhesions are quite different. In the case of FXIIIa, alpha vbeta3 and alpha5beta1 integrins are involved and consequently the RGD peptide substantially inhibited the adhesion. On the other hand, the cell adhesion to TGc is mediated by alpha4beta1 integrin but not alpha5beta1; a CS-1 peptide, which represents the binding site of fibronectin to alpha4beta1 integrin, completely inhibited the cell adhesion to TGc. It is possible that TGc and FXIIIa may mediate cell adhesion under different physiological and pathological situations.     

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.     

Characterizing the specificity of activated Factor XIII for glutamine-containing substrate peptides

Activated Factor XIII (FXIIIa) is a transglutaminase that catalyzes the formation of gamma-glutamyl-varepsilon-lysine crosslinks in the fibrin network. To better understand the source of FXIIIa substrate specificity, Q-containing substrates based on beta-casein, K9-peptide, and alpha(2)-antiplasmin were characterized. alpha(2)AP (1-15, Q2, Q4) and alpha(2)AP (1-15, Q2, Q4N, K12R) are highly promising peptide models since they exhibited k(cat)/K(m) values comparable to intact beta-casein. In the absence of a lysine-like donor, FXIIIa could promote deamidation of a reactive Q to an E and solution NMR served as an effective strategy for monitoring this reaction. A tendency toward deamidation allowed greater investigations of the alpha(2)-antiplasmin based peptides. FXIIIa preferentially selects the Q2 residue for carrying out crosslinking processes. The E3 and Q4 provide supporting roles in binding. When a crosslinking reaction occurs at Q2, the Q4 position is sterically blocked from reactivity. By contrast, deamidation of Q2 to E2 allows, for the first time, observation of reactivity at Q4. The K12 position provides an additional favorable site of interaction with the FXIIIa surface. The sensitivity of alpha(2)AP (1-15, Q2, Q4) to amino acid changes at Q2, Q4, and K12 suggests the importance of individual FXIIIa subsites that are controlled by chemical environment and sterics.     

Perturbations in factor XIII resulting from activation and inhibition examined by solution based methods and detected by MALDI-TOF MS

Factor XIII can be activated proteolytically by thrombin cleavage of the activation peptide or non-proteolytically by exposure to 50 mM Ca2+. The resultant transglutaminase cross-links Q and K residues within the noncovalently associated fibrin clot. Hydrogen deuterium exchange coupled with MALDI-TOF MS demonstrated that FXIII activation protects regions within the beta sandwich (98-104) and the beta barrel 1 (526-546) from deuterium, while exposing the potential Q substrate recognition site (220-230) to deuteration (Turner, B. T., Jr., and Maurer, M. C. (2002) Biochemistry 41, 7947-7954). Chemical modification indicated the availability of several residues upon activation including K73, K221, C314, and C409 (Turner, B. T., Jr., Sabo, T. M., Wilding, D., and Maurer, M. C. (2004) Biochemistry 43, 9755-9765). In the current work, activations of FXIII by IIa and by Ca2+ as well as FXIIIa inhibition by the K9 DON peptide (with the Q isostere 6-diazo-5-oxo-norleucine) and iodoacetamide were further examined. New findings unique for FXIIIaIIa included alkylation of C238 and C327, acetylation of K68, and increased proteolysis of 207-214. By contrast, FXIIIaCa led to increased proteolysis of 73-85 and 104-125 and to a loss of K129 acetylation. The FXIIIa inhibitors K9 DON and iodoacetamide both promoted even greater protection from deuteration for the beta sandwich (98-104) and beta barrel 1 (526-546). Interestingly, only K9 DON was able to block modification of catalytic core C409 near the dimer interface. The solution based approaches reveal that activation and inhibition lead to local and long range effects to FXIII(a) and that many are influenced by Ca2+ binding. Important glimpses are being provided on FXIIIa allostery and the presence of putative FXIIIa exosites.     

PEGylation of lysine residues reduces the pro-migratory activity of IGF-I

Background

The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some of these functions involve regulation by the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, the functions and exact nature of these interactions remain incomplete.

