Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have characterized the insulin-like growth factor-binding proteins (IGF-BPs) released by isolated sheep thyroid epithelial cells. Thyroid follicles were isolated with collagenase and cultured in Coon's modified F-12 M (0H medium) supplemented with insulin, cortisol, transferrin, glycyl-histidyl-lysine and somatostatin (5H medium) and TSH (6H medium). Conditioned 0H medium specifically bound both 125I-labelled IGF-I and -II, although binding capacity was reduced following acid-gel filtration to separate endogenous IGF-BP complexes, suggesting some destruction of BPs. The binding of 125I-labelled IGF-I or -II to conditioned (0H) medium was progressively displaced by increasing amounts of unlabelled homologous peptides, while fractionation on concanavalin A-Sepharose showed that the IGF-BPs consisted of both glycoprotein and non-glycoprotein components. The molecular sizes of the IGF-BPs were resolved by separation of 0H medium on SDS-PAGE and ligand blot analysis with 125I-labelled IGF-I or -II. Conditioned medium contained four specific binding species for IGF-II of 19, 30, 38 and 46 kDa; all but the smallest also binding radiolabelled IGF-I. Prior fractionation on concanavalin A-Sepharose showed that the 46 kDa binding species was a glycoprotein. Competition studies with increasing concentrations of unlabelled IGF-I or -II during ligand blotting suggested that the 46 and 30 kDa binding species had a greater affinity for IGF-II than IGF-I, while the 38 kDa had a greater relative affinity for IGF-I. Incubation of cells in 5H medium reduced the abundance of the 46 kDa binding protein, while incubation in 6H medium decreased the release of all binding protein species. Results show that isolated thyroid follicles released several forms of IGF-BP with differing relative affinities for IGF-I and -II. Gross changes seen in the presence of BPs between 0H, 5H and 6H media suggest acute hormonal control of release.
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PMID:Characterization of insulin-like growth factor-binding proteins secreted by isolated sheep thyroid epithelial cells. 169 63

Insulin-like growth factors (IGF-I and IGF-II) are endocrine and autocrine factors affecting bone growth and metabolism. Binding proteins for IGFs (IGFBPs) are synthesized by the target tissues of IGF actions. Thus, IGFBPs may act as modulators for the biological functions of IGFs. We have characterized the rat IGFBPs (rIGFBPs) and studied their regulation by 17 beta-estradiol (beta E2) and human GH (hGH) in rat osteoblast-like (ROB) cell cultures. ROB cells were prepared from 19- to 20-day fetal rat calvariae by sequential collagenase digestion and studies were performed on the serum and phenol red-free conditioned medium of confluent cultures. [125I]IGF-I ligand blots showed that the major rIGFBP in the ROB is a nonglycosylated protein of 31 kilodaltons. This protein was immunoprecipitated by a specific antibody to rIGFBP-2 and messenger RNA for rIGFBP-2 was detected by RNA hybridization indicating that the rIGFBP-2 is the major rIGFBP of ROB. A minor band at 24 kilodaltons is likely to be the rat homologue of the newly isolated inhibitory IGFBP-4. The predominant glycosylated adult form of rIGFBPs of rat serum, rIGFBP-3, was undetectable. When cultures were treated with beta E2 for 2 days, there was a dose-dependent biphasic response which showed an inhibition of the rIGFBP-2 at low doses of 10(-11) to 10(-9) M and a stimulation at 10(-6) M. These changes in rIGFBP-2 parallel the changes in the endogenous IGF-I level. rIGFBP-2 level was not affected by 17 alpha-estradiol at the same concentration range. hGH, on the other hand stimulated the levels of rIGFBP-2 and rIGFBP-4 at doses ranging from 10(-11) to 10(-9) M without changing the IGF-I secretion. The alteration of the rIGFBPs by beta E2 and hGH suggests a role for these hormones in bone by modulating the biological functions of IGFs via their binding proteins.
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PMID:Further characterization of insulin-like-growth factor binding proteins in rat osteoblast-like cell cultures: modulation by 17 beta-estradiol and human growth hormone. 170 37

