Addition Of Platelet-derived Growth Factor Research Articles

Stage-specific effects of bone morphogenetic proteins on the oligodendrocyte lineage

Oligodendrocyte maturation is regulated by multiple secreted factors present in the brain during critical stages of development. Whereas most of these factors promote oligodendrocyte proliferation and survival, members of the bone morphogenetic protein family (BMPs) recently have been shown to inhibit oligodendrocyte differentiation in vitro. Oligodendrocyte precursors treated with BMPs differentiate to the astrocyte lineage. Given that cells at various stages of the oligodendrocyte lineage have distinct responses to growth factors, we hypothesized that the response to BMP would be stage-specific. Using highly purified, stage-specific cultures, we found that BMP has distinct effects on cultured oligodendrocyte preprogenitors, precursors, and mature oligodendrocytes. Oligodendrocyte preprogenitors (PSA-NCAM+, A2B5-) treated with BMP2 or BMP4 developed a novel astrocyte phenotype characterized by a morphological change and expression of glial fibrillary acidic protein (GFAP) but little glutamine synthetase expression and no labeling with A2B5 antibody. In contrast, treating oligodendrocyte precursors with BMPs resulted in the accumulation of cells with the traditional type 2 astrocyte phenotype (GFAP+, A2B5+). However, many of the cells with an astrocytic morphology did not express GFAP or glutamine synthetase unless thyroid hormone was present in the medium. The addition of fibroblast growth factor along with BMP to either oligodendrocyte preprogenitor or the oligodendrocyte precursor cells inhibited the switch to the astrocyte lineage, whereas platelet-derived growth factor addition had no effect. Treatment of mature oligodendrocytes with BMP elicited no change in morphology or expression of GFAP. These data suggest that as cells progress through the oligodendrocyte lineage, they show developmentally restricted responses to the BMPs.

Platelet-derived Growth Factor-stimulated Migration of Murine Fibroblasts Is Associated with Epidermal Growth Factor Receptor Expression and Tyrosine Phosphorylation

Previous studies have shown that epidermal growth factor (EGF) synergizes with various extracellular matrix components in promoting the migration of B82L fibroblasts expressing wild-type EGF receptors and that functional EGF receptors are critical for the conversion of B82L fibroblasts to a migratory cell type (). In the present study, we examined the effects of platelet-derived growth factor (PDGF) on the motility of B82L fibroblasts using a microchemotaxis chamber. We found that PDGF can enhance fibronectin-induced migration of B82L fibroblasts expressing wild-type EGF receptors (B82L-clone B3). However, B82L cells that lack the EGF receptor (B82L-parental) or that express an EGF receptor that is kinase-inactive (B82L-K721M) or C-terminally truncated (B82L-c'973) exhibit little PDGF-stimulated migration. In addition, none of these three cell lines exhibit the capacity to migrate to fibronectin alone. These observations indicate that, similar to cell migration toward fibronectin, PDGF-induced cell migration of B82L fibroblasts is augmented by the expression of an intact EGF receptor kinase. The loss of PDGF-stimulated motility in B82L cells that do not express an intact EGF receptor does not appear to result from a gross dysfunction of PDGF receptors, because ligand-stimulated tyrosine phosphorylation of the PDGF-beta receptor and the activation of mitogen-activated protein kinases are readily detectable in these cells. Moreover, an interaction between EGF and PDGF receptor systems is supported by the observation that the EGF receptor exhibits an increase in phosphotyrosine content in a time-dependent fashion upon the addition of PDGF. Altogether, these studies demonstrate that the expression of EGF receptor is critical for PDGF-stimulated migration of murine B82L fibroblasts and suggest a role for the EGF receptor downstream of PDGF receptor activation in the signaling events that lead to PDGF-stimulated cell motility.

Prevention of v-Ha-Ras-Dependent Apoptosis by PDGF Coordinates in Phosphorylation of ERK and Akt

In some v-Ha-ras-transfected cell lines, serum deprivation results in apoptosis. Clarification of the molecular mechanisms by which oncogenic Ras controls susceptibility to apoptosis may assist in the development of effective therapies against human cancer with oncogenic ras gene. In this report, we established a v-Ha-ras-transfected human fibroblast clone, R1. In R1 cells, induction of v-Ha-Ras enhanced susceptibility to cell death under serum-deprived conditions. Ladders of cellular DNA were identified only when oncogenic ras was induced under serum-deprived conditions. Platelet-derived growth factor (PDGF) precluded DNA fragmentation of serum-deprived v-Ha-ras-transformed cells. Under serum-depleted conditions, the amounts of activated ERK and Akt decreased as compared with those under serum-containing conditions. The decreased levels of activated ERK and Akt were restored by the addition of PDGF. Inhibition of phosphorylated-ERK and Akt resulted in renewed susceptibility to cell death. These results indicate that failure of signal transduction of oncogenic Ras by the deficiency of growth factors such as PDGF causes v-Ha-Ras-dependent apoptosis.