Methods

IGF-I variants PEGylated at lysines K27, K65 and K68, were assessed for binding to IGFBPs using BIAcore, and for phosphorylation of the IGF-IR. Furthermore, functional consequences of PEGylation were investigated using cell viability and migration assays. In addition, downstream signaling pathways were analyzed using phospho-AKT and phospho-ERK1/2 assays.

Results

IGF-I PEGylated at lysines 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) was employed. Receptor phosphorylation was similarly reduced 2-fold with PEG-K65 and PEG-K68 in 3T3 fibroblasts and MCF-7 breast cancer cells, whereas PEG-K27 showed a more than 10- and 3-fold lower activation for 3T3 and MCF-7 cells, respectively. In addition, all PEG-IGF-I variants had a 10-fold reduced association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes, whereas cell viability was fully preserved. Analysis of downstream signaling revealed that AKT was preferentially affected upon treatment with PEG-IGF-I variants whereas MAPK signaling was unaffected by PEGylation.

Conclusion

PEGylation of IGF-I has an impact on cell migration but not on cell viability.

General significance

PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on receptor interaction as well as key extracellular proteins such as VN and IGFBPs.     

Tissue transglutaminase acylation: Proposed role of conserved active site Tyr and Trp residues revealed by molecular modeling of peptide substrate binding

Transglutaminases (TGases) catalyze the cross-linking of peptides and proteins by the formation of gamma-glutamyl-epsilon-lysyl bonds. Given the implication of tissue TGase in various physiological disorders, development of specific tissue TGase inhibitors is of current interest. To aid in the design of peptide-based inhibitors, a better understanding of the mode of binding of model peptide substrates to the enzyme is required. Using a combined kinetic/molecular modeling approach, we have generated a model for the binding of small acyl-donor peptide substrates to tissue TGase from red sea bream. Kinetic analysis of various N-terminally derivatized Gln-Xaa peptides has demonstrated that many CBz-Gln-Xaa peptides are typical in vitro substrates with K(M) values between 1.9 mM and 9.4 mM, whereas Boc-Gln-Gly is not a substrate, demonstrating the importance of the CBz group for recognition. Our binding model of CBz-Gln-Gly on tissue TGase has allowed us to propose the following steps in the acylation of tissue TGase. First, the active site is opened by displacement of conserved W329. Second, the substrate Gln side chain enters the active site and is stabilized by hydrophobic interaction with conserved residue W236. Third, a hydrogen bond network is formed between the substrate Gln side chain and conserved residues Y515 and the acid-base catalyst H332 that helps to orient and activate the gamma-carboxamide group for nucleophilic attack by the catalytic sulphur atom. Finally, an H-bond with Y515 stabilizes the oxyanion formed during the reaction.     

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.     

Cloning and Sequence Analysis of a cDNA Encoding Salmon (Onchorhynchus keta) Liver Transglutaminase

We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys²⁷², His³³¹, and Asp³⁰¹) were found in the highly conserved region, but the region surrounding Tyr⁵¹¹, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These findings suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.     

Characterization of insulin-like growth factor (IGF) binding proteins and their role in modulating IGF-I action in BHK cells.

We have found that over one-half of the total cell surface 125I-insulin-like growth factor I (IGF-I) binding to BHK cells represents binding to IGF binding proteins (IGFBPs) rather than to the IGF-I receptor. In addition to a number of secreted IGFBPs, we have now characterized two cell-associated IGFBPs with unique characteristics. The cell-associated IGFBPs have molecular weights of 30,000 (30K) and 25,000 (25K), as determined by the Western ligand blot technique. IGFBP-30K is located at the cell surface and can be readily labeled by affinity cross-linking with 125I-IGF-I. Surface expression of IGFBP-30K increases 5.4 +/- 1.2-fold (n = 11) with serum starvation. This induction is fully evident by 4 h, plateauing by 24 h, and is completely inhibitable by cycloheximide. The fasting-induced increase in IGFBP-30K is inhibited by IGF-I and by des-IGF-I and, to a lesser extent, by insulin. Unlike cell-associated IGFBP-30K, secretion of IGFBP was stimulated (6.8 +/- 0.5-fold, n = 2) by IGF-I, whereas IGFBP secretion was inhibited 54% by insulin. These results demonstrate coordinate regulation of IGFBP by serum starvation and IGF-I, such that at low concentrations of IGF-I, cell surface binding protein increases whereas binding protein secretion decreases. At high concentrations of IGF-I, IGFBP secretion increases and cell surface IGF-I receptor, as well as IGFBP, decreases. Taken together, these regulatory events regulate the availability of IGF-I for biologic signalling.     

Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I

Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF- I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors.     

Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis

Introduction

The microenvironment surrounding inflamed synovium leads to the activation of fibroblast-like synoviocytes (FLSs), which are important contributors to cartilage destruction in rheumatoid arthritic (RA) joints. Transglutaminase 2 (TG2), an enzyme involved in extracellular matrix (ECM) cross-linking and remodeling, is activated by inflammatory signals. This study was undertaken to assess the potential contribution of TG2 to FLS-induced cartilage degradation.

Methods

Transglutaminase (TGase) activity and collagen degradation were assessed with the immunohistochemistry of control, collagen-induced arthritic (CIA) or TG2 knockdown (shRNA)-treated joint tissues. TGase activity in control (C-FLS) and arthritic (A-FLS) rat FLSs was measured by in situ 5-(biotinamido)-pentylamine incorporation. Invadopodia formation and functions were measured in rat FLSs and cells from normal (control; C-FLS) and RA patients (RA-FLS) by in situ ECM degradation. Immunoblotting, enzyme-linked immunosorbent assay (ELISA), and p3TP-Lux reporter assays were used to assess transforming growth factor-β (TGF-β) production and activation.

Results

TG2 and TGase activity were associated with cartilage degradation in CIA joints. In contrast, TGase activity and cartilage degradation were reduced in joints by TG2 knockdown. A-FLSs displayed higher TGase activity and TG2 expression in ECM than did C-FLSs. TG2 knockdown or TGase inhibition resulted in reduced invadopodia formation in rat and human arthritic FLSs. In contrast, increased invadopodia formation was noted in response to TGase activity induced by TGF-β, dithiothreitol (DTT), or TG2 overexpression. TG2-induced increases in invadopodia formation were blocked by TGF-β neutralization or inhibition of TGF-βR1.

Conclusions

TG2, through its TGase activity, is required for ECM degradation in arthritic FLS and CIA joints. Our findings provide a potential target to prevent cartilage degradation in RA.     

Transglutaminase 2 mediates polymer formation of I-kappaBalpha through C-terminal glutamine cluster

Recently we reported that transglutaminase 2 (TGase 2) activates nuclear factor-kappaB (NF-kappaB) independently of I-kappaB kinase (IKK) activation, by inducing cross-linking and protein polymer formation of inhibitor of nuclear factor-kappaBalpha (I-kappaBalpha). TGase 2 catalyzes covalent isopeptide bond formation between the peptide bound-glutamine and the lysine residues. Using matrix-assisted laser desorption ionization time-of-flight mass spectra analysis of I-kappaBalpha polymers cross-linked by TGase 2, as well as synthetic peptides in an in vitro competition assay, we identified a glutamine cluster at the C terminus of I-kappaBalpha (amino acids 266-268) that appeared to play a key role in the formation of I-kappaBalpha polymers. Although there appeared to be no requirement for specific lysine residues, we found a considerably higher preference for the use of lysine residues at positions 21, 22, and 177 in TGase 2-mediated cross-linking of I-kappaBalpha. We demonstrated that synthetic peptides encompassing the glutamine cluster at amino acid positions 266-268 reversed I-kappaBalpha polymerization in vitro. Furthermore, the depletion of free I-kappaBalpha in EcR/TG cells was completely rescued in vivo by transfection of mutant I-kappaBalphas in glutamine sites (Q266G, Q267G, and Q313G) as well as in a lysine site (K177G). These findings provide additional clues into the mechanism by which TGase 2 contributes to the inflammatory process via activation of NF-kappaB.     