Isolated sheep thyroid follicles release specific insulin-like growth factor-binding proteins (IGFBPs). Since IGFBPs can modulate IGF bioactivity, at least in vitro, their presence in thyroid tissue may influence synergistic interactions between TSH and endogenous IGF-I or -II which are known to control both thyroid growth and function. We have examined the hormonal control of IGFBP release in relation to iodine organification. Sheep thyroid follicles were isolated by incubation with collagenase and differential centrifugation, grown in Coon's modified Ham's F12M medium with the addition of transferrin, glycylhistidyl-lysine, somatostatin (3H), TSH, cortisol and insulin (6H), and maintained in OH (hormone-free) or 3H medium with or without further supplements for 48 h. Conditioned culture medium was separated by 8% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 125I-labelled IGF-II followed by autoradiography (ligand blot). Additionally, the radioactive bands were cut from the filters and quantified by gamma-spectrometry. Iodine organification was assessed by incubation of follicles with 10(6) c.p.m. Na125I for 3 h before washing, solubilization in 0.1 mol NaOH/l and the precipitation of organified radioisotope with 10% (v/v) trichloroacetic acid. Cells conditioned in OH or 3H medium released specific IGFBPs of 46, 34, 28 and 19 kDa on ligand blot analysis. The proteins of 34 and 19 kDa were immunopositive on Western blot analysis using anti-bovine IGFBP-2 antiserum. The 46-kDa IGFBP was retained by Concanavalin A-Sepharose chromatography and demonstrated to be glycoprotein. This is probably ovine IGFBP-3. The addition of TSH, or TSH plus cortisol to OH or 3H medium significantly decreased the 125I-labelled IGF-II associated with the 34- and 28-kDa IGFBP species. All IGFBP species were substantially reduced in 6H medium, which was predominantly due to the effects of TSH and cortisol. When total 125I-labelled IGF-II associated with IGFBPs was considered, a significant (P less than 0.01) inverse correlation existed between IGFBP activity and iodine organification in the same cultures; the latter being greatest in OH or 3H medium supplemented with TSH and cortisol. None of these hormone additions altered the endogenous release of IGF-II by the cells. These results suggest that endogenous IGFs, under hormonal control, may modulate the action of endogenous IGF in the regulation of thyroid function.
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PMID:Hormonal regulation of insulin-like growth factor (IGF)-binding proteins secreted by isolated sheep thyroid epithelial cells: relationship with iodine organification. 171 78

Insulin is a major regulatory hormone for optimal tissue growth and function in utero. Its continued availability to the growing fetus depends on increasing islet cell mass. The purpose of the study was to examine the interactions between nutrient availability and insulin-like growth factor (IGF) release and action during DNA synthesis by isolated fetal rat islets of Langerhans. Specifically, we wished to determine (a) whether the availability of glucose or total amino acids altered the release of endogenous IGF-I or -II, (b) if both IGF-I and -II were effective mitogens for pancreatic beta-cells, (c) whether islets released IGF-binding proteins (IGFBPs) and their possible regulation by nutrient availability and (d) how IGFBPs might regulate the ability of IGFs to alter islet DNA synthesis. Islets of Langerhans were isolated from fetal rat pancreata on day 22 of gestation by collagenase digestion. Islets enriched in beta-cells following a 5-day preincubation regime were maintained in various concentrations of glucose (1.4-16.7 mmol/l) or amino acids (x1- x3 total concentrations), with or without exogenous IGF-I, -II, IGFBP-1 or IGFBP-2. The release of insulin and endogenous IGF-I and -II were each determined by radioimmunoassay, and IGFBP release characterized by Western ligand blot analysis. DNA synthesis was measured by the incorporation of [3H]thymidine. Isolated islets demonstrated an increased release of insulin in response to increasing amounts of both glucose and amino acids, demonstrating functional viability. Both classes of nutrients also increased the DNA synthetic rate of islets. Islets released almost twice as much IGF-II (0.22 +/- 0.08 nmol/l, mean +/- S.E.M., n = 4) as IGF-I (0.14 +/- 0.03 nmol/l) in cultures containing 8.7 mmol glucose/l and x1 amino acids. Lesser or greater concentrations of glucose did not alter the release of either IGF, but the release of IGF-II was significantly increased (0.53 +/- 0.08 nmol/l, P < 0.01) in the presence of x2 amino acids. Exogenous IGF-I was fivefold more active in stimulating DNA synthesis by islets (half maximal concentration (ED50) 1.6 +/- 0.4 nmol/l, n = 3) than was IGF-II (ED50 8.1 +/- 0.6 nmol/l), regardless of glucose concentration. Isolated islets released four species of IGFBP with molecular sizes of approximately 19, 25, 35 and 46 kDa respectively. The 35 kDa form was identified by Western immunoblot as IGFBP-2. Increasing the glucose concentration between 1.4 mmol/l and 16.7 mmol/l caused a dose-related increase in the release of the 19, 25 and 35 kDa IGFBP species.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interactions of nutrients, insulin-like growth factors (IGFs) and IGF-binding proteins in the regulation of DNA synthesis by isolated fetal rat islets of Langerhans. 750 87