The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures.

Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-beta. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. The addition of PDGF or TGF-beta to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP- 1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.

Phenotypic and functional characteristics of porcine peritoneal mesothelial cells.

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.

Effects of activated macrophages and fibroblast growth factor on random skin flap survival in swine.

To examine the effect of application of activated autologous macrophages and basic fibroblast growth factor (FGF) on random skin flap survival in swine. A randomized nonblinded controlled trial. According to a standard design, six dorsally based, random-pattern skin flaps were raised in each of 12 Yorkshire pigs. Twenty-five milliliters of blood is harvested from each animal 20 to 24 hours prior to flap creation. Monocytes are isolated, placed in culture medium, and then activated by the addition of platelet-derived growth factor (PDGF) and tissue growth factor beta (TGF-alpha). Following an 18-hour incubation period, the monocytes are decanted and quantified, and their viability confirmed. These cells are then infused into the wound bed of the treatment flaps immediately following flap creation, and FGF is added prior to flap closure. The position of treatment and control flaps is systematically varied with regard to anterior-to-posterior and side-to-side flap positions within each animal. The area of superficial flap necrosis is evaluated on postoperative day 7, digitally scanned, and analyzed using graphics software. Control flaps are elevated similarly, but receive no placebo treatment. Two-way analysis of variance (ANOVA) demonstrated nonsignificant differences between pig side and anterior, middle, and posterior flap positions within treatment and control flap groups. Using side and position pooled data, a one-way ANOVA showed no statistically significant differences between treatment and control flaps. The cellular and biochemical events following creation of a surgical wound are complex and incompletely understood. Our attempt to augment the natural role of the macrophage in wound healing by employing cytokines to activate these cells and to accelerate their arrival by implanting them into the wound bed failed to enhance flap survival. Further study is warranted to ascertain the details of wound healing, particularly with respect to cytokine concentrations and the timing of their roles, if we are to find a clinically applicable means of enhancing flap survival.

Functions of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor.

During liver fibrogenesis, hepatic stellate cells (HSC) proliferate and migrate under the influence of growth factors, including platelet-derived growth factor (PDGF) and basic-fibroblast growth factor (b-FGF). The plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We evaluated the expression and biological functions of the plasminogen activation system in human HSC and its interaction with PDGF and b-FGF. Urokinase-plasminogen activator receptors (u-PAR) were measured by radioligand binding, cell cross-linking, immunoassay, and RNAse protection assay. u-PA and plasminogen activator inhibitors (PAIs) expression and activities were analyzed by zymography, immunoassay, and RNase protection assay. Cell migration and proliferation, studied in Boyden chambers and by microscopic counting, were evaluated after the addition of PDGF, b-FGF, and blockade with anti-u-PA, anti-u-PAR antibodies, and antisense oligodeoxynucleotides (aODN) against u-PAR mRNA. We have shown that HSC produce u-PAR, u-PA, and PAI-1. PDGF and b-FGF up-regulate u-PA and u-PAR, but not PAI-1, and exogenous addition of u-PA stimulates HSC proliferation, chemotaxis, and chemoinvasion. Inhibition of u-PA/u-PAR with antibodies against u-PA or u-PAR and with u-PAR aODN inhibit the proliferative, chemotactic, and chemoinvasive activity of PDGF and b-FGF. These findings indicate that u-PA and u-PAR are required for the mitogenic and chemoinvasive activity of PDGF and b-FGF on HSC.

