Absence Of Insulin-like Growth Factor Research Articles

Ligustrazine prevents basilar artery remodeling in two-kidney-two-clip renovascular hypertension rats via suppressing PI3K/Akt signaling

ObjectiveIn the present study, we used a two-kidney-two-clip (2k2c) stroke-prone renovascular hypertension rat model (RHRSP) to investigate the protective effects of ligustrazine (TMP) on cerebral arteries and to examine PI3K/Akt pathway behavior under this protection. MethodsThe cerebral artery remodeling was induced by 2k2c-induced renovascular hypertension. Brain basilar artery tissues were isolated and their histological changes were detected through H&E and EVG staining, α-SMA IHC staining, and transmission electron microscopy at four, eight, and twelve weeks after 2k2c surgery, both with and without TMP treatment. Meanwhile, the ET-1, Ang II, and NO levels in basilar arteries and plasma were determined. Furthermore, the PTEN expression and the activation of PI3K/Akt in basilar artery tissues were detected through IHC and Western Blot. In addition, the primary basilar artery smooth muscle cells (BASMCs) were cultured and TMP protection of BASMCs stimulated with ET-1/Ang II in the presence or absence of insulin-like growth factor 1 (IGF-1) was determined. ResultsTMP attenuated basilar artery remodeling, decreased ET-1 and Ang II levels and increased NO level in basilar arteries and plasma of RHRSP rats. Moreover, TMP reduced BASMCs proliferation upon ET-1/Ang II stimulation. We also found that TMP could effectively suppress the activation of PI3K/Akt in 2k2c-RHRSP rat basilar artery and ET-1/Ang II stimulated BASMCs. Most importantly, IGF-1, as an activator of PI3K/Akt, could damage the protective effect of TMP. ConclusionsTMP exerts its protective effects and prevents basilar artery remodeling in RHRSP rats at least partly through the inhibition of PI3K/Akt pathway.

Trans-10,cis-12 Conjugated Linoleic Acid Inhibits Insulin-Like Growth Factor-I Receptor Signaling in TSU-Pr1 Human Bladder Cancer Cells

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid; the major isomers are trans-10,cis-12 CLA (t10c12) and cis-9,trans-11 CLA (c9t11). CLA has been demonstrated to exert strong anticarcinogenic effects in a variety of experimental cancer models. We previously observed that CLA (a mixture of isomers) and t10c12 decreased the growth of TSU-Pr1 cells, whereas linoleic acid and c9t11 exerted no effects. In the current study, the mechanisms underlying the t10c12-mediated regulation of the growth of these bladder cancer cells were evaluated. TSU-Pr1 cells were incubated in serum-free medium with various concentrations of t10c12 or c9t11 in the presence or absence of insulin-like growth factor (IGF)-I. The incorporation of [(3)H]thymidine into DNA was decreased, and the number of annexin V-stained cells was increased after t10c12 treatment, whereas c9t11 had no effect on apoptosis or [(3)H]thymidine incorporation. Treatment with exogenous IGF-I alone increased the numbers of viable cells but did not counteract the t10c12-induced growth inhibition of TSU-Pr1 cells. t10c12 effected a dose-dependent reduction in IGF-I receptor (IGF-IR) transcripts and protein levels, whereas c9t11 exerted no effects. Additionally, t10c12 inhibited the IGF-I-induced phosphorylation of IGF-IR, the recruitment of the p85 regulatory subunit of phosphoinositide 3-kinase to IGF-IR, and the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. These results indicate that the inhibition of IGF-IR signaling and the activation of Akt and ERK-1/2 contributed to decreased cell proliferation and increased apoptosis in TSU-Pr1 cells treated with t10c12.

