The v-sis oncogene encodes a protein structurally and functionally related to human platelet-derived growth factor (PDGF). In the present studies, we show that the primary translational product of the human sis proto-oncogene is a 26-kd protein, p26c-sis. This product is processed to yield a disulfide-linked homodimer, p56c-sis, which is further processed to a 35,000-dalton dimer, p35c-sis. Like the v-sis gene product, the PDGF-2 precursor undergoes N-linked glycosylation, implying its processing through the endoplasmic reticulum. The PDGF-2 product was shown to possess functional properties of PDGF. Whereas lysates of control COS-1 cells lacked mitogenic activity, lysates of COS-1 cells transfected with a c-sis/PDGF-2 expression vector specifically stimulated DNA synthesis of quiescent fibroblasts. Moreover, this activity was completely inhibitable by PDGF antibody. Identical forms of the sis/PDGF-2 product were identified in human tumor cells that expressed c-sis/PDGF-2 transcripts. These proteins were shown to be specifically associated with the membrane component of the tumor cells and were not detectably secreted into the culture medium. These findings support the concept that expression of the sis/PDGF-2 product in human cells responsive to its proliferative actions can be an important step in the processes leading to malignancy.

Vascular smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of atherosclerosis and post-angioplasty restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting SMC contraction and proliferation, yet the exact mechanism is unknown. We therefore investigated the effect of berberine on SMC growth after mechanic injury in vitro. DNA synthesis and cell proliferation assay were performed to show that berberine inhibited serum-stimulated rat aortic SMC growth in a concentration-dependent manner. Mechanical injury with sterile pipette tip stimulated the regrowth of SMCs. Treatment with berberine prevented the regrowth and migration of SMCs into the denuded trauma zone. Western blot analysis showed that activation of the MEK1/2 (mitogen-activated protein kinase kinase 1/2), extracellular signal-regulated kinase (ERK), and up-regulation of early growth response gene (Egr-1), c-Fos and Cyclin D1 were observed sequentially after mechanic injury in vitro. Semi-quantitative reverse-transcription PCR assay further confirmed the increase of Egr-1, c-Fos, platelet-derived growth factor (PDGF) and Cyclin D1 expression in a transcriptional level. However, berberine significantly attenuated MEK/ERK activation and downstream target (Egr-1, c-Fos, Cyclin D1 and PDGF-A) expression after mechanic injury in vitro. Our study showed that berberine blocked injury-induced SMC regrowth by inactivation of ERK/Egr-1 signaling pathway thereby preventing early signaling induced by injury in vitro. The anti-proliferative properties of berberine may be useful in treating disorders due to inappropriate SMC growth.

Artificial vessel grafts are often used for the treatment of occluded blood vessels, but neointimal lesions commonly occur. To both elucidate and quantify which cell types contribute to the developing neointima, we established a novel mouse model of restenosis by grafting a decellularized vessel to the carotid artery. Typically, the graft developed neointimal lesions after 2 weeks, resulting in lumen closure within 4 weeks. Immunohistochemical staining revealed the presence of endothelial and smooth muscle cells, monocytes, and stem/progenitor cells at 2 weeks after implantation. Explanted cultures of neointimal tissues displayed heterogeneous outgrowth in stem cell medium. These lesional cells expressed a panel of stem/progenitor markers, including c-kit, stem cell antigen-1 (Sca-1), and CD34. Furthermore, these cells showed clonogenic and multilineage differentiation capacities. Isolated Sca-1(+) cells were able to differentiate into endothelial and smooth muscle cells in response to vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF)-BB stimulation in vitro. In vivo, local application of VEGF to the adventitial side of the decellularized vessel increased re-endothelialization and reduced neointimal formation in samples at 4 weeks after implantation. A population of stem/progenitor cells exists within developing neointima, which displays the ability to differentiate into both endothelial and smooth muscle cells and can contribute to restenosis. Our findings also indicate that drugs or cytokines that direct cell differentiation toward an endothelial lineage may be effective tools in the prevention or delay of restenosis.