DECREASE IN P53 PROTEIN IN CULTURED CARDINAL LIGAMENT FIBROBLASTS FROM PATIENTS WITH PROLAPSUS UTERI

The authors examined the growth response of cardinal ligamental fibroblasts derived from patients with prolapsus uteri (HPLiF) and compared it with the response of those from control subjects (HCLiF). The growth rate during the logarithmic growth phase was not different between HPLiF and HCLiF, while the cell density at confluence (saturation density) was significantly higher in HPLiF than in HCLiF. When added alone, platelet-derived growth factor (PDGF), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) produced minimal effects on DNA synthesis in HCLiF. The simultaneous addition of PDGF, IGF-I and EGF synergistically stimulated the DNA synthesis. In contrast, PDGF alone was able to initiate DNA synthesis in HPLiF. The combination of PDGF, IGF-I, and EGF significantly stimulated the DNA synthesis of HPLiF compared with HCLiF. p53 protein and p53 gene transcripts decreased by 50% in HPLiF. The anti-WAF1 antibody reacted intensely with a 21-kDa protein in the homogenates of control fibroblasts, while the immunoreactive band in prolapsus fibroblasts was clearly reduced. These results indicate that the higher proliferative activity at near confluency in prolapsus fibroblasts may result from the decreased expression of p53 protein and p53 mRNA followed by the decrease in p21 protein. Furthermore, the failure of cells to enter quiescence may lead to a decrease in the synthesis and deposition of elastin and thus may contribute to the loss of supportive function in uterine connective tissues.

Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae.

The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete. Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts. Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae. The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.

Ablation of Goalpha overrides G1 restriction point control through Ras/ERK/cyclin D1-CDK activities.

We have generated stable IIC9 cell lines, Goa1 and Goa2, that overexpress full-length antisense Goalpha RNA. As shown previously, expression of antisense Goalpha RNA ablated the alpha subunit of the heterotrimeric G protein, Go, resulting in growth in the absence of mitogen. To better understand this change in IIC9 phenotype, we have characterized the signaling pathway and cell cycle events previously shown to be important in control of IIC9 G1/S phase progression. In this paper we clearly demonstrate that ablation of Goalpha results in growth, constitutively active Ras/ERK, elevated expression of cyclin D1, and constitutively active cyclin D1-CDK complexes, all in the absence of mitogen. Furthermore, these characteristics were abolished by the transient overexpression of the transducin heterotrimeric G protein alpha subunit strongly suggesting the transformation of Goalpha-ablated cells involves Gobetagamma subunits. This is the first study to implicate a heterotrimeric G protein in tumor suppression.

Upregulation of CD44s in c-sis-transformed balb/c 3T3 cells by autocrine growth factor mechanisms, including PDGF

Regulation of CD44s by the c-sis oncogene product was investigated. Both CD44s protein and mRNA were comparably upregulated, consistent with some degree of transcriptional regulation; its ligand binding was also activated in confluent but not in sparse cultures of sis-transformed Balb/c 3T3 cells. CD44s was also elevated in confluent cultures of parental 3T3 cells treated with conditioned media from confluent c-sis transformants (but not from 3T3 cells); these media (but not media from ras transformants or parental 3T3 cells) contained platelet-derived growth factor (PDGF)-immunoreactive material. CD44s upregulation by these media could be partially blocked by anti-PDGF antibodies. These media also induced activation via tyrosine autophosphorylation and activation-dependent downregulation of PDGF beta-receptors. sis-transformed 3T3 cells contained low levels of PDGF receptor, but CD44 levels could still be increased in these cells by addition of PDGF. These results suggest that CD44s is upregulated in confluent cultures of c-sis-transformed cells by autocrine growth factors, including PDGF, secreted into the cell's microenvironment.

D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor.

Despite extensive analysis of phosphoinositide 3-hydroxykinases (PI 3-kinases) at the molecular level, comparatively little is known about the mechanisms by which products of these enzymes exert their expected second-messenger functions. This study examines the metabolism of D-3 phosphoinositides in mouse Ph-N2 fibroblasts lacking the platelet-derived growth factor (PDGF) alpha-receptor. Treatment of these cultures with BB PDGF, but not AA PDGF, resulted in transient activation of PI 3-kinase activity measured in vitro. Treatment of myo-[3H]inositol-labelled Ph-N2 cells with BB PDGF resulted in the rapid induction of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and, to a smaller extent, PtdIns3P. The appearance of PtdIns(3,4,5)P3 preceded that of PtdIns(3,4)P2 and PtdIns3P after the addition of PDGF, suggesting that PtdIns(4,5)P2 is the preferred substrate of the agoniststimulated PI 3-kinase in intact cells. Treatment of both resting and PDGF-stimulated cells with the fungal metabolite wortmannin resulted in pronounced, selective effects on the levels of all D-3 phosphoinositides. Kinetic studies with this PI 3-kinase inhibitor revealed the presence of at least two independent routes for the biosynthesis of D-3 phosphoinositides in PDGF-treated cells.