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells

Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5′AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10 μM each), but not ALA (100 μM), increased progesterone secretion ( P < 0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10 -8 M) or FSH (5 × 10 −8 M). The different AMPK subunits, except for γ3, are present in the goat ovary. Treatment with metformin (10 mM), an activator of AMPK, increased AMPK phosphorylation ( P < 0.05) and reduced progesterone secretion by 50% ( P < 0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation ( P < 0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion ( P < 0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

Bile salts regulate proliferation and apoptosis of liver cells by modulating the IGF1 system

Background In different cell types, the insulin-like growth factor 1 and its receptor modulate growth, apoptosis and damage repair in cooperation with estrogen receptors. Aim To evaluate the involvement of the insulin-like growth factor 1 system and estrogen receptors in bile salts modulation of apoptosis/proliferation of hepatocytes and cholangiocytes. Primary cultures of rat hepatocytes and cholangiocytes were exposed to glycochenodeoxycholate or tauro-CDC in the presence or absence of insulin-like growth factor 1 receptor blocking antibody (αIR3), small interfering RNA for insulin-like growth factor 1, 17β-estradiol or estrogen receptor antagonist (ICI 182,780). Proliferation was evaluated by proliferating cell nuclear antigen Western blot and apoptosis by measuring caspase-3 activity or annexin-V. Results In hepatocytes, the insulin-like growth factor 1 receptor blocker enhanced glycochenodeoxycholate-induced apoptosis and caused tauro-CDC to promote apoptosis. 17β-Estradiol or the estrogen receptor antagonist (ICI 182,780) did not influence the apoptotic effect of glycochenodeoxycholate. In cholangiocytes, both glycochenodeoxycholate and tauro-CDC induced proliferation at 100 μM, while they induced apoptosis at 1 mM with a more pronounced effect of glycochenodeoxycholate. Apoptosis induced by 1 mM glycochenodeoxycholate or tauro-CDC in cholangiocytes was enhanced by blocking insulin-like growth factor 1 receptor or by silencing insulin-like growth factor 1. 17β-Estradiol counteracts glycochenodeoxycholate-induced cholangiocyte apoptosis by enhancing insulin-like growth factor 1 secretion and activating the insulin-like growth factor 1 system. Conclusions Modulation of the IGF1 system could represent a potential strategy for the management of bile salts-induced liver injury.

Effects of Hepatocyte Growth Factor on Cyclic Nucleotide-Dependent Signaling and Steroidogenesis in Rat Ovarian Granulosa Cells In Vitro1

Hepatocyte growth factor (HGF) down-modulates FSH-dependent estradiol-17beta (E(2)) production in ovarian granulosa cells in vitro. The mechanisms of action underlying the antiestrogenic effects of HGF are vague, although evidence indicates that HGF may affect cAMP signal transduction in rat granulosa cells. The present study investigated the effects of HGF on FSH-induced steroidogenesis in the presence and absence of insulin-like growth factor I (IGF-I), as well as the actions of HGF within cyclic nucleotide-dependent signal transduction cascades in granulosa cells. Immature rat granulosa cells were incubated with FSH, IGF-I, and HGF. HGF impaired the production of FSH-stimulated and FSH + IGF-I-stimulated E(2) synthesis, as well as FSH + IGF-I-dependent estrone production. Progesterone synthesis was not altered by HGF. HGF suppressed FSH-dependent cAMP content at 24 h, but not at 36 h; cGMP content was stimulated by HGF with and without FSH at 24 h. In the presence of the cyclic nucleotide phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX), FSH-dependent cAMP accumulation was not affected by HGF. The suppressive effect of HGF on FSH-dependent E(2) production was alleviated by IBMX, whereas the HGF-dependent block in FSH + IGF-I-supported E(2) production was not prevented by IBMX. The effects of HGF on cyclic nucleotide PDE activities were manifested in a time-dependent and hormone-dependent manner. FSH-induced cAMP PDE was suppressed by HGF at 24 h but not at 36 h, whereas FSH-dependent cGMP PDE was impaired at 36 h, but not at 24 h. HGF prevented the IGF-I-dependent reduction in FSH-stimulated cAMP-PDE activity at 24 and 36 h, and lowered FSH + IGF-I-stimulated cGMP-PDE activity at 36 h, concomitant with an HGF-dependent increase in cGMP content at 24 h. These data indicate that HGF affects cAMP-directed and cGMP-directed signaling pathways at multiple sites in granulosa cells. These HGF-dependent effects may provide insight for mechanisms of action whereby HGF reduces E(2) secretion by granulosa cells.