To detect altered gene expression associated with mouse lung tumor progression, we compared the gene expression profile of lung adenocarcinomas with that of lung adenomas and normal lungs. Autoradiographic analysis showed that among the 588 genes surveyed, 152 genes were detected and the remaining 436 genes did not give any signals. A gene-specific semiquantitative reverse transcription polymerase chain reaction method was used to confirm the expression profile. A total of 29 genes was found to be differentially expressed in mouse lung tumors when compared to normal lungs. The pattern of expression, either underexpression or overexpression, was the same for 10 genes between adenocarcinomas and adenomas. Among them, seven genes were overexpressed, two genes were underexpressed and one gene was lost. Interestingly, 19 genes showed differential expression or increased incidence or difference in level of change between lung adenomas and adenocarcinomas, including Stat1, ADAP, IGFBP-6, PDGF-A, TGF-beta2, Int-3, VEGFR2, BAX, BAG-1, c-Jun, FasL, TRAIL, YB-1, CD31, Cdccan be designated as candidate 'lung tumor progression' (LTP) genes because their expression changes may specifically affect lung tumor progression in mice. Further analyses of these candidate LTP genes may provide new leads for elucidation of lung tumor progression in mice.

Endothelin-1 (ET-1), a 21-amino acid peptide released from the endothelium, elicits a variety of biological effects that include vascular smooth muscle cell (VSMC) contraction, release of secondary mediators, and cell proliferation. The present study was undertaken to examine the proliferative potential of ET-1 toward pulmonary artery VSMC in culture. In the presence of low serum and epidermal growth factor (EGF), ET-1 stimulated marked DNA synthesis and proliferation of VSMC. The contributing factor from serum appeared to be platelet-derived growth factor (PDGF) because the antibody to PDGF eliminated the stimulatory activity. The antibody to EGF also prevented the stimulation, suggesting that both PDGF and EGF are required for the full expression of the VSMC growth-promoting activity of ET-1. A paradoxical aspect of ET-1 effect on VSMC was the ability of ET-1 to inhibit the EGF-stimulated DNA synthesis when the two factors were added together to a high baseline DNA synthetic activity. The inhibition was prevented if ET-1 was added 12-18 h after the addition of EGF or if ET-1 and EGF were added to a protein kinase C-depleted VSMC. The inhibition by ET-1 may be mediated by protein kinase C activation followed by inhibition of EGF binding to its receptor. The results indicate that ET-1 under appropriate conditions can modulate the growth of pulmonary artery VSMC in both positive and negative directions.

The transcription factor NF-kappaB regulates cell cycle progression and proliferation in a number of cell types. An important unresolved issue is the potential role of NF-kappaB in the proliferation of vascular smooth muscle cells (VSMCs) as a basis for the development of vascular disease. To investigate the contribution of NF-kappaB to mitogen-induced proliferation of VSMCs, a knock-in mouse model expressing the NF-kappaB superrepressor IkappaBalphaDeltaN (c(IkappaBalphaDeltaN)) was used. Comparing wild-type and IkappaBalphaDeltaN-expressing VSMCs, we found that proliferation rates did not differ after mitogenic stimulation by platelet-derived growth-factor-BB (PDGF-BB) or serum. In line with this, NF-kappaB activation was not observed in VSMCs derived from transgenic mice expressing an NF-kappaB-dependent lacZ reporter (c((Igk)3conalacZ)). We further show, that classical mitogenic signaling pathways (namely mitogen-activated protein kinase [MAPK] and the phosphatidyl-inositol-3-OH-kinase [PI3K] pathways) control VSMC proliferation, but independently of NF-kappaB activation. In contrast to VSMCs, mouse embryonic fibroblasts (MEFs) derived from IkappaBalphaDeltaN-expressing mice showed significantly impaired proliferation rates after mitogenic stimulation. This was reflected by strongly impaired cyclin D1 expression in serum-stimulated MEFs derived from (c(IkappaBalphaDeltaN)) mice. These results implicate that essential pathogenetic functions of NF-kappaB in the development of atherosclerosis involve apoptotic and inflammatory signaling of VSMCs rather than proliferation. They further provide genetic evidence for a cell-type restricted requirement of NF-kappaB in the control of cellular proliferation.

