Relaxin-induced deoxyribonucleic acid synthesis in porcine granulosa cells is mediated by insulin-like growth factor-I

Relaxin stimulates in vitro DNA synthesis and cell proliferation of porcine granulosa cells (GC) and theca cells. The objective of the study reported here was to determine whether components of the ovarian insulin-like growth factor (IGF) system mediate relaxin's growth-promoting effects on porcine GC in vitro. In small follicle GC, relaxin (1-100 ng/ml) significantly (p < 0.05) increased IGF-I secretion to 25-34% above control. Hormonal responsiveness of GC was shown by incubation with FSH (200 ng/ml), which resulted in 125% stimulation of IGF-I secretion relative to that in cells incubated alone. When IGF-I activity in the GC cultures was neutralized with a specific IGF-I antibody, relaxin (10 and 100 ng/ml)-induced [3H]thymidine incorporation was inhibited (p < 0.05). Coincubation with IGF-I antibody also suppressed basal and IGF-I (10 ng/ml)-induced [3H]thymidine incorporation into GC DNA, but had no effect on insulin (1 microgram/ml)-induced DNA synthesis, demonstrating the specificity and lack of toxicity of the IGF-I antibody. Ligand blot analysis showed no change in secretion of GC IGF binding protein (IGFBP) in response to relaxin (1, 10, and 100 ng/ml). In contrast, IGF-I (10 ng/ml) increased secretion of IGFBP-3 and -5, whereas FSH (200 ng/ml) decreased IGFBP-3 secretion and increased IGFBP-4 secretion (p < 0.05). In IGF-I receptor competition studies, IGF-I, but not relaxin, displaced [125I]IGF-I from the GC IGF-I receptor. These studies provide direct evidence for an interaction of relaxin and the ovarian IGF system. They are the first to show 1) a stimulatory effect of relaxin on IGF-I secretion; 2) the necessity of IGF-I activity for relaxin-induced GC DNA synthesis; and 3) the absence of an effect of relaxin on GC IGFBPs or IGF-I receptor. These findings support a paracrine role for relaxin in the porcine follicle and show that relaxin acts indirectly to promote follicle growth by stimulating GC IGF-I secretion.     

Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells

GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ganglioside GM1 activates the mitogen-activated protein kinase Erk2 and p70 S6 kinase in U-1242 MG human glioma cells

Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [3H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

Skeletal cell stresses and bone adaptation

BACKGROUND: We have previously shown that growth hormone (GH) consistently stimulates proliferation of human osteoblasts in vitro. In rat osteoblasts, GH augments the effects of insulin-like growth factor (IGF) I on cell proliferation and differentiation. We therefore investigated the effects of IGF-I and -II alone and in combination with GH on human osteoblasts in vitro. METHODS: Human osteoblast-like cells (HOB) were established from trabecular explants (n = 18) and human marrow stromal cells (HMS) from marrow aspiration (n = 21). The cell cultures were stimulated with IGF-I or IGF-II (1, 10 or 100 ng mL-1) alone, in combination with hGH (100 ng mL-1) or after prestimulation with hGH. RESULTS: IGF-I alone, in combination with hGH and after pretreatment with hGH, increased proliferation of HOB and HMS by 49-190% (P < 0.05-0.01). IGF-II alone, in combination with hGH and after pretreatment with hGH increased proliferation of HOB by 57-158% (P < 0.01). In HMS only IGF-II in combination with hGH and after prestimulation with hGH increased proliferation. IGF-I alone and in combination with hGH decreased alkaline phosphatase (AP) in both cell types. IGF-II did not affect AP in HOB, but increased AP in HMS, this effect was abolished by hGH. In HOB, collagen production (PICP) was increased by IGF-II but unaffected by IGF-I. In HMS, PICP was decreased by IGF-I and -II but increased by hGH. Co-stimulation further increased PICP. CONCLUSION: IGF-I and -II exerted proliferative effects on both HOB and HMS. Co-stimulation with GH exhibited synergism in enhancing the proliferative response. In HMS prestimulation improved the proliferative response significantly. The effects of the IGFs on differentiation are more complex and dependent on cell maturation and of the IGF used.     