Cross-linking of wild-type and mutant alpha 2-antiplasmins to fibrin by activated factor XIII and by a tissue transglutaminase

Human alpha(2)-antiplasmin (alpha(2)AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in alpha(2)AP, only Gln(2) is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (k(cat)/K(m)((app))) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type alpha(2)AP (WT-alpha(2)AP) and Q2A mutant alpha(2)AP (Q2A-alpha(2)AP). [(14)C]Methylamine incorporation showed the k(cat)/K(m)((app)) of FXIIIa to be 3-fold greater than that of tTG for WT-alpha(2)AP. With FXIIIa or tTG catalysis, [(14)C]methylamine was incorporated into Q2A-alpha(2)AP, indicating that WT-alpha(2)AP has more than one Gln cross-linking site. To identify transglutaminase-reactive sites in WT-alpha(2)AP or Q2A-alpha(2)AP, each was labeled with 5-(biotinamido)pentylamine by FXIIIa or tTG catalysis. After each labeled alpha(2)AP was digested by trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gln residues were labeled with the following reactivities: Gln(2) > Gln(21) > Gln(419) > Gln(447). Q(2)A-alpha(2)AP was also labeled at Gln(21) > Gln(419) > Gln(447), but became cross-linked to fibrin by FXIIIa or tTG at approximately one-tenth the rate for WT-alpha(2)AP. These results show that alpha(2)AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gln substrate sites in alpha(2)AP and yields the same order of reactivities.     

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.     

Liver mRNA expression of IGF-I and IGFBPs in adult undernourished diabetic rats.

To investigate the role played by factors other than GH, such as nutrients and insulin, on IGF-I secretion, adult male rats of 200 g.b.w. were food-restricted for 7 days and then made diabetic by streptozotocin administration (UD). Different groups of UD rats were submitted to the following four day treatments: left untreated (UD), refed (UD+R), treated with insulin (UD+I), or a combination of both refeeding and insulin (UD+R+I). Serum concentration of IGF-I and liver mRNA expression of IGF-I, IGF-binding proteins and GH receptor were measured. Insulin treatment alone partially recovered liver IGF-I and IGFBPs mRNA expression, while refeeding alone had no effect. Only a combination of both insulin and refeeding recovered both parameters. Contrary to the results obtained with a longer period of recovery, these experiments show that serum and mRNA expression of IGF-I and IGFBPs in adult undernourished diabetic rats can be restored by insulin and nutrients administration with no prior restoration of serum and pituitary GH to control values and no compensatory changes in GH receptor gene expression.     

Endogenous IGF-I regulates IGF binding protein production in human intestinal smooth muscle cells

Human intestinal smooth muscle in culture produces insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3, IGFBP-4, and IGFBP-5, which modulate the effects of IGF-I. This study examined the regulation of IGFBP production by endogenous IGF-I. R3-IGF-I, an agonist unaffected by IGFBPs, elicited concentration-dependent increase in growth, measured by [(3)H]thymidine incorporation, and production of IGFBP-3, IGFBP-4, and IGFBP-5, measured by Western blot. Antagonists of the IGF-I receptor, IGF-I Analog or monoclonal antibody 1H7, elicited concentration-dependent inhibition of growth and decrease in IGFBP-3, IGFBP-4, and IGFBP-5 production, implying that endogenous IGF-I stimulated growth and IGFBP production. R3-IGF-I-induced increase in IGFBP-3, IGFBP-4, and IGFBP-5 production was partially inhibited by a mitogen-activated protein (MAP) kinase or a phosphatidylinositol-3-kinase (PI 3-kinase) inhibitor and abolished by the combination. We conclude that endogenous IGF-I stimulates growth and IGFBP-3, IGFBP-4, and IGFBP-5 production in human intestinal smooth muscle cells. Regulation of IGFBP production by IGF-I is mediated by activation of distinct MAP kinase and PI 3-kinase pathways, the same pathways through which IGF-I stimulates growth.     

Structural determinants for binary and ternary complex formation between insulin-like growth factor-I (IGF-I) and IGF binding protein-3.