Src-like adapter protein (Slap) is a recently identified protein that negatively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V. M. Dixit, S. A. Courtneidge, and A. Pandey, Curr. Biol. 8:975-978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate whether Slap could be an antagonist of all Src functions. Like Src, Slap was found to be myristylated in vivo and largely colocalized with Src when coexpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis, and the inhibition was rescued by the transcription factor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression overrides the G(1)/S block induced by both SrcK- and a Slap mutant with a deletion of its C terminus (SlapDeltaC), but not the block induced by Slap or SlapDeltaSH3, implying that the C terminus is a noncompetitive inhibitor of Src mitogenic function. Furthermore, a chimeric adapter comprising SrcDeltaK fused to the Slap C terminus (Src/SlapC) also inhibited Src function during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling. Slap, however, did not reverse deregulated Src-induced cell transformation, as it was unable to inhibit depolymerization of actin stress fibers while still being able to inhibit SrcY527F-induced DNA synthesis. This was attributed to a distinct Slap SH3 binding specificity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber formation. Indeed, three amino acids important for ligand binding in Src SH3 were replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 partners p85alpha, Shc, and Sam68 in vitro, and the chimeric tyrosine kinase Slap/SrcK, composed of SlapDeltaC fused to the SH2 linker kinase sequence of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is required for signaling during mitogenesis and since Slap/SrcK behaved as a dominant negative in the PDGF mitogenic response when microinjected into quiescent fibroblasts. We conclude that Slap is a negative regulator of Src during mitogenesis involving both the SH2 and the C terminus domains in a noncompetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.

The vast complexity of platelet-derived growth factor (PDGF)-induced downstream signaling pathways is well known, but the precise roles of critical players still elude us due to our lack of specific and temporal control over their activities. Accordingly, although Src family members are some of the better characterized effectors of PDGFbeta signaling, considerable controversy still surrounds their precise functions. To address these questions and limitations, we applied a chemical-genetic approach to study the role of c-Src at the cellular level, in defined signaling cascades; we also uncovered novel phosphorylation targets and defined its influence on transcriptional events. The spectacular control of c-Src on actin reorganization and chemotaxis was delineated by global substrate labeling and transcriptional analysis, revealing multiple cytoskeletal proteins and chemotaxis promoting genes to be under c-Src control. Additionally, this tool revealed the contrasting roles of c-Src in controlling DNA synthesis, where it transmits conflicting inputs via the phosphatidylinositol 3 kinase and Ras pathways. Finally, this study reveals a mechanism by which Src family kinases may control PDGF-mediated responses both at transcriptional and translational levels.

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. The PDGF B-chain (c-sis proto-oncogene) homodimer (PDGF BB) and v-sis, its viral counterpart, activate both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and mediate anchorage-independent growth in NIH3T3 cells. In contrast, the PDGF A chain homodimer (PDGF AA) activates alpha-PDGFR only and fails to induce phenotypic transformation. In the present study, we investigated alpha- and beta-PDGFR specific signaling pathways that are responsible for the differences between the transforming ability of PDGF AA and BB. To study PDGF BB activation of beta-PDGFR, we established NIH3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. Here, we demonstrate that beta-PDGFR activation alone is sufficient for PDGF BB-mediated anchorage-independent cell growth. More importantly, inhibition of alpha-PDGFR signaling enhanced PDGF BB-mediated phenotypic transformation, suggesting that alpha-PDGFR antagonizes beta-PDGFR-induced transformation. While both alpha- and beta-receptors effectively activate ERKs, alpha-PDGFR, but not beta-PDGFR, activates stress-activated protein kinase-1/c-Jun NH(2)-terminal kinase-1 (JNK-1). Inhibition of JNK-1 activity using a dominant-negative JNK-1 mutant markedly enhanced PDGF BB-mediated anchorage-independent cell growth, demonstrating an antagonistic role for JNK-1 in PDGF-induced transformation. Consistently, overexpression of wild-type JNK-1 reduced PDGF BB-mediated transformation. Taken together, the present study showed that alpha- and beta-PDGFRs differentially regulate Ras-mitogen-activated protein kinase pathways critical for regulation of cell transformation, and transformation suppressing activity of alpha-PDGFR involves JNK-1 activation.