Prostaglandin E2 (PGE2) can stimulate collagen synthesis in bone at low concentrations or in the presence of cortisol. Moreover, cortisol inhibits and PGE2 stimulates the production of insulin-like growth factor (IGF-I) in cultured osteoblastic cells. Therefore, we examined the role of IGF-I in the response to PGE2. In 96-h fetal rat calvarial organ cultures, PGE2 increased, and cortisol and indomethacin decreased the medium IGF-I concentration, suggesting that both exogenous and endogenous PGs regulate IGF-I production. In the presence of cortisol, the stimulatory effects of PGE2 on medium IGF-I and incorporation of [3H] proline into collagenase-digestible protein were highly correlated (r = 0.95). When exogenous IGF-I (30 nM) was added, the stimulatory effect of PGE2 was abrogated in the absence, but not the presence, of cortisol. When we added IGF-binding proteins, which blocked the effects of IGF-I and IGF-II, collagenase-digestible protein labeling was decreased in control and cortisol-treated cultures, whereas the stimulatory effect of PGE2 was reduced, but not abrogated. We conclude that endogenous IGFs play a role in maintaining bone formation in cultured fetal rat calvariae and may mediate in part the anabolic response to PGE2. However, the PGE2 response probably involves additional IGF-independent pathways.
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PMID:Effects of prostaglandin E2 on bone formation in cultured fetal rat calvariae: role of insulin-like growth factor-I. 769 77

Insulin-like growth factor-I (IGF-I) and IGF-II are among the most prevalent growth factors secreted by bone cells and are presumed to act as autocrine regulators of bone formation. We recently demonstrated that IGFs inhibit bone collagen degradation, and we postulated that they may either inhibit the expression of interstitial collagenase or stimulate the synthesis of tissue inhibitors of metalloproteinase-1 (TIMP-1), -2, or -3. We tested the effects of IGF-I and -II on collagenase and TIMP-1, -2, and -3 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Steady state messenger RNA (mRNA) levels were determined by Northern blot analysis, and collagenase concentrations were determined in the culture medium by a specific immunoassay. After 2-6 h of treatment, IGF-I and -II decreased collagenase transcripts by up to 80%. IGF-I was a more potent inhibitor than IGF-II, because it was active at doses as low as 10 nM, whereas a dose of 100 nM was required to observe the IGF-II effect. In addition, IGF-I and -II opposed the stimulatory effect of retinoic acid on collagenase transcripts. Immunoreactive collagenase levels were not detectable in control or IGF-treated cultures, but IGF-I and -II decreased the levels induced by retinoic acid by 70-90%. The protein synthesis inhibitor cycloheximide superinduced collagenase transcripts, and IGF-I or -II decreased this mRNA induction to levels similar to, but not lower than, those observed in control cultures. The effects of IGF-I and -II on collagenase transcripts were not modified by the DNA synthesis inhibitor hydroxyurea at 1 mM. Neither IGF-I nor IGF-II modified the expression of TIMP-1, -2, or -3 mRNA in Ob cells. TIMP protein levels were not determined, and our study does not exclude a translational or posttranslational effect of IGF. In conclusion, IGF-I and -II decrease interstitial collagenase transcripts as well as induced protease levels in Ob cells, and this effect may contribute to their inhibitory actions on bone collagen degradation.
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PMID:Insulin-like growth factors inhibit interstitial collagenase synthesis in bone cell cultures. 789 45