Interaction between acid-labile subunit and insulin-like growth factor binding protein 3 expressed in Xenopus oocytes.

The acid-labile subunit (ALS) associates with the insulinlike growth factor (IGF)-I or II, and the IGF binding protein-3 (IGFBP-3) in order to form a 150-kD complex in the circulation. This complex may regulate the serum IGFs by restricting them in the vascular system and promoting their endocrine actions. Little is known about how ALS binds to IGFBP3, which connects the IGFs to ALS. Xenopus oocyte was utilized to study the function of ALS in assembling IGFs into the ternary complexes. Xenopus oocyte was shown to correctly translate in vitro transcribed mRNAs of ALS and IGFBP3. IGFBP3 and ALS mRNAs were injected in a mixture, and their products were immunoprecipitated by antisera against ALS and IGFBP3. Contrary to traditional reports that ALS interacts only with IGF-bound IGFBP3, this study shows that ALS is capable of forming a binary complex with IGFBP3 in the absence of IGF. When cross-linked by disuccinimidyl suberate, the band that represents the ALSIGFBP3 complex was evident on the PAGE. IGFBP3 movement was monitored according to the distribution between the hemispheres. Following a localized translation in the vegetal hemisphere, IGFBP3 remained in the vegetal half in the presence of ALS. However, the mutant IGFBP3 freely diffused into the animal half, despite the presence of ALS, which is different from the wild type IGFBP3. This study, therefore, suggests that ALS may play an important role in sequestering IGFBP3 polypeptides via the intermolecular aggregation. Studies using this heterologous model will lead to a better understanding of the IGFBP3 and ALS that assemble into the ternary structure and circulate the IGF system.

Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors

BACKGROUNDActivated hepatic stellate cells (HSC) are central to the pathogenesis of liver fibrosis, both as a source of fibrillar collagens that characterise fibrosis and matrix degrading metalloproteinases and their tissue...

Insulin-like Growth Factor-binding Protein 5 Complexes with the Acid-labile Subunit: ROLE OF THE CARBOXYL-TERMINAL DOMAIN

We have recently shown that insulin-like growth factor (IGF)-binding protein 5 forms ternary complexes with IGF-I or IGF-II and the acid-labile subunit (ALS) (Twigg, S. M., and Baxter, R. C. (1998) J. Biol. Chem. 273, 6074-6079). Because IGF-binding protein 3 (IGFBP-3) binds to ALS through its basic carboxyl-terminal domain, we tested whether a homologous region present in IGFBP-5 is involved in IGFBP-5 binding to ALS. Chimeric peptides were generated by carboxyl-terminal domain interchange between recombinant human IGF-BP-5 and IGFBP-6, producing two IGFBP peptides designated 5-5-6 and 6-6-5. Determined by immunoprecipitation and by Superose chromatography, 6-6-5 formed ternary complexes, albeit less potently than IGF-BP-5. In contrast, 5-5-6, like IGFBP-6, did not form ternary complexes by these methods. Whereas 6-6-5, like IGFBP-6, had a marked preference for binary complex formation with IGF-II rather than IGF-I, it formed ternary complexes more efficiently with IGF-I, like IGF-BP-5. The glycosaminoglycans heparin and heparan sulfate bind to IGFBP-5 through its basic carboxyl-terminal domain. At high concentrations, these glycosaminoglycans inhibited ALS binding to binary complexed IGF-BP-5. In addition, in the absence of IGFs, IGFBP-5, a synthetic peptide representing the basic carboxyl-terminal sequence IGFBP-5(201-218), and the corresponding IGFBP-3 basic sequence IGFBP-3(215-232), competed weakly for ALS binding to covalent IGF-IGFBP-5 complex, as did a random-sequence synthetic peptide with the same composition as IGFBP-5(201-218). These findings are consistent with the basic carboxyl-terminal domain on IGFBP-5 being the principal site in IGFBP-5 that binds to ALS.

Regulation of rat Schwann cell Po expression and DNA synthesis by insulin-like growth factors in vitro.