Differential expression and biological effects of insulin-like growth factor-binding protein-4 and -5 in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis. The bioactivity of IGF-I is modulated by a group of high affinity, specific binding proteins (IGF-binding proteins; IGFBPs) that are present in the interstitial fluid. Previously, we have reported that porcine VSMCs synthesize and secrete IGF-I and several forms of IGFBPs, including IGFBP-2, IGFBP-4, and IGFBP-5. In this study, we examined the role of autocrine/paracrine secreted IGF-I in controlling the expression of IGFBP-4 and IGFBP-5 as well as the effects of these IGFBPs in modulating the cellular replication response to IGF-I. The concentrations of IGFBP-4 in the conditioned medium increased significantly from <50 ng/ml to 742 +/- 105 ng/ml. This increase was associated with a decrease in the activity of an IGF-I-regulated IGFBP-4 protease. In contrast, the synthesis of IGFBP-5 was inversely correlated with culture density, and its concentration decreased from 792 +/- 91 to 44 +/- 14 ng/ml. IGFBP-5 mRNA in sparse cultures was 3-fold higher compared with those in confluent cultures. This culture density-dependent change in IGFBP-5 mRNA correlated closely with endogenous IGF-I levels. Since treatment of VSMC with exogenous IGF-I increased IGFBP-5 mRNA levels, we neutralized the effect of endogenously secreted IGF-I with an anti-IGF-I antibody to determine if it would alter IGFBP-5 mRNA abundance. This resulted in a 4.4-fold decrease in IGFBP-5 mRNA levels. When added together with IGF-I, exogenous IGFBP-4 inhibited IGF-I-induced DNA synthesis in a concentration-dependent manner. IGFBP-5, on the other hand, potentiated the effect of IGF-I. Therefore, IGFBP-4 and IGFBP-5 appear to be differentially regulated by autocrine/paracrine IGF-I through distinct mechanisms. These two proteins, in turn, play opposing roles in modulating IGF-I action in stimulating VSMC proliferation.     

The integration of object properties

Growth plate cartilage cell express receptors for, and are affected by both IGF-I and 1 alpha, 25(OH)2D3. The studies were undertaken to investigate interaction between these two hormone systems, that is, (i) to study effects of 1 alpha, 25(OH)2D3 on IGF-type 1 receptors (IGFIR), on IGF-I stimulated cell replication, colony formation, and on alkaline phosphatase activity (AP), and conversely, (ii) to study the effect of IGF-I on vitamin D receptor (VDR) expression on 1 alpha, 25(OH)2D3 stimulated growth parameters and on AP activity. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures, (serum-free) or in agarose stabilized suspension cultures (0.1% FCS). Vitamin D receptor and IGFIR were visualized by immunostaining with the monoclonal antibody (mAb) 9A7 gamma and mAb alpha IR3, respectively, and quantitated by RT-PCR for mRNA and by Scatchard analysis using [3H]-1,25(OH)2D3 and [125I]-alpha IR3. Cell proliferation was measured by [3H]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I dose-dependently increased [3H]-thymidine incorporation. 1 alpha, 25(OH)2D3, but not 1 beta, 25(OH)2D3 was stimulatory at low ((10-12 M) and slightly inhibitory at high (10-8 M) concentrations. The effect of IGF-I was additive to that of 1 alpha, 25 (OH)2D3 [IGF-I 60 ng/ml, 181 +/- 12.7; 1 alpha, 25(OH)2D3 10(-12) M, 181 +/- 9.8%, IGF-I + 1 alpha, 25(OH)2D3, 247 +/- 16.7%, P < 0.05 by ANOVA] and specifically obliterated by polyclonal IGF-I antibody (AB-1). Interaction could also be confirmed in suspension cultures. IGFIR mRNA and [125I]-alphaIR3 binding was increased by low (10(-12) m) but not by high (10(-8) M) concentrations of 1 alpha, 25(OH)2D3. Homologous up-regulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly amplified by coincubation with 1 alpha, 25(OH)2D3 (10(-12)m). Immunostaining with alpha IR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1 alpha, 25(OH)2D3 or IGF-I suggested some increase of receptor expression in single cells, but the predominant effect was increased recruitment of receptor positive cells, Vitamin D receptor expression was markedly stimulated (fourfold) by IGF-I (60 ng/ml), but not IGF-II and inhibited by actinomycin D. This study shows that IGF-I and 1 alpha, 25(OH)2D3 mutually up-regulate their respective receptors in growth plate chondrocytes. In parallel, they have additive effects on cell proliferation and colony formation suggesting independent effector pathways.     

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon's horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10(-14)-10(-8) M) and des(1-3) IGF-I (10(-10)-10(-8) M) were found to inhibit whereas IGF-II (10(-14)-10(-8) M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.     