The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts. PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min. PLD-catalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation. Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation. Collectively, these data indicate that PDGF activates both PLD and PC-PLC. In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly. DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC. Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling. In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD. D609 (2-10 microg/ml), an inhibitor of PC-PLC, blocked PDGF- but not epidermal growth factor-induced MAPK activation. However, D609 should be used with caution since it also affects PLD activity. The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.

OBJECTIVE

Migration of smooth muscle cells into the neointima has been implicated in atherogenesis. Vitronectin, a serum factor that promotes cell spreading and attachment, accumulates in atherosclerotic human tissues. The aim of this study was to determine the role of vitronectin and its receptor (integrin alpha V beta 3) in migration of smooth muscle cells.

METHODS

Human aortic smooth muscle cell migration was studied in modified Boyden chambers. Expression of vitronectin receptor was determined by northern blotting of receptor mRNA and immunoprecipitation of receptor protein.

RESULTS

Vitronectin dose dependently increased smooth muscle cell migration by an amount comparable to that induced by platelet derived growth factor, (PDGF)-BB. Antiserum to alpha V beta 3 diminished vitronectin driven migration. Northern blot analysis showed low constitutive expression of alpha V and beta 3 mRNA by smooth muscle cell and rapid induction with transforming growth factor beta (TGF-beta) and thrombin. Immunoprecipitation confirmed increased synthesis of the alpha V beta 3 vitronectin receptor complex by TGF-beta or thrombin. Smooth muscle cells pretreated with TGF-beta or thrombin showed increased vitronectin driven migration. cAMP suppressed induction of migration, but inhibition of protein kinase C increased it.

CONCLUSIONS

These results show that vitronectin-induced human aortic smooth muscle cell migration is mediated by alpha V beta 3 vitronectin receptor and expression of the receptor is induced by TGF-beta and thrombin, which in turn induce vitronectin driven, vitronectin receptor modulated smooth muscle cell migration.

Platelet-derived growth factor (PDGF) is a potent mitogenic and chemotactic cytokine, and PDGF-C is a novel growth factor belonging to the PDGF family. In this study, to determine whether this growth factor can contribute to fibrosis and tissue remodeling, we examined the effect of PDGF-CC on the expression of fibrogenic/fibrolytic genes such as type I collagen, fibronectin (FN), matrix metalloproteinases (MMPs), and their inhibitors (TIMPs) in normal human dermal fibroblasts in vitro. PDGF elevated the levels of MMP-1 or TIMP-1 protein as well as mRNA, whereas this cytokine had no influence on the expression of type I collagen, FN, or TIMP-2. PDGF-CC also increased the levels of MMP-1 catalytic activity in the cultured media and mRNA expression, which was paralleled that on the levels of promoter activation. Additionally, PDGF-CC induced the mitogenic and migratory activity of human dermal fibroblasts in a dose-dependent manner. On the other hand, we also determined the specificity of the inhibitory effect of monoclonal antibodies against PDGF-CC generated by immunizing balb/c mice with recombinant human PDGF-CC. This antibody could inhibit the regulatory effects of MMP-1 or TIMP-1 synthesis as well as the mitogenic effects on human dermal fibroblasts induced by PDGF-CC, whereas this antibody did not affect those induced by other PDGF forms such as PDGF-AA, -AB, or -BB. These results suggest that this cytokine plays a role in the tissue remodeling.