The purpose of this study was to develop a primary culture system using serum-free medium for rat placental trophoblast cells and to investigate the factors that control rat placental lactogen-II (rPL-II) secretion in vitro. The placentae of day 13 pregnant rats were dissociated in Medium 199 containing 0.1% collagenase and 0.002% DNAase. Dissociated cells were fractionated into five segments by centrifugation through a 40% Percoll density gradient and incubated on rat tail collagen bed in medium SFM-101 for up to 7 days. Fraction B at the Percoll gradient density of 1.05 g ml-1 was enriched with rPL-II-producing cells and the time course of rPL-II secretion was characterized by a rapid increase in the first 2 days, remaining at high values (mean: 14-16 ng micrograms-1 DNA) for the following 2-3 days and decreasing thereafter. The rPL-II-producing cells from faction B identified by immunocytochemical examination accounted for approximately 69% of total cultured cells and consisted of a few giant cells and polygonal cells. Growth factors (bovine insulin, 0.1-20 micrograms ml-1; recombinant human insulin-like growth factor (IGF)-I, IGF-II, 0.1-1.0 micrograms ml-1; murine epidermal growth factor (EGF), 0.001-10 micrograms ml-1), rat pituitary hormones (rat growth hormone, rat prolactin, 0.1-10 micrograms ml-1) and hypothalamic hormones (human growth hormone-releasing hormone (GHRH), corticotrophin-releasing hormone (CRH), LHRH, 0.1-10 micrograms ml-1) were individually added to the culture medium to investigate the putative factors that directly control rPL-II secretion by the trophoblast cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stimulation of rat placental lactogen-II (rPL-II) secretion by cultured trophoblasts by insulin: development of a rat placental cell culture system and effects of peptide hormones on rPL-II secretion in vitro. 810 35

Insulin-like growth factors I and II are peptides with a structural homology for proinsulin, and are involved in hepatocyte proliferation. IGF-I and IGF-II, however, have different metabolic roles, and their mechanisms of action are incompletely known. We hypothesized that IGF-I and IGF-II act by different signal transduction pathways. To test this hypothesis, hepatocytes from 200 g male Sprague-Dawley rats were isolated by a two-step collagenase perfusion technique and plated at a density of 10(5) cells/16 mm Primaria plate. Proliferation was measured by [3H]thymidine ([3H]thy) incorporation into DNA, and an autoradiographic nuclear labeling index (LI). To analyze signal transduction, cyclic AMP (cAMP) levels were measured 5 min after addition of reagents by a radioimmunoassay. Reagents (doses) used were: IGF-I (2 nM), IGF-II (2 nM), the inhibitory peptide somatostatin-14 (SS14) (10 nM), and the adenylyl cyclase antagonist dideoxyadenosine (DDA) (10 microM). A summary of the findings is as follows: (1) IGF-I stimulates [3H]thy, LI and cAMP accumulation. (2) IGF-II stimulates [3H]thy and LI but not cAMP; (3) IGF-I but not IGF-II effects are inhibited by SS14 and DDA. We conclude that the hepatotrophic effects of IGF-I and IGF-II occur by different mechanisms: IGF-I is cAMP-dependent, IGF-II is cAMP-independent.
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PMID:Divergent mechanisms of insulin-like growth factor I and II on rat hepatocyte proliferation. 857 Aug 60