Myelination by Schwann cells is likely to be regulated in vivo by positive and negative epigenetic factors. In vitro, the positive regulation of myelin differentiation, in particular expression of the major myelin protein Po, can be mimicked by cAMP elevating agents, while serum, transforming growth factor (TGF) beta s, and fibroblast growth factor (FGF)2 have been shown to exert a negative effect on this differentiation. Growth factors which promote Po induction have not, however, been identified previously. Using a forskolin concentration (0.4 microM) which alone produces little Po mRNA or protein induction, we show that insulin-like growth factor (IGF)-I, IGF-II and high concentrations of insulin promote high levels of Po induction, although in the absence of forskolin they have no effect. Another event related to Schwann cell differentiation, induction of galactocerebroside expression in response to cAMP analogues, is also potentiated by IGFs. In a different context, IGFs regulate Schwann cell DNA synthesis. We find that in defined medium forskolin plus FGF2, TGF beta or platelet-derived growth factor (PDGF) BB causes minimal DNA synthesis in the absence of IGFs and that IGFs act as potent mitogens under these conditions. IGFs also potentiate DNA synthesis induced by beta isoforms of neu-differentiation factors (NDFs), although in this case considerable DNA synthesis occurs even in the absence of IGF. These results show that IGFs can act as powerful stimulators of both proliferation and differentiation in Schwann cells, and that the total growth factor input determines which of these pathways IGFs will promote.

Purified rat acid-labile subunit and recombinant human insulin-like growth factor (IGF)-binding protein-3 can form a 150-kilodalton binary complex in vitro in the absence of IGFs

Insulin-like growth factors (IGFs) circulate in plasma mainly as part of a 150-kilodalton (kDa) complex with 40- to 45-kDa IGF-binding protein-3 (IGFBP-3) and an approximately 85-kDa acid-labile subunit (ALS) that does not bind IGFs directly. This complex sequesters IGFs in plasma, thereby providing a potential reservoir of the growth factors for tissues while constraining their potential hypoglycemic effects. Although it has been thought that IGFBP-3 must first bind IGF-I or IGF-II before it can complex with ALS to form the 150-kDa complex, we recently showed that unoccupied 150-kDa binary complexes of IGFBP-3 and ALS are abundant in adult rat serum. We now demonstrate that IGFBP-3 and rat (r)ALS can form 150-kDa complexes in the absence of IGFs. ALS was purified from rat serum by anion exchange chromatography and affinity chromatography on an IGF-I-Sepharose column to which human (h) IGFBP-3 had been noncovalently bound. The preparation contained less than 0.1 ng IGF-I/microgram(s) purified ALS. In the absence of IGF, radiolabeled (r)ALS and recombinant hIGFBP-3 formed complexes that could be immunoprecipitated by antiserum to hIGFBP-3; these complexes were identified by direct quantitation of the precipitated radioactivity or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE). Inclusion of IGF-I in the incubation increased complex formation. Complex formation also was demonstrated by incubation of unlabeled rALS with hIGFBP-3, followed by affinity cross-linking. Complexes were fractionated by SDS-PAGE, blotted, and shown to contain unoccupied IGF-binding sites by their ability to bind radioiodinated IGF-II. In addition, when rALS was subjected to SDS-PAGE and blotted, radioiodinated recombinant hIGFBP-3 bound to the 85-kDa protein. In this experiment IGFBP-3 binding was slightly increased by coincubation with IGF-I. Thus, purified rALS can form a 150-kDa complex with hIGFBP-3 in the absence of IGF in vitro. The efficiency of complex formation was increased to variable extents by coincubation with IGF-I depending on the assay method.

Purified rat acid-labile subunit and recombinant human insulin-like growth factor (IGF)-binding protein-3 can form a 150-kilodalton binary complex in vitro in the absence of IGFs.