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.

Inhibition of mesangial cell proliferation by platelet factor 4.

Platelet factor 4(PF4), an abundant platelet secretory product, is a strong candidate for modulating glomerular pathology. Because PF4 might be released from platelets and influence intrinsic cell growth during glomerular injury, the effect of PF4 on fetal calf serum- and platelet-derived growth factor (PDGF)-induced mesangial cell mitogenesis was examined. Mitogenesis was measured as the amount of 3H-thymidine incorporated into acid-precipitable material as well as by autoradiography. The effect of PF4 on mesangial cell expression of mRNA for PDGF A chain and transforming growth factor-beta (TGF-beta 1) was also examined. Fetal calf serum (10%)- and PDGF (10 ng/mL)-stimulated increases in mesangial cell 3H-thymidine incorporation were inhibited by incremental concentrations of PF4 (1 to 25 micrograms/mL) showing a maximum reduction of approximately 80% at 25 micrograms/mL of PF4. PF4 was effective when added 24 h before and 1, 4, and 8 h, but not 16 h after the addition of PDGF, indicating that inhibition occurred at delayed events in cell-cycle regulation. PF4 inhibited PDGF-induced increments in mRNA encoding PDGF A chain and TGF-beta 1. Also, PF4 did not interfere with PDGF receptor binding. The results of this study show that PF4 is a negative regulator of mesangial cell proliferation and suggest an interference in cell growth by pathways associated with modulation of the autocrine growth factors PDGF and TGF-beta 1.

Effects of platelet-derived growth factor (PDGF) on the in vitro production of bovine embryos in protein-free media

The purpose of our experiments was to explore the effects of platelet-derived growth factor (PDGF)-supplementation at the various steps of in vitro production of bovine embryos using protein-free media. Cumulus-oocyte-complexes (COC) were collected by slicing abattoir ovaries and then dividing the COC into 2 morphological categories. After maturation for 24 h in TCM-199 supplemented with hormones and either 20% estrous cow serum (ECS) or 1 mg/ml polyvinyl-alcohol (PVA), oocytes were co-incubated for 19 h with frozen/thawed spermatozoa from bull of proven fertility. The semen was diluted in Fert-Talp supplemented with heparin, hypotaurine and epinephrine and either 6 mg/ml bovine serum albumin (BSA) or 1 mg/ml PVA. Presumptive zygotes were transferred into embryo culture medium containing either 20% ECS or 1 mg/ml PVA for a total of 10 d. The PDGF was added at concentrations of 1, 10 or 100 ng/ml to the maturation medium (Experiment 1), fertilization medium (Experiment 2) or culture medium from Day 1 on (Experiment 3), respectively, or at 1 ng/ml PDGF to both the fertilization and culture medium from Day 3 on (Experiment 4), with each medium supplemented with PVA. Oocytes/embryos incubated in the absence of PDGF in media supplemented with either ECS or PVA served as controls. An average of 20 COC was incubated in 1 droplet under silicone oil, and each experiment contained 4 to 6 replicates. No significant differences were found among the various concentrations of PDGF, nor did PDGF-supplementation during maturation (Experiment 1) or embryo culture on Day 1 (Experiment 3) significantly affect development of oocytes/embryos (34.7 ± 3.5 to 40.4 ± 2.5% morulae, 11.9 ± 2.4 to 18.8 ± 2.5% blastocysts; and 23.2 ± 2.3 to 27.5 ± 3.4% morulae, 11.5 ± 2.6 to 12.7 ± 2.3% blastocysts, respectively; x ± SEM). In the presence of 10 ng/ml PDGF in the fertilization medium development to morulae and blastocysts was similar to that of the ECS-group, and was higher (P < 0.05) than that of the PVA-control (ECS: 32.1 ± 4.6 and 13.8 ± 2.7%; PVA: 17.5 ± 0.8 and 6.1 ± 1.3%; PDGF: 30.6 ± 3.0 and 14.0 ± 2.2%, respectively). Development to morulae/blastocysts was increased, and was at the same level as in the ECS-group when the fertilization and/or embryo culture medium on Day 3 contained PDGF compared with the PVA-control group (morulae: ECS 25.3 ± 4.4%, PVA 13.9 ± 2.2% [P < 0.05], PDGF 16.7 ± 3.2 to 19.1 ± 1.1%; blastocysts: ECS 5.3 ± 2.1%, PVA 5.0 ± 1.7%, PDGF 7.1 ± 1.6 to 9.1 ± 1.7%, respectively). These results indicate that under our laboratory conditions PDGF can elevate low rates of development and the addition of PDGF to the fertilization medium enhances bovine preimplantation embryonic development. Thus, PDGF can be potentially an important factor in a completely defined medium to substitute the effects of serum.