Structural analogs of recombinant human insulin-like growth factor-I (IGF-I), with alterations to each of the B, C, A, and D domains, have been tested for their ability to form binary complexes with IGF-binding protein-3 (IGFBP-3) and ternary complexes with IGFBP-3 and the acid-labile subunit (alpha-subunit). Two functionally distinct regions of IGF-I have been identified. The first, involving residues 3 and 4 and the alpha-helix between residues 8 and 18 of the B-domain, as well as residues 49-51 in the A-domain, appears important for IGFBP-3 binding, such that substitution of these residues results in decreased binary complex available for alpha-subunit binding. The second region, distal to the IGFBP-3-binding epitope and primarily involving the D-domain and B-domain near residue 24, with some involvement of the C-domain, appears slightly inhibitory to binary complex formation, such that analogs with a truncated D-domain or with a Gly4 bridge substituted for the C-domain show enhanced binding to IGFBP-3. However, binary complexes formed from these analogs bind the alpha-subunit with reduced affinity, the effect being most marked when substitution of the C-domain, or replacement of Tyr24, is superimposed on D-domain truncation. It is concluded that although the alpha-subunit does not itself bind IGF-I, its interaction with IGFBP-3 in the ternary complex is dependent on structural determinants on IGF-I distal to the IGFBP-3 binding domain.     

Characterization of pituitary IGF-I receptors: modulation of prolactin and growth hormone

There have been no studies in any vertebrate that have localized insulin-like growth factor (IGF)-I receptors in prolactin (PRL) cells or that have correlated pituitary binding to the potency of IGF-I in regulating both PRL and growth hormone (GH) secretion. We show that IGF-I binds with high affinity and specificity to the pituitary gland of hybrid striped bass (Morone saxatilis x M. chrysops). IGF-I and IGF-II were equipotent in inhibiting saturable (125)I-IGF-I binding, whereas insulin was ineffective. IGF-I binds with similar affinity to the rostral pars distalis (>95% PRL cells) as the whole pituitary gland and immunohistochemistry colocalizes IGF-I receptors and PRL in this same region. Des(1-3)IGF-I, a truncated analog of IGF-I that binds with high affinity to IGF-I receptors but weakly to IGF-I binding proteins (IGFBPs), showed a similar inhibition of saturable (125)I-IGF-I binding, but it was more potent than IGF-I in stimulating PRL and inhibiting GH release. These results are the first to localize IGF-I receptors to PRL cells, correlate IGF-I binding to its efficacy in regulating GH and PRL secretion, as well as demonstrate that IGFBPs may play a significant role in modulating the disparate actions of IGF-I on PRL and GH secretion.     

Mammary Specific Transgenic Over-expression of Insulin-like Growth Factor-I (IGF-I) Increases Pig Milk IGF-I and IGF Binding Proteins,with no Effect on Milk Composition or Yield

IGF-I regulates lactation by stimulating mammary mitogenesis, inhibiting apoptosis, and partially mediating the effects of growth hormone on lactogenesis. Herein, lactation performance during first and second parity was assessed in transgenic swine (TG) that over-expressed human IGF-I in milk under the control of the bovine α-lactalbumin promoter, regulatory regions and signal peptide coding sequence. Milk samples were collected throughout lactation (farrowing to d24) from TG sows and non-transgenic littermates (CON) and IGF-I, IGF-II, and IGFBP determined. Colostral (<24 h postpartum) IGF-I content was 26-fold greater (p < 0.001) in TG sows (949 ± 107 μg/L; range 228–1600 μg/L) than CON (36 ± 17.8 μg/L) and was 50- to 90-fold greater (p < 0.001) in mature milk (d2-24 postpartum). There was no effect of parity on milk IGF-I content. Milk IGF-II concentration was unaffected by IGF-I over-expression. Low molecular weight IGFBP (IGFBP-2 and -5) in the milk of TG sows were higher (p = 0.02) than CON in the early postpartum period, but did not differ in mature milk. Milk yield, determined by weigh-suckle-weigh, was similar in TG and CON as was litter weight gain. Milk nutrient composition was not significantly affected by IGF over-expression. Thus, mammary specific transgenic over-expression of IGF-I significantly increased milk IGF-I and IGFBP content, but did not impact lactation performance in swine.