We recently demonstrated upregulation of insulin-like growth factor I (IGF-I) binding sites in the smooth muscle layer of inflamed rat colon. The increase in binding sites was due to increased expression of IGF binding proteins (IGFBPs), which modulate the effects of IGF. To further study the role of IGF in the colon, we investigated whether cultured colonic smooth muscle cells (SMC) express IGF-I receptors and IGFBPs. SMC were isolated by collagenase digestion from rat colonic smooth muscle and grown in primary culture. Equilibrium binding experiments using (125)I-labeled IGF-I showed the presence of an IGF-I receptor with a dissociation constant of 1.96 nM and a maximal binding constant of 53,000 receptors/cell. Competition binding studies with IGF-II and insulin, together with chemical cross-linking experiments, corroborated this conclusion. Western ligand blotting of conditioned medium and Northern analysis of total RNA demonstrated that the cells expressed and secreted IGFBP-4, -5, and -3 with molecular masses of 25, 31, and 45 kDa, respectively. These results, together with our in vivo studies in the rat, support a role for IGF in tissue fibrosis and stricture formation during chronic intestinal inflammation.
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PMID:Expression of insulin-like growth factor I receptors and binding proteins by colonic smooth muscle cells. 912 68

We examined and compared the actions of IGF-I and -II on the release of insulin from isolated, intact rat islets of Langerhans within a perifusion system. Islets were isolated from adult male rats by collagenase digestion and Ficoll gradient separation, and were maintained in tissue culture for 48 h before perifusion. Following an equlibration period, islets were perifused with medium containing 2.7 mM glucose from 0 to 30 min. and 2.7, 11.1 or 16.7 mM glucose from 30 to 90 min. All chambers then received medium with 2.7 mM glucose from 90 to 120 min. Various doses (6.7-53 nM) of IGF-1, des(1-3) IGF-I or IGF-II were given either as a pulse between 30 and 35 min, or continuously from 30 to 90 min. Insulin was measured in effluent medium by RIA. When 11.1 mM glucose was administered after 30 min an immediate increase in insulin release occurred, from a baseline of 1-3 pmol/fraction to approximately 7 pmol/ fraction. The elevated rate of release was maintained until 90 min, and fell when the glucose concentration was lowered. Glucose at 16.7 mM was a less effective insulin secretogogue than was 11.1 mM. When islets received a pulse infusion of IGF-I (13.3 nM) at 30 min in the presence of 11.1 mM glucose, a statistically significant increase (p < 0.005) in insulin release occurred, of approximately 10 pmol/fraction in excess of that seen with glucose alone. The IGF-1-stimulated insulin release was still higher than controls at 115 min. When the concentration of IGF-I was altered between 6.7 nM and 53 nM, maximum insulin release was achieved with 13.3 nM IGF I, both lower and higher concentrations being less effective. A significant inhibition of insulin release occurred with 53 nM IGF-I compared with glucose alone. IGF-II (13.3 nM) did not significantly increase insulin release, while 53 nM IGF-II significantly inhibited release of insulin relative to controls. Des(1-3) IGF-I (13.3 nM), which has a reduced binding affinity for IGF-binding proteins (IGFBPs), administered with 11.1 mM glucose caused an immediate increase in insulin release, which fell to control values within 30 min. Western ligand blot analysis identified four IGFBP species in perifused islets, of 46 kDa, 35 kDa, 28 kDa, and 19 kDa respectively, of which the 28 kDa species was identified immunologically as IGFBP-1. When IGF-I was administered continuously from 30 to 90 min it inhibited glucose-stimulated insulin release at all concentrations used. The results suggest that under perfusion conditions, IGF-I can act both as a potent insulin secretogogue, augmenting the actions of glucose, and as an inhibitor of insulin release, depending on concentration and kinetics of administration.
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PMID:IGF-I has a dual effect on insulin release from isolated, perifused adult rat islets of Langerhans. 913 65


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