Characterization of insulin-like growth factor binding proteins and regulation of IGFBP3 in human mesangial cells

IGF-I regulates renal growth and development. Insulin-like growth factor binding proteins (IGFBPs) are synthesized by the kidney and may modulate the local autocrine and/or paracrine actions of IGF-I. We have previously demonstrated that mesangial cells (MC) release IGF-I and IGF-binding activity; however, the specific IGFBPs produced by these cells and the factors involved in their regulation are unknown. We examined MC for expression of IGFBP-1 to -6 mRNAs and proteins. RNase protection assays using total RNA demonstrated that MC express all of the IGFBPs. [125I]IGF-I Western ligand blot of conditioned medium demonstrated that MC release IGFBPs of 24, 29, 32 kDa, and a doublet at 46 kDa, consistent with IGFBP-4, -5, -2 and -3, respectively. IGFBP species of 28 and 34 kDa were also detected. Since IGF-I and TGF-beta are implicated in glomerular hypertrophy and matrix expansion, we tested their effect on IGFBPs released by MC. IGF-I (100 ng/ml), TGF-beta (2 ng/ml) and forskolin (10(-5) M) differentially regulated the abundance of IGFBPs released in the conditioned medium in a time-dependent manner. IGF-I and TGF-beta were potent inducers of the release of IGFBP3 protein; however, TGF-beta, but not IGF-I, increased IGFBP3 mRNA levels. Recombinant IGFBP3 was tested for its effect on IGF-I-induced mitogenesis. IGFBP3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner with a peak effect observed at 50 nM IGFBP3. Although TGF-beta is a potent inhibitor of IGF-I-stimulated DNA synthesis, this effect is not mediated via IGFBPs. Expression of IGFBP-1 to -6 by MC suggests that these proteins may modulate IGF-I bioavailability in the glomerulus. IGF-I itself, TGF-beta and cAMP agonists may indirectly modulate the effects of IGF-I via the release of IGFBPs by MC.     

Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist

The transfer of cholesterol from the outer to the inner mitochondrial membrane, where side-chain cleavage occurs to form pregnenolone, is a crucial event in the regulation of steroidogenesis and recently has been demonstrated to be mediated by steroidogenic acute regulatory protein (StAR). We generated a partial porcine StAR complementary DNA (280 bp) by RT-PCR and used the corresponding antisense riboprobe to quantify the control of StAR gene expression by FSH and insulin-like growth factor I (IGF-I) in hormonally responsive swine granulosa cells, which typically manifest synergistic steroidogenic stimulation by these two dominant intrafollicular regulators. RNase protection assays were implemented to investigate the time course of the actions of FSH (100 ng/ml), IGF-I (100 ng/ml), and FSH plus IGF-I on StAR messenger RNA accumulation in serum-free cultures granulosa cells. Treatment with FSH (1.6-fold) or IGF-I (2.7-fold) alone had a small but consistent stimulatory effect on StAR message accumulation (corrected for 18S ribosomal RNA in each lane) at 48 h, whereas only IGF-I stimulated StAR protein expression (at least 6-fold as assessed by Western blot). Notably, the combined effect of FSH plus IGF-I was strongly synergistic and already significant by 24 h and maximal at 48 h (P < 0.001). Protein kinase A agonist, 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) (1 mM) alone elicited a 3.5-fold increase in StAR message and more than 3.7-fold increase in StAR protein expression by 48 h. The combination of IGF-I and FSH or 8-bromo-cAMP evoked a 26- to 40-fold (P < 0.001) synergistic rise in StAR message accumulation. StAR protein also showed a similar synergistic pattern of expression driven by IGF-I and FSH or 8-bromo-cAMP, namely a greater than 56- to 60-fold increase. In summary, two distinct first messenger regulatory molecules, FSH and IGF-I, interact synergistically to induce amplification of StAR messenger RNA and protein expression in serum-free monolayer cultures of immature (swine) granulosa cells.     

Effect of phospholipase C inhibitor U-73122 on antigen-induced airway smooth muscle contraction in guinea pigs

The importance of phospholipase C (PLC) in airway smooth muscle contraction was studied, using an inhibitor of PLC, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Tracheas from ovalbumin (OA)-sensitized guinea pigs contracted rapidly after exposure to low concentrations of antigen (OA). However, tracheas treated with U-73122 for 10 min prior to the addition of antigen, demonstrated a 3 log rightward shift in the OA dose-response curve with an IC50 of 7 microM. The analogue of U-73122, 1-[6[[17 beta-3-methoxyestra-1,3,5 trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73433), was approximately 5-fold less active in inhibiting smooth muscle contraction. In addition to the inhibition of antigen-induced smooth muscle contraction, U-73122 inhibited carbachol- and leukotriene D4-induced smooth muscle contraction. Furthermore, U-73122 inhibited in a dose-dependent manner antigen-induced histamine release from guinea pig tracheal tissue. The inhibition of smooth muscle contraction by U-73122 correlated well with the inhibition of polyphosphoinositide mediates smooth muscle contractile responses to muscarinic agonists and leukotrienes as well as antigenic-induced contraction.     