Most mammalian cells have the capacity to migrate. When placed into culture, cells will generally display a set rate of basal, unstimulated locomotion. The cells will begin to move in one direction and, after some time, change directions resulting in a random walk. External stimuli can influence cell motility in several ways to either enhance or retard the rate of migration (chemokinesis), to change the average amount of cell migration observed before the cell turns (persistence), or to increase the directionality of movement by limiting the number of turns made by the cells. Several factors have been identified that stimulate cell movement, but the signaling mechanisms that mediate this induced cell movement have only recently begun to be studied. In this review, we discuss the signals that support the directional movement of fibroblasts and epithelial cells in response to chemoattractant gradients. The work will emphasize studies carried out by our laboratory and others on the stimulation of cell motility by the PDGF. These results indicate that at least two sets of signaling molecules cooperate to regulate cell motility in vivo. These include phospholipase C-gamma, phosphoinositide-3' kinase and the Ras-GTPase activating protein Ras-GAP. The first set are those which bind to the intracellular domain of the receptor tyrosine kinase and bring about the phosphorylation and/or activation of intracellular effectors proximal to the receptor. The second is a set of down-stream effectors that regulate either the rate of cell movement or the directionality of that movement depending on the cell type. These include Ras and the Ras-related GTPase Rac along with free phosphoinositides and calcium ions that regulate the actin polymerization machinery. Signals that mediate nuclear changes leading to cell proliferation, such as elements of the MAP kinase pathway, do not appear to play a role in PDGF-stimulated cell migration. Current work thus suggests that a coordinated spatial regulation of signaling elements that interact with the cell membrane and cytoskeleton but not necessarily with nuclear elements is the controlling mediator of directional cell motility.

Vascular insufficiency and retinal ischaemia precede many proliferative retinopathies and stimulate secretion of vasoactive growth factors. Vascular endothelial growth factor (VEGF) plays a major role and we therefore investigated the other members of the VEGF family: Placental growth factor (PlGF), VEGF-B, -C, and -D, and platelet derived growth factors (PDGF) A and B. Neonatal mice were exposed to hyperoxia for 5 days and then returned to room air (resulting in acute retinal ischaemia). RT-PCR demonstrated that all the members of the VEGF family are expressed in the retina and in situ hybridization (ISH) located their mRNAs primarily in ganglion cells. Similarly to VEGF itself, VEGF-C, PDGF-A, and PDGF-B were upregulated during retinal ischaemia (P < 0.05). Only PlGF gene expression increased during hyperoxia (P < 0.01). The expression pattern of these growth factors suggests a role in the normal retina and during vaso-obliterative and ischaemic phases.

Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of 125I-labeled epidermal growth factor (125I-EGF). PDGF, like phorbol diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with [32P]phosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of [32P]phosphoserine and [32P]phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654, previously shown to be phosphorylated in response to phorbol diesters in intact cells or by purified protein kinase C in vitro. Further, it was observed that PDGF mimicked the action of phorbol diesters to inhibit the EGF-dependent tyrosine phosphorylation of the EGF receptor in [32P]phosphate-labeled fibroblasts. These results are consistent with the hypothesis that increases in diacylglycerol and Ca2+ levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF.

Platelet-derived growth factor (PDGF) stimulates expression of the c-myc, c-fos, JE, and KC genes in BALB/c/3T3 cells. Here we show that these genes respond differentially to at least two distinct intracellular second messengers generated by PDGF. A broad body of data support the view that response of the c-myc and c-fos genes to PDGF is channeled, at least in part, through activation of protein kinase C. Both PDGF and protein kinase C agonists stimulate transcription of c-myc and c-fos. Down regulation of protein kinase C inhibits the transcriptional induction of c-fos and c-myc by PDGF. In contrast, protein kinase C agonists do not stimulate transcription of JE and KC. Down regulation of protein kinase C does not inhibit the ability of PDGF to stimulate transcription of JE and KC. The differential response of these four genes to PDGF and 12-O-tetradecanoylphorbol-13-acetate correlates with differential phosphorylation of specific intracellular proteins. PDGF treatment stimulates phosphorylation of intracellular proteins at 31, 32, and 80 kilodaltons. Down regulation of protein kinase C prevents phosphorylation of the 80-kDa protein in response to PDGF, but phosphorylation of the smaller proteins is not affected. Finally, PDGF which induces all four genes (and stimulates phosphorylation of three proteins) is a much more potent mitogen than protein kinase C agonists which regulate only some of these events.