Insulin-like growth factors (IGFs) circulate in plasma mainly as part of a 150-kilodalton (kDa) complex with 40- to 45-kDa IGF-binding protein-3 (IGFBP-3) and an approximately 85-kDa acid-labile subunit (ALS) that does not bind IGFs directly. This complex sequesters IGFs in plasma, thereby providing a potential reservoir of the growth factors for tissues while constraining their potential hypoglycemic effects. Although it has been thought that IGFBP-3 must first bind IGF-I or IGF-II before it can complex with ALS to form the 150-kDa complex, we recently showed that unoccupied 150-kDa binary complexes of IGFBP-3 and ALS are abundant in adult rat serum. We now demonstrate that IGFBP-3 and rat (r)ALS can form 150-kDa complexes in the absence of IGFs. ALS was purified from rat serum by anion exchange chromatography and affinity chromatography on an IGF-I-Sepharose column to which human (h) IGFBP-3 had been noncovalently bound. The preparation contained less than 0.1 ng IGF-I/microgram(s) purified ALS. In the absence of IGF, radiolabeled (r)ALS and recombinant hIGFBP-3 formed complexes that could be immunoprecipitated by antiserum to hIGFBP-3; these complexes were identified by direct quantitation of the precipitated radioactivity or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE). Inclusion of IGF-I in the incubation increased complex formation. Complex formation also was demonstrated by incubation of unlabeled rALS with hIGFBP-3, followed by affinity cross-linking. Complexes were fractionated by SDS-PAGE, blotted, and shown to contain unoccupied IGF-binding sites by their ability to bind radioiodinated IGF-II. In addition, when rALS was subjected to SDS-PAGE and blotted, radioiodinated recombinant hIGFBP-3 bound to the 85-kDa protein. In this experiment IGFBP-3 binding was slightly increased by coincubation with IGF-I. Thus, purified rALS can form a 150-kDa complex with hIGFBP-3 in the absence of IGF in vitro. The efficiency of complex formation was increased to variable extents by coincubation with IGF-I depending on the assay method.

Insulin-like Growth Factor (IGF)-binding Protein-3 (IGFBP-3) Functions as an IGF-reversible Inhibitor of IGFBP-4 Proteolysis

Previous studies have shown that insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is degraded only in the presence of exogenous IGFs; however, we found that cation-dependent proteinase activity present in conditioned medium of MC3T3-E1 osteoblasts degrades 125I-recombinant human (rh)IGFBP-4 in the absence of IGFs. Addition of IGF-I, IGF-II, or insulin to conditioned medium had little affect on 125I-rhIGFBP-4 proteolysis, while extraction of IGFs resulted in only a approximately 10% reduction in proteinase activity. Since factors other than IGFs appeared to be involved in regulating IGFBP-4 proteolysis, we hypothesized that IGFBP-3, an IGFBP produced by many cell lines, but not MC3T3-E1 cells, might function as an inhibitor of IGFBP-4 proteolysis. Addition of rhIGFBP-3 to conditioned media inhibited 125I-rhIGFBP-4 proteolysis by 90%, while IGF-I and IGF-II reversed the inhibitory effects of rhIGFBP-3 in a dose-dependent manner. 125I-rhIGFBP-4 proteolysis was not inhibited by N-terminal rhIGFBP-3 fragments that bind IGFs, but was inhibited by two synthetic peptides corresponding to sequences contained in the mid-region or C-terminal region of IGFBP-3. Both inhibitory peptides contain highly basic, putative heparin-binding domains and heparin partially reversed the inhibitory effects of rhIGFBP-3 on 125I-rhIGFBP-4 proteolysis. These data demonstrate that rhIGFBP-3 inhibits IGFBP-4-degrading proteinase activity and binding of IGFs or glycosaminoglycans to IGFBP-3 may induce conformational changes in the binding protein, causing disinhibition of the proteinase.

Regulation of insulin-like growth factor-binding protein-4 in human endometrial stromal cell cultures: evidence for ligand-induced proteolysis.