Role for membrane and secreted insulin-like growth factor-binding protein-2 in the regulation of insulin-like growth factor action in lung tumors

The insulin-like growth factors (IGFs) have been implicated in the autocrine and/or paracrine growth of a number of tumor types, including lung tumors. Importantly, insulin-like growth factor-binding proteins (IGFBPs), which both enhance and inhibit the physiological and biological actions of the IGFs, have been shown to be secreted in vitro by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actions of IGF-I and IGF-II. Affinity cross-linking studies demonstrated expression of type-I and type-II IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membrane-associated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity cross-linking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGF-II bound predominantly to IGF-I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. Soluble IGFBP-2 inhibited the binding of radiolabeled IGF-I and -II to both SCLC and NSCLC cells in a concentration-dependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-I receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC.     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

p70 S6 kinase activation is not required for insulin-like growth factor-induced differentiation of rat, mouse, or human skeletal muscle cells

Insulin-like growth factors (IGFs) are potent stimulators of muscle differentiation, and phosphatidylinositol 3-kinase (PI 3-kinase) is an essential second messenger in this process. Little is known about the downstream effectors of the IGF/PI 3-kinase myogenic cascade, and contradictory observations have been reported concerning the involvement of p70 S6 kinase. In an attempt to clarify the role of p70 S6 kinase in myogenesis, here we have studied the effect of rapamycin on rat, mouse, and human skeletal muscle cell differentiation. Both insulin and IGF-II activated p70 S6 kinase in rat L6E9 and mouse Sol8 myoblasts, which was markedly inhibited at 1 ng/ml rapamycin concentrations. Consistent with previous observations in a variety of cell lines, rapamycin exerted a potent inhibitory effect on L6E9 and Sol8 serum-induced myoblast proliferation. In contrast, even at high concentrations (20 ng/ml), rapamycin had no effect on IGF-II-induced proliferation or differentiation. Indeed, neither the morphological differentiation, as assessed by myotube formation, nor the expression of muscle-specific markers such as myogenin, myosin heavy chain, or GLUT4 (glucose transporter-4) glucose carriers was altered by rapamycin. Moreover, here we extended our studies on IGF-II-induced myogenesis to human myoblasts derived from skeletal muscle biopsies. We show that, as observed for rat and mouse muscle cells, human myoblasts can be induced to form multinucleated myotubes in the presence of exogenous IGF-II. Moreover, IGF-II-induced human myotube formation was totally blocked by LY294002, a specific PI 3-kinase inhibitor, but remained unaffected in the presence of rapamycin.     

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.     

Lifetime comorbidity, lifetime history of psychosis and suicide attempts, and current symptoms of patients with deteriorated affective disorder

Expression of insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/Man6P) receptors in cultured bovine alveolar macrophages (BAM) was demonstrated by competitive binding studies and crosslinking experiments. Western blotting of protein extracts from cultured BAM using an anti bovine IGF-II/Man6P receptor antiserum (#66416) confirmed the presence of IGF-II/Man6P receptors on BAM. The effects of IGFs and Man6P on generation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labelled cultured BAM. IGF-I at nanomolar concentrations and Man6P (10[-8]-10[-3] M) stimulated the accumulation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after 30 sec. IGF-II (up to 2.3 x 10[-8] M) had no significant effect on inositol phosphate accumulation under the same conditions. Both IGFs and Man6P induced a rise in [Ca2+]i in cultured BAM. In addition, using the fluorescent dye SNARF-1/AM we could demonstrate rapid but small IGF-II (10[-9] M) triggered acidification (0.07 pH units) of cultured BAM. Taken together, our results indicate not only the presence of both IGF-I and IGF-II/Man6P receptors on BAM, but also provide evidence of the linkage of the IGF-I receptor to the inositol phosphate system.     