The platelet-derived growth factor (PDGF) family consists of subunits A and B and receptors alpha and beta. This paper evaluates the potential role of the homodimer PDGF-BB as a growth factor in meningiomas. It analyzes the expression of messenger RNA in members of the PDGF family in these tumors, measures the growth response of meningiomas to exogenous PDGF-BB in culture, and examines the induction of the c-fos proto-oncogene by PDGF-BB. Northern blot analysis was carried out on tissue from 20 meningiomas to measure the expression of PDGF-A, PDGF-B, PDGF-alpha receptor (PDGF-alpha-R) and PDGF-beta receptor (PDGF-beta-R). All tumors expressed PDGF-A and PDGF-B subunits. Nineteen of the 20 tumors expressed PDGF-beta-R and none expressed PDGF-alpha-R as measured by this technique. Because the beta receptor is selectively sensitive to stimulation by the PDGF-B subunit, these data suggest that meningiomas might be susceptible to stimulation by PDGF-BB. To test this hypothesis, the effect of exogenous PDGF-BB on meningioma growth was evaluated by incubating cells from 10 human meningiomas. Tritiated thymidine incorporation was used to evaluate stimulation of growth over a 48-hour period using PDGF-BB concentrations of 1, 3, or 6 ng/ml. Linear regression analysis and multiple-factor analysis of variance were used to measure PDGF-BB effects. Three of the 10 tumor specimens responded significantly to PDGF-BB, with a three- to sixfold increase in thymidine incorporation over 72 hours of exposure, and there was a significant overall growth-stimulating effect of PDGF-BB in the 10 tumor specimens tested. In the last set of experiments, the functionality of the PDGF-beta-R was determined by examining the induction of the proto-oncogene c-fos by PDGF-BB in meningioma cell cultures. A significant increase in c-fos protein was observed 3 hours after PDGF-BB addition. These findings demonstrate that PDGF-A, PDGF-B, and PDGF-beta-R are expressed in meningiomas and suggest that the beta receptor is functional: when it is activated, c-fos levels are increased, and an increase in meningioma cell division is observed after the addition of PDGF-BB. These studies support the hypothesis that PDGF acts as a growth factor in meningiomas.

Transforming growth factor-beta (TGF-beta) is thought to regulate ductal and lobuloalveolar development as well as involution in the mammary gland. In an attempt to understand the role TGF-beta plays during normal mammary gland development, and ultimately cancer, we previously generated transgenic mice that express a dominant-negative TGF-beta type II receptor under control of the metallothionine promoter (MT-DNIIR). Upon stimulation with zinc sulfate, the transgene was expressed in the mammary stroma and resulted in an increase in ductal side branching. In this study, mammary gland transplantation experiments confirm that the increase in side branching observed was due to DNIIR activity in the stroma. Development during puberty through the end buds was also accelerated. Cbl is a multifunctional intracellular adaptor protein that regulates receptor tyrosine kinase ubiquitination and downregulation. Mice with a targeted disruption of the c-Cbl gene displayed increased side branching similar to that observed in MT-DNIIR mice; however, end bud development during puberty was normal. Transplantation experiments showed that the mammary stroma was responsible for the increased side branching observed in Cbl-null mice. Cbl expression was reduced in mammary glands from DNIIR mice compared to controls and TGF-beta stimulated expression of Cbl in cultures of primary mammary fibroblasts. In addition, both TGF-beta and Cbl regulated platelet-derived growth factor receptor-alpha (PDGFR alpha) expression in vivo and in isolated mammary fibroblasts. The hypothesis that TGF-beta mediates the levels of PDGFR alpha protein via regulation of c-Cbl was tested. We conclude that TGF-beta regulates PDGFR alpha in the mammary stroma via a c-Cbl-independent mechanism. Finally, the effects of PDGF-AA on branching were determined. Treatment in vivo with PDGF-AA did not affect branching making a functional interaction between TGF-beta and PDGF unlikely.

PDGF-C is a new member of the PDGF-family and has recently been identified as a rat mesangial cell mitogen. Its expression and function in human kidneys is unknown. Localization of PDGF-C protein was analyzed by immunohistochemistry using a rabbit polyclonal antibody directed against the core-domain of PDGF-C in human fetal kidneys (n = 8), normal adult human kidneys (n = 9), and in renal biopsies of patients with IgA nephropathy (IgAN, n = 31), membranous nephropathy (MGN, n = 8), minimal change disease (MC, n = 7), and transplant glomerulopathy (TxG, n = 12). Additionally, PDGF-C mRNA was detected in microdissected glomeruli by real-time RT-PCR in cases of normal adult kidneys (n = 7), IgAN (n = 27), MGN (n = 11), and MC (n = 13). In the fetal kidney, PDGF-C localized to the developing mesangium, ureteric bud epithelium, and the undifferentiated mesenchyme. In the adult kidney, PDGF-C was constitutively expressed in parietal epithelial cells of Bowman's capsule, tubular epithelial cells (loops of Henle, distal tubules, collecting ducts), and in arterial endothelial cells. A marked upregulation of glomerular PDGF-C protein was seen in MGN and TxG with a prominent positivity of virtually all podocytes. In MC, PDGF-C localized to podocytes in a more focal distribution. In MGN, increased glomerular PDGF-C protein expression was due to increased mRNA synthesis as a 4.3-fold increase in PDGF-C mRNA was detected in microdissected glomeruli from MGN compared with normal. PDGF-C protein was additionally expressed in individual mesangial cells in TxG. Finally, upregulated PDGF-C protein expression was detected within sclerosing glomerular and fibrosing tubulointerstitial lesions in individual cases from all analyzed groups. We conclude that PDGF-C is constitutively expressed in the human kidney and is upregulated in podocytes and interstitial cells after injury/activation of these cells.

Prostate carcinoma cell lines DU-145 and PC-3 express both platelet derived growth factor (PDGF)-1 and PDGF-2/sis genes. Concomitantly, these cells synthesize and secrete PDGF-like proteins, as judged by indirect immunofluorescence and by direct immunoprecipitation with specific PDGF antiserum. Conditioned media derived from DU-145 and PC-3 cells stimulated the incorporation of [3H]thymidine by 3T3 cells and competed with 125I-labeled PDGF for its binding to cell surface receptors of 3T3 cells. The biological activity was stable to heating at 100 degrees C for 10 min, sensitive to reducing agents, and neutralized by the IgG fraction of PDGF antiserum, properties similar to those of authentic PDGF. Both DU-145 and PC-3 cell lines appear to lack receptors for PDGF as indicated by their inability to mitogenically respond to PDGF and receptor binding of 125I-labeled PDGF. Production of PDGF-like proteins by human prostate carcinoma cells may play an important role in a paracrine mode in the organization of the extracellular matrix of the malignant tissue.

To evaluate the role of protein kinase C-mediated pathways in the proliferation of malignant gliomas, this study examined the effect of a protein kinase C (PKC)-activating phorbol ester (12-O-tetradecanoyl-13-phorbol acetate or TPA) and a protein kinase C inhibitor (polymyxin B) on deoxyribonucleic acid (DNA) synthesis of malignant glioma cells in vitro. A serum-free chemically defined medium, MCDB 105, was employed for all studies. Two established human malignant glioma cell lines (T98G and U138), two rat glioma lines (9L and CCPDGF). Inhibition of PKC using the antibiotic polymyxin B (20 micrograms/ml) abolished the TPA-induced mitogenic response in the five responsive lines tested. In two tumors (U138 and 9L), polymyxin B also eliminated EGF-, PDGF-, and serum-induced DNA synthesis as well as abolishing baseline DNA synthesis. These cells remained viable, however, as assessed by trypan blue exclusion; after removal of polymyxin B from the medium, they were able to resume DNA synthesis in response to TPA and serum. In the three other tumors (T98G and the two low-passage human glioma lines), growth factor-induced and serum-induced DNA synthesis were inhibited by approximately 25% to 85%. It is concluded that PKC-mediated pathways affect DNA synthesis in the human malignant glial tumors studied. The response of the glioma cells to TPA is similar to the responses seen in fetal astrocytes, but differs significantly from those reported for normal adult glial cultures. Because the response of the 9L glioma to TPA is similar to the responses seen in the human tumors, the 9L rat glioma model may prove useful for examining the role of PKC-mediated pathways in controlling glioma growth in vivo.

Platelet-derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B-chain is encoded by the c-sis proto-oncogene, which is expressed in several malignant and non-malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A-chain has been reported to occur in human solid tumor cell lines independently of c-sis expression. We report here the non-coordinate expression of the A- and B-chains in human leukemia cell lines. The PDGF-A and B-chain (c-sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c-sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor-beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL-60 does not express PDGF-A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A-chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF-A expression in HL-60 cells is also observed after treatment with the tumor necrosis factor-alpha but granulocytic differentiation of HL-60 cells induced with dimethyl sulfoxide or the granulocyte colony-stimulating factor is not associated with PDGF gene expression.

Fibroblasts are heterogeneous with respect to surface markers, morphology, and participation in fibrotic responses. This study was undertaken to determine whether Thy-1(-) and Thy-1(+) rat lung fibroblasts, which have distinct and relevant phenotypes, differ in their proliferative responses to platelet-derived growth factor (PDGF) isoforms. Homogeneous populations of Thy-1(-) and Thy-1(+) fibroblasts were found to proliferate equally in the presence of PDGF-BB, but PDGF-AA-mediated proliferation occurred only in Thy-1(-) cells. This differential activity correlated with significantly higher expression of PDGF-alpha receptor in Thy-1(-) fibroblasts as shown by immunoblotting, immunofluorescence, and Northern blotting. There was a rapid increase in c-myc mRNA in Thy-1(-) but not in Thy-1(+) fibroblasts on stimulation with PDGF-AA and PDGF-BB. The PDGF-alpha receptor, which mediates signaling by all PDGF isoforms, has been implicated in numerous clinical and experimental forms of fibrosis and regulates lung morphogenesis. Differential expression of the PDGF-alpha receptor supports distinct roles for Thy-1(-) and Thy-1(+) fibroblast populations in developmental and fibrotic processes in the lung.

We have examined the constitutive and inducible secretion of platelet-derived growth factor (PDGF)-like proteins in a variety of human hemopoietic cell lines. The highest levels of secreted protein were noted in four human erythroleukemia lines which, in addition to erythroid lineage markers, express one or more megakaryocytic lineage markers. Induction of these lines by 12-O-tetradecanoylphorbol-13-acetate enhanced the expression of megakaryocytic markers and increased secretion of PDGF-like proteins several fold. In concert with these changes, there was significant induction of c-sis/PDGF-B messenger RNA (mRNA) expression in all lines, whereas one line showed significant concurrent induction of PDGF-A mRNA expression. Whether PDGF-like secretion is part of the stem cell-like phenotype displayed by these lines or is secondary to their leukemic transformation remains to be determined. Nevertheless, these lines provide new cellular models for studying the expression and function of PDGF analogs in hemopoietic cells.