The present study investigates the regulation of IGFBP-4 levels by insulin-like growth factor (IGF) peptides in human endometrial stromal cell cultures. A 24-kilodalton (kDa) IGF-binding protein (IGFBP) secreted by stromal cells was identified as IGFBP-4 by immunoprecipitation and Western ligand blotting. Western ligand blot analysis of conditioned medium showed that treatment of stromal cells with IGF-I or IGF-II induced a dose-dependent reduction of detectable IGFBP-4 levels. Two IGF analogs that bind type I IGF receptors, but have reduced affinity for IGFBPs, increased detectable levels of IGFBP-4, and their ability to reduce IGFBP-4 at high concentrations was positively correlated with their affinity for this binding protein. LR3-IGF-I, which has 1000-fold lower binding affinity than IGF-I, increased detectable IGFBP-4 at all concentrations tested. Des-(1-3)-IGF-I, whose affinity for IGFBP-4 is 30-fold lower than that of IGF-I, increased detectable IGFBP-4 at low concentrations (0.1-10 ng/mL), but reduced its levels at 100 ng/mL, consistent with the significant binding to IGFBP-4 at this concentration. In contrast, IGFBP-4 was undetectable in cultures receiving Leu27-IGF-II, which has reduced affinity for the type IIGF receptor but unaltered affinity for IGFBPs and the type II receptor. High concentrations of insulin (100 ng/mL), which interacts with type I IGF receptors without binding IGFBPs, also increased detectable levels of IGFBP-4 in stromal cultures. The addition of IGF-I or IGF-II to cell-free conditioned medium from stromal cells cultured in the absence of IGFs resulted in the reduction of detectable endogenous IGFBP-4 levels. The effects of IGFs on IGFBP-4 levels in this cell-free system were time, temperature, and pH dependent and were prevented by the serine proteinase inhibitor, aprotinin, by the divalent cation chelator, EDTA, and by the metal ion chelator, 1,10-phenanthroline. Western immunoblotting showed that the IGF-induced reduction of intact 24-kDa IGFBP-4 was accompanied by the generation of an immunoreactive fragment of approximately 16 kDa, which was not detectable by Western ligand blotting. Cell-free conditioned medium from endometrial stromal cultures proteolyzed covalently cross-linked [125I]IGF-II-IGFBP-4 complexes in the absence of added IGFs, generating an 18-kDa radiolabeled fragment, and addition of free IGF peptide did not enhance the degradation of IGF-II-IGFBP-4 complexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological effects of prostate specific antigen as an insulin-like growth factor binding protein-3 protease.

Prostate specific antigen (PSA) is an insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) protease found in seminal plasma and produced by prostatic epithelial cells (PC-E) in vivo. We examined the effects of PSA-proteolysis of IGFBP-3 on the affinity of IGFBP-3 fragments for IGFs and on the mitogenic action of IGFs on PC-E. Recombinant human IGFBP-3 was cleaved by PSA, then incubated with 125I-IGF-I or -II in the presence of varying concentrations of unlabelled peptides, and then cross-linking electrophoresis and densitometric analysis were performed. While the affinity of IGF-II for the PSA-generated IGFBP-3 fragments fell slightly compared to intact IGFBP-3, the affinity of the PSA-generated IGFBP-3 fragments for IGF-I fell by ten fold. The addition of IGF-I or -II to PC-E in serum-free culture conditions resulted in a two-fold stimulation of cell number compared to control. The presence of IGFBP-3 in the media blocked the IGF-induced stimulation, but had no independent effect in the absence of IGFs. When PSA was added to PC-E cultures to which both IGF-I or -II and IGFBP-3 were added, the inhibitory effects of IGFBP-3 on IGF mitogenesis were reversed. We conclude that PSA decreases the affinity of IGFBP-3 for IGF and can potentiate IGF action in the presence of inhibitory IGFBP-3. This phenomenon may contribute to normal and malignant prostate growth.

Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts.