Immune responses to intestinal parasites: protection, pathology and prophylaxis

It has been suggested that adjunctive growth hormone (GH) therapy improves ovarian response and in vitro fertilization (IVF) outcome in specific groups of patients. The correlation between insulin-like growth factor (IGF) and GH is well established. The aim of this study was to determine whether changes in plasma GH correlate with IGF blood levels in patients during IVF treatment. Thirty-six women undergoing IVF and embryo transfer (ET) were examined. Ovarian stimulation was carried out by gonadotropin-releasing hormone agonists (GnRHa) and gonadotropins. Blood was drawn at the early and late follicular phase, on the day of human chorionic gonadotropin (hCG) injection and at the mid- and the late luteal phases. The samples were assayed for IGF-I, IGF-II, IGF-binding protein-3 (IGF BP-3), GH and estradiol. According to the IGF-I and GH plasma levels, patients were divided into three major groups: Group I consisted of patients in whom peak levels of GH reached more than 4 ng/ml and IGF-I decreased significantly. In this group, estradiol levels were 1863 +/- 149 pg/ml. Group II consisted of patients in whom peak blood GH levels did not exceed 2.5 ng/ml and the IGF-I level remained unchanged. In this group estradiol levels were 630 +/- 57 pg/ml. Group III consisted of patients in whom blood GH levels were low and remained unchanged while estradiol levels were 1600 +/- 420 pg/ml. In this group no significant increase in IGF-levels were observed. There was no significant change in the levels of either IGF-II or IGF BP-3 in any of the groups. We can conclude that (1) there is a negative correlation between GH and IGF-I plasma levels in patients undergoing controlled ovarian hyperstimulation (COH)-IVF, when levels of estradiol and GH are elevated; (2) plasma levels of IGF-I under ovarian hyperstimulation are probably regulated by a multifactorial system; and (3) no correlation was found between the plasma levels of IGF-I and those of IGF-II and IGF BP-3 in all patient groups.     

Rapid and long-lasting suppression of the ATF-2 transcription factor is a common response to neuronal injury

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [3H]thymidine into DNA and [14C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M(r) 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M(r) approximately 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro.     

Extracellular signal-regulated kinase-1 and -2 respond differently to mitogenic and differentiative signaling pathways in myoblasts

In this report we show that extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) respond differently to signals that elicit proliferation and/or differentiation of myoblasts using the C2C12 cell line and nondifferentiating mutant NFB4 cells derived from them. Induction of differentiation by withdrawal of serum rendered ERKs in C2C12 myoblasts relatively insensitive to restimulation by serum. Instead, myogenic differentiation of C2C12 cells was associated with sustained activation of ERK-2 dependent on the insulin-like growth factor II (IGF-II) autocrine loop. By contrast, mutant NFB4 cells cultured under the same conditions remained proliferative and demonstrated robust activation of ERKs in response to serum. Similarly, a Gi-dependent signaling pathway induced activation of ERKs in NFB4 cells, but not in C2C12 cells, after stimulation by lysophosphatidic acid (LPA). In NFB4 cells partially rescued by prolonged IGF-I treatment, ERK activity remained responsive to Gi-dependent LPA stimulation, whereas rescue of NFB4 cells by constitutive expression of myogenin or MyoD, associated with activation of the IGF-II autocrine loop, rendered the Gi-signaling pathway refractory to LPA stimulation. Relatively high levels of G(alpha i2) were detected in NFB4 cells and IGF-I treated NFB4 cells, which correlated with responsive Gi signaling. Activation of the IGF-II autocrine loop in C2C12 and NFB4 myoblasts or treatment with IGF-II was associated with loss of G(alpha i2) and inhibition of Gi-dependent signaling. Thus, IGF-I and IGF-II activate distinct signaling cascades, with IGF-II eliciting a stronger differentiation effect correlated with down-regulation of G(alpha i2) protein. Short-term stimulation of NFB4 cells with IGF-I, a mitogenic signal for myoblasts, also induced ERK-1 and -2 activation. Transient stimulation of NFB4 cells with IGF-I while blocking activation of Gi-proteins is with pertussis toxin resulted in preferential activation of ERK-2 characteristic of differentiated C2C12 cells, suggesting that proliferation induced by IGF-I is Gi-dependent and separable from the IGF-I-signaling pathway that leads to differentiation.     

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001-1 microg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01-10 microg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01-10 microg/ml), estradiol output by rabbit granulosa cells (at 1 microg/ml) and porcine ovarian follicles (at 10 microg/ml), as well as cAMP production by bovine (at 0.001-1 microg/ml) and rabbit (at 1 microg/ml) granulosa cells. No effects of genistein (at 10 microg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 microg/ml), as well as the preimplantation development of rabbit zygotes (at 1 microg/ml). Lavendustin A (0.001-1 microg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 microg/ml). Lavendustin (at 1 microg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 microg/ml). Inhibitory actions of lavendustin (at 10 microg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.