An 11-Amino Acid Sequence from c-met Initiates Epithelial Chemotaxis via Phosphatidylinositol 3-Kinase and Phospholipase C

Interaction of hepatocyte growth factor with its high affinity receptor c-met initiates a cascade of intracellular events leading to epithelial motility. An 11-amino acid sequence from the c-met receptor has been found to cause cell transformation in transfected fibroblasts (Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., Dalla, Z. P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M.(1994) Cell 77, 261-271). We inserted this sequence into a mutant platelet-derived growth factor receptor (F5) to determine if this region of c-met can initiate cell motility and which signaling pathways it activates. The platelet-derived growth factor (PDGF) receptor/c-met hybrid (F5 met) initiated PDGF-dependent chemotaxis in renal epithelial cells (8.0 +/- 2.3 versus 70.5 +/- 4.8 cells/mm2), while the parental construct, F5, did not. Addition of PDGF to cells expressing F5 met caused activation of the phosphatidylinositol (PI) 3-kinase (control 2.0 +/- 0.8, +PDGF 17.1 +/- 5.1, n = 3, p < 0.05) and phospholipase C (control 478.5 +/- 67 dpm/well, +PDGF 1049.3 +/- 93, n = 4, p = 0.003), while neither pathway was activated in cells expressing F5. The chemotactic response of F5 met was inhibited by both the PI 3-kinase inhibitor wortmannin and the phospholipase C inhibitor U-71322. Selective activation of the PI 3-kinase utilizing a PDGF receptor mutant (F3) containing the native high affinity PI 3-kinase binding site also resulted in PDGF stimulated chemotaxis, although less than that generated by the c-met sequence. These findings demonstrate that the 11-amino acid sequence from c-met initiates epithelial motility via coincident activation of the PI 3-kinase and phospholipase C and that selective activation of the PI 3-kinase can initiate a partial chemotactic response.

Roles of growth factors in the development of bovine embryos fertilized in vitro and cultured singly in a defined medium

Bovine embryos at the 8- or 16-cell stage were cultured singly, or in groups (10-12 embryos), in the presence or absence of bovine oviduct epithelial cells (BOEC) in a defined medium which was used as a basic culture medium. A higher (P < 0.05) proportion of 8-cell embryos (48.3-50.8%) cultured singly developed beyond the 8-cell stage after the addition of platelet-derived growth factor (PDGF)-AB (1 ng mL-1) only, or with PDGF-AB + basic fibroblast growth factor (bFGF; 1 ng ml-1) + transforming growth factor (TGF)-beta 1 beta 2 (1 ng mL-1) than in basic medium alone (30.3%). In contrast, a significantly (P < 0.02) higher percentage (62.6-65.8%) of 16-cell embryos developed to the morula stage after the addition of TGF-beta 1 beta 2 only, or the addition of TGF-beta 1 beta 2 + bFGF + PDGF-AB than in basic medium alone (30.2%). These proportions were not significantly (P > 0.05) different from the proportions obtained when embryos were cultured in groups, but were significantly (P < 0.005) lower than the proportions obtained when embryos were cultured in groups on BOEC monolayers. Arachidonic acid (50 ng mL-1), beta-mercaptoethanol (10 microM) and glutathione (10-1000 microM) stimulated the development of 8-cell embryos in the presence of PDGF and TGF-beta 1 beta 2; blastocyst formation was observed for the first time in 8-cell embryos cultured singly in the presence of these embryotrophic substances (2.2-6.2%).

The 3-phosphorylated phosphoinositide response of 3T3-L1 preadipose cells exposed to insulin, insulin-like growth factor-1, or platelet-derived growth factor.

We compared the pattern of 3-phosphorylated phosphoinositides produced by confluent 3T3-L1 preadipose cells upon exposure to growth factors that either induce differentiation (insulin, insulin-like growth factor-1) or do not (platelet-derived growth factor). Following addition of insulin or insulin-like growth factor-1, PI(3,4,5)P3 rapidly rose, on average, to levels tenfold over basal. PI(3,4)P2 either did not change (after insulin) or slightly increased (1.5 fold). Time course studies with insulin demonstrated that the rise in PI(3,4,5)P3 peaked by 1 minute, and levels then remained steady over 30 minutes. Dose-response experiments showed that insulin at a concentration of 1 nM was sufficient for the PI(3,4,5)P3 response. Insulin failed to increase PI(3,4)P2 at any of the time points or at any of the doses used. In contrast, after addition of platelet-derived growth factor, both PI(3,4)P2 and PI(3,4,5)P3 rose concurrently and to comparable extents. These data suggest that one possible mechanism contributing to insulin/insulin-like growth factor-1-induced 3T3-L1 preadipose cell differentiation is a distinct pattern of 3-phosphorylated phosphoinositide accumulation, defined by a prominent increase in PI(3,4,5)P3 with no (in the case of insulin), or a minimal (in the case of IGF-1), rise in PI(3,4)P2.

Platelet-derived Growth Factor Stimulates Protein Kinase A through a Mitogen-activated Protein Kinase-dependent Pathway in Human Arterial Smooth Muscle Cells

The abilities of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-I) to regulate cAMP metabolism and mitogen-activated protein kinase (MAP kinase) activity were compared in human arterial smooth muscle cells (hSMC). PDGF-BB stimulated cAMP accumulation up to 150-fold in a concentration-dependent manner (EC50 approximately 0.7 nM). The peak of cAMP formation and cAMP-dependent protein kinase (PKA) activity occurred approximately 5 min after the addition of PDGF and rapidly declined thereafter. Incubating cells with PDGF and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) enhanced the accumulation of cAMP and PKA activity by an additional 2.5-3-fold, whereas IBMX alone was essentially without effect. The PDGF-stimulated increase in cAMP was prevented by addition of the cyclooxygenase inhibitor indomethacin, consistent with release of prostaglandins stimulating cAMP. PDGF, but not IGF-I, stimulated MAPK activity, cytosolic phospholipase A2 (cPLA2) phosphorylation, and cAMP synthesis which indicated a key role for MAP kinase in the activation of cPLA2. Further, PDGF stimulated the rapid release of arachidonic acid and synthesis of prostaglandin E2 (PGE2) which could be inhibited by a cPLA2 inhibitor (AACOCF3). Calcium mobilization was required for PDGF-induced arachidonic acid release and PGE2 synthesis but not for MAPK activation, whereas PKC was required for PGE2-mediated activation of PKA. In summary, these results demonstrated that PDGF increases cAMP formation and PKA activity through a MAP kinase-mediated activation of cPLA2, arachidonic acid release, and PGE2 synthesis in human arterial smooth muscle cells.

Tyrosine Kinase Inhibitors Inhibit Multiple Steps of the Cell Cycle of Vascular Smooth Muscle Cells

Protein tyrosine kinase (PTK) inhibitors have been reported to inhibit proliferation of vascular smooth muscle cells (SMC). To elucidate the made of this inhibition, the effects on the cell cycle of cultured vascular SMC of three PTK inhibitors with different modes of action (methyl 2,5-dihydroxyeinnamate, genistein, and herbimycin A) were studied. Rat aortic SMC were synchronized to the G0 phase of the cell cycle and then released to proceed through the cell cycle by the addition of platelet-derived growth factor (PDGF), and [3H]thymidine incorporation into DNA was measured. The three PTK inhibitors all inhibited PDGF-induced DNA synthesis in a dose-dependent fashion, with IC50 values of 4.7 ± 1.4 μM for methyl 2,5.dihydroxycinnamate, 6.7 ± 2.5 μM for genistein, and 0.17 ± 0.07 μM for herbimycin A. Time course studies suggested that the agents inhibited early G1 phase but not the G0-G1 transition. The lack of effect on the G0-G1 transition was also supported by the finding that the agents did not inhibit the ligand-induced autophosphorylation of PDGF receptor nor the induction of c-fos mRNA at concentrations which were sufficient to inhibit DNA synthesis. PTK inhibitors inhibited progression of the S phase when they were added to SMC that had been arrested at the G1-S border with hydroxyurea. Methyl 2,5-dihydroxyeinnamate also blocked the M phase when it was added to SMC cultured in the presence of 10% fetal calf serum, while genistein and herbimycin A did not inhibit the M phase under the same experimental conditions. In accordance with our previous observation, methyl 2,5-dihydroxycinnamate impaired microtubule networks and formation of the mitotic spindle during the M phase. Our findings indicated that PTK inhibitors inhibit multiple steps of the vascular SMC cell cycle.