The mechanisms by which insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) levels in cellular conditioned media are poorly understood. The effect of IGFs on IGFBP-4 levels in fibroblast conditioned media is not mediated via the type 1 or type 2 cellular IGF receptors, and the IGFs exert little or no effects on IGFBP-4 messenger RNA levels in human adult fibroblasts or in rat neuroblastoma cells. To determine whether the effects of IGFs on IGFBP-4 might be exerted through alterations in IGFBP-4 degradation, we incubated cell-free, fibroblast-conditioned media from either sheep or human dermal fibroblasts with or without IGF-I, IGF-II (each 1 microgram/ml), or insulin (10 micrograms/ml) for 72 h at 37 C. Samples were then analyzed by Western ligand blot using radiolabeled IGFs and by immunoblotting using a polyclonal antisera to human IGFBP-4. In the absence of IGFs, no apparent changes in the basal concentrations of the various IGFBPs were observed. In contrast, incubation of media with IGFs caused a 70-80% reduction in levels of both sheep and human IGFBP-4, whereas incubation with insulin was without effect. Similarly, incubation of cell-free conditioned media containing recombinant human IGFBP-4 with IGF-I caused a reduction in detectable levels of the 28K protein. The decrease in IGFBP-4 levels was accompanied by the appearance of an immunoreactive approximate 17-20K fragment that did not bind radiolabeled IGFs by ligand blot. The IGF-dependent decrease in IGFBP-4 was prevented by coincubation of the media with serine protease inhibitors, EDTA, or 1,10-phenanthrolene, suggesting that IGFs may activate an IGFBP-4 specific metallo-serine protease present in fibroblast conditioned media. Alternatively, binding of IGF-I or -II to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. The demonstration that IGF-I and IGF-II can promote directly the proteolytic degradation of IGFBP-4 into fragments that do not bind IGFs provides a novel mechanism by which the IGFs may increase their own availability and/or activity in biological fluids.

Regulation of 11β- and 17α-Hydroxylases in Cultured Bovine Adrenocortical Cells: 3′,5′-Cyclic Adenosine Monophosphate, Insulin-Like Growth Factor-I, and Activators of Protein Kinase C*

The induction of steroid 11 beta-hydroxylase and 17 alpha-hydroxylase was studied in bovine adrenocortical cell cultures in serum-free medium. In the absence of insulin-like growth factor (IGF)-I or insulin, cholera toxin failed to increase 11 beta-hydroxylase enzyme activity or messenger RNA (mRNA) levels; cholera toxin increased 11 beta-hydroxylase activity and mRNA only in the presence of 10 nM IGF-I or of higher concentrations of insulin. 17 alpha-Hydroxylase enzyme activity and mRNA, in contrast, were increased maximally by cholera toxin in the absence of insulin or IGF. We also compared the induction of 11 beta-hydroxylase and 17 alpha-hydroxylase by intracellular second messengers. When cultures were incubated with cholera toxin, cAMP analogs, forskolin, ACTH, or prostaglandin E1 in defined medium with insulin, all agents increased the mRNA levels for 11 beta-hydroxylase and 17 alpha-hydroxylase. 11 beta-Hydroxylase enzyme activity was detectable in control (insulin only) cultures and was increased to varying extents by the different agents. 17 alpha-Hydroxylase enzyme activity was undetectable in control cultures and was increased more than 50-fold by all agents. We compared the sensitivity of induction of 11 beta-hydroxylase and 17 alpha-hydroxylase enzyme activities by cAMP using serial dilutions of an equimolar mixture of N6-monobutyryl cAMP and 8-bromo cAMP. For both enzymes, the response curve was biphasic, with a maximal response in the range of 20 to 100 microM each analog, but the decline in response at higher cAMP concentrations was much more marked for 11 beta-hydroxylase than for 17 alpha-hydroxylase. The effects of activation of protein kinase C were studied in cultures incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA) together with a cAMP analog mixture. TPA decreased cAMP-induced 11 beta-hydroxylase mRNA; TPA also decreased the induction of 17 alpha-hydroxylase mRNA, as previously reported. TPA caused a dose-dependent decrease in cAMP-induced 11 beta-hydroxylase enzyme activity. Angiotensin II at 0.1 to 10 microM also decreased induction of 11 beta-hydroxylase. Induction of 11 beta-hydroxylase and 17 alpha-hydroxylase is coordinately regulated by cAMP, protein kinase C, and IGF-I/insulin, but responses to these regulators differ in various respects between these two cytochrome P450 